{"gene":"RPS6","run_date":"2026-04-28T20:42:06","timeline":{"discoveries":[{"year":1988,"finding":"A mitogen-activated S6 kinase of Mr 70,000 (p70 S6K) was purified to homogeneity from Swiss 3T3 cells stimulated by EGF, serum, or orthovanadate. The purified kinase phosphorylates ribosomal protein S6 of the 40S subunit in vitro, exhibits autophosphorylation activity, and is inactivated by phosphatase 2A treatment, establishing that the kinase itself is regulated by phosphorylation.","method":"Biochemical purification (Mono Q/Mono S chromatography), in vitro kinase assay with 40S subunit, phosphatase 2A inactivation","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 — purification to homogeneity with in vitro reconstitution and phosphatase inactivation","pmids":["3257566"],"is_preprint":false},{"year":1989,"finding":"A hepatic 70 kDa S6 kinase purified from cycloheximide-treated rats phosphorylates ribosomal protein S6 and additional substrates including histone H2b, glycogen synthase, and ATP citrate lyase (the latter on the same serine phosphorylated by insulin in intact cells), demonstrating broad substrate specificity beyond S6.","method":"50,000-fold purification using peptide affinity column (C-terminal S6 peptide), in vitro kinase assays with multiple substrates","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 — near-homogeneous purification with defined substrate mapping","pmids":["2760046"],"is_preprint":false},{"year":1989,"finding":"An insulin-stimulated S6 kinase purified from rabbit liver has a molecular weight of ~70,000 and its activity is abolished by phosphatase 2A treatment, confirming phosphorylation-dependent activation. The inactivated enzyme can be re-activated ~5-fold by preincubation with partially purified MAP-2 kinase, placing a MAP kinase-like activity upstream.","method":"Multi-step chromatographic purification, phosphatase 2A inactivation, reactivation by MAP-2 kinase","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 1 methods but single lab, upstream kinase identification partial","pmids":["2553707"],"is_preprint":false},{"year":1991,"finding":"The purified 70K S6 kinase phosphorylates S6 exclusively on five sites (Ser235, Ser236, Ser240, Ser244, and a fifth low-stoichiometry site) within a 19-amino acid peptide at the carboxyl terminus of S6, matching the in vivo phosphorylation pattern. Mn²⁺ and Zn²⁺ inhibit the kinase with IC₅₀ ~2 µM for Zn²⁺, and autophosphorylation (~1.2 mol/mol) reduces activity ~25%.","method":"In vitro kinase assay with purified 70K S6K and 40S subunit, tryptic phosphopeptide mapping, site identification by sequencing, metal ion inhibition studies","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 — in vitro reconstitution with direct site identification matching in vivo data","pmids":["1939282"],"is_preprint":false},{"year":1981,"finding":"S6 phosphorylation in HeLa cells peaks rapidly (~30 min) after serum stimulation, preceding the slower increase in translation elongation/initiation rates, and correlates temporally with mRNA recruitment into polysomes, suggesting rpS6 phosphorylation may facilitate mRNA recruitment rather than globally controlling elongation.","method":"Polysome profiling, kinetic analysis of S6 phosphorylation vs. protein synthesis rates in suspension culture","journal":"European journal of biochemistry","confidence":"Medium","confidence_rationale":"Tier 2 — kinetic correlation study in intact cells, single lab","pmids":["7333277"],"is_preprint":false},{"year":1984,"finding":"Extensive phosphorylation of ribosomal protein S6 is associated with conformational changes in the 40S ribosomal subunit, specifically altered accessibility of proteins S3, S4, S7, S23/24 (small subunit) and L9, L10, L12, L18, L27, L34, L36 (large subunit) to reductive methylation.","method":"Reductive methylation accessibility assay comparing ribosomes with unphosphorylated vs. phosphorylated S6","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 — direct biochemical comparison, single lab, single method","pmids":["6693389"],"is_preprint":false},{"year":1986,"finding":"Insulin, IGF-1, and TPA activate a cytosolic S6 kinase in rat astrocytes, demonstrating that S6 kinase responds to tyrosine kinase receptors and protein kinase C activation in neural cells. The effect is insensitive to cycloheximide, indicating activation occurs through post-translational modification of a pre-existing kinase.","method":"S6 kinase activity assay in cytosolic fractions of cultured astrocytes after hormone treatment; cycloheximide control","journal":"FEBS letters","confidence":"Medium","confidence_rationale":"Tier 2 — direct kinase activity measurement, multiple stimuli tested","pmids":["3530808"],"is_preprint":false},{"year":1992,"finding":"Rapamycin selectively inhibits p70 S6 kinase activity and blocks the appearance of the hyperphosphorylated form of p70 S6K in COS cells, but does not affect p85 Rsk S6 kinase or MAP kinase, demonstrating that a rapamycin-sensitive signaling element is specifically required for p70 S6K activation.","method":"Kinase assays with 40S subunits, SDS-PAGE mobility shift, selective inhibitor treatment in hepatoma cells and transfected COS cells","journal":"Science","confidence":"High","confidence_rationale":"Tier 2 — replicated across cell lines with selective pharmacological dissection","pmids":["1380182"],"is_preprint":false},{"year":1994,"finding":"The p70/p85 S6 kinase signaling pathway is independent of p21ras: dominant-negative p74raf and p21ras block p44MAPK but not p70S6K activation. Furthermore, activation of p70S6K by PDGFR requires autophosphorylation at tyrosine 751 within the kinase-insert domain of the receptor, bifurcating the signaling from the ras-MAPK pathway at the receptor level.","method":"Dominant-negative Raf and Ras mutant expression, epitope-tagged kinase assays, PDGFR kinase-insert domain mutants","journal":"Nature","confidence":"High","confidence_rationale":"Tier 2 — genetic epistasis with multiple mutants, replicated in multiple cell lines","pmids":["8090223"],"is_preprint":false},{"year":1995,"finding":"FRAP/mTOR kinase activity is required in vivo for activation of p70 S6K: kinase-dead FRAP variants fail to activate p70S6K, and FRAP autophosphorylates in vitro; autophosphorylation is inhibited by the FKBP12-rapamycin complex. An N-terminal domain of FRAP beyond its kinase domain is also required for p70S6K regulation.","method":"FRAP variant overexpression, in vitro autophosphorylation, rapamycin-FKBP12 inhibition of autophosphorylation, kinase-dead mutant analysis","journal":"Nature","confidence":"High","confidence_rationale":"Tier 1 — in vitro kinase assay combined with genetic epistasis, replicated","pmids":["7566123"],"is_preprint":false},{"year":1996,"finding":"S6 kinase contains an autoinhibitory domain (codons 58–77) that overrides mitogen-dependence; deletion of the first 77 amino acids produces a constitutively active, rapamycin-insensitive S6K. A separate N-terminal domain (codons 1–58) is required for mitogen-induced activation and rapamycin sensitivity, defining two functionally distinct N-terminal regulatory regions.","method":"Deletion mutagenesis of S6K, expression in Rat1 cells, kinase activity assays, morphological phenotype analysis","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 1 — systematic deletion analysis with reconstitution and functional readout","pmids":["8524322"],"is_preprint":false},{"year":1998,"finding":"RAFT1/mTOR directly phosphorylates p70 S6K on Thr-389 (a rapamycin-sensitive site essential for S6K activity) in vitro and in vivo, and also phosphorylates 4E-BP1 and 4E-BP2. Thr-389 phosphorylation is necessary for S6K activity, and RAFT1 phosphorylates p70S6K much more efficiently than 4E-BP1.","method":"In vitro kinase assay with immunoprecipitated RAFT1/mTOR, phospho-specific site mapping, rapamycin treatment, serum stimulation kinetics","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 — direct in vitro phosphorylation with site mapping and functional validation","pmids":["9465032"],"is_preprint":false},{"year":1998,"finding":"Amino acid withdrawal rapidly inactivates p70 S6K and promotes 4E-BP1 dephosphorylation in CHO cells through a pathway sensitive to rapamycin and PI3K inhibitors but independent of protein kinase B (PKB/Akt), demonstrating that amino acids regulate p70 S6K via a distinct upstream pathway from insulin/PKB.","method":"Amino acid deprivation/repletion in CHO cells, kinase activity assays, PI3K and mTOR inhibitor treatments, PKB activity measurements","journal":"The Biochemical journal","confidence":"High","confidence_rationale":"Tier 2 — multiple inhibitors dissecting pathway, replicated by multiple groups","pmids":["9693128"],"is_preprint":false},{"year":1999,"finding":"Drosophila S6 kinase (dS6K) regulates cell size in a cell-autonomous manner: dS6K null flies display extreme developmental delay, severe body size reduction due to smaller cells (not fewer cells), a phenotype distinct from ribosomal protein mutations (Minutes). This genetically establishes S6K as a cell-size regulator.","method":"Drosophila dS6K null mutant analysis, cell counting, cell size measurement, mosaic analysis","journal":"Science","confidence":"High","confidence_rationale":"Tier 2 — clean genetic loss-of-function with specific cellular phenotype, highly cited","pmids":["10497130"],"is_preprint":false},{"year":1999,"finding":"Leucine activation of p70S6K in amino acid-depleted cells requires strict structural features: the correct chirality, the four-branched hydrocarbon structure, and a primary amine. The effect is rapamycin-sensitive but less sensitive to wortmannin, indicating that leucine acts via mTOR rather than PI3K to activate p70S6K.","method":"p70S6K activity assays with leucine analogues in H4IIE cells, pharmacological inhibition with rapamycin and wortmannin","journal":"FEBS letters","confidence":"Medium","confidence_rationale":"Tier 2 — structure-activity relationship with pharmacological dissection, single lab","pmids":["10214966"],"is_preprint":false},{"year":1999,"finding":"Osmotic stress (sorbitol) inactivates p70/p85 S6K and causes dephosphorylation of Thr412 in the COOH-terminus via a calyculin A-sensitive phosphatase, similar to rapamycin. Rapamycin-resistant truncation mutants of S6K are also resistant to osmotic stress-induced inactivation, demonstrating that both rapamycin and osmotic stress converge on a common calyculin A-sensitive phosphatase-dependent mechanism.","method":"S6K kinase assays, phospho-specific antibodies, pharmacological inhibitors of phosphatases, rapamycin-resistant S6K truncation mutants","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1-2 — genetic (truncation mutants) and pharmacological epistasis converging on same mechanism","pmids":["10455142"],"is_preprint":false},{"year":2000,"finding":"TGF-β induces association of its receptor with PP2A-Bα subunit, leading to assembly of a PP2A complex (Bα, Aβ, Cα subunits) with p70S6K, dephosphorylation and inactivation of p70S6K, and G1 arrest. This is a second pathway for TGF-β-induced cell cycle arrest parallel to Smad2/3 activation.","method":"Co-immunoprecipitation of PP2A subunits with TGF-β receptor and p70S6K, kinase activity assays, G1 arrest rescue experiments","journal":"Genes & development","confidence":"High","confidence_rationale":"Tier 2 — reciprocal co-IP plus functional epistasis with two independent pathways dissected","pmids":["11124802"],"is_preprint":false},{"year":1999,"finding":"S6K1 and S6K2 activation requires sequential phosphorylation: PI3K-mediated phosphorylation of C-terminal sites permits PDK1 to phosphorylate the activation loop residue, and mTOR acts as a checkpoint for amino acid availability. At least eight phosphorylation sites mediate S6K1 activation in a hierarchical fashion.","method":"Review synthesizing phosphorylation site mutant data, PI3K/mTOR pathway dissection, PDK1 phosphorylation of activation loop","journal":"Experimental cell research","confidence":"Medium","confidence_rationale":"Tier 2 — synthesis of multiple labs' mutagenesis and epistasis data","pmids":["10579915"],"is_preprint":false},{"year":2002,"finding":"S6K2 activation requires mTOR kinase activity, amino acid sufficiency, and phosphatidic acid, similar to S6K1. Manipulation of mTOR nuclear/cytoplasmic distribution (using NLS/NES tags) showed that optimal mTOR shuttling is required for maximal S6K2 activation, with S6K2 being a predominantly nuclear protein.","method":"mTOR NLS/NES tagging constructs, kinase activity assays, subcellular fractionation, leptomycin B treatment","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 — direct manipulation of localization with functional consequence, single lab","pmids":["12087098"],"is_preprint":false},{"year":2002,"finding":"Loss of TSC2/tuberin leads to constitutive hyperphosphorylation of ribosomal protein S6 and constitutive activation of p70S6K in LAM patient-derived smooth muscle cells and TSC2-/- cells. Re-introduction of wild-type tuberin abolishes S6 phosphorylation and inhibits p70S6K, demonstrating that TSC2 negatively regulates the p70S6K–rpS6 axis.","method":"Immunoblotting of S6 phosphorylation, p70S6K kinase assays, tuberin re-expression in TSC2-null cells, rapamycin treatment","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 — genetic rescue experiment in patient-derived and TSC2-null cells with multiple readouts","pmids":["12045200"],"is_preprint":false},{"year":2003,"finding":"TSC1/2 acts as a GAP for the small GTPase Rheb, and insulin-activated Rheb (via PI3K) drives S6K1 phosphorylation; loss-of-function TSC1/2 mutations phenocopy Rheb overexpression in activating S6K1. A disease-associated GAP-domain point mutation in TSC2 fails to stimulate Rheb GTPase or block Rheb-induced S6K1 activation.","method":"GTPase assay with purified Rheb, epistasis by co-expression, TSC2 disease mutant analysis, S6K1 phosphorylation assays","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 1-2 — biochemical GAP assay plus genetic epistasis with disease mutant validation","pmids":["12820960"],"is_preprint":false},{"year":2003,"finding":"M-CSF, RANKL, and TNFα promote osteoclast survival through convergent mTOR/S6K signaling; rapamycin blocks cytokine-dependent osteoclast survival and induces apoptosis. Inhibitors of PI3K, Akt, ERK, and geranylgeranylation suppress cytokine-induced S6K activation and osteoclast survival, and inhibition of translation by other mechanisms also induces apoptosis, demonstrating that mTOR/S6K-dependent protein synthesis is essential for osteoclast survival.","method":"Rapamycin and signaling inhibitor treatment, S6K activity assays, apoptosis assays, in vitro bone resorption","journal":"Cell death and differentiation","confidence":"Medium","confidence_rationale":"Tier 2 — pharmacological pathway dissection with functional cell death readout","pmids":["14502240"],"is_preprint":false},{"year":2004,"finding":"Apelin (65-77) activates p70 S6K in CHO cells and endothelial cells via dual pathways: a PI3K→Akt pathway (blocked by wortmannin) leading to T389 and T421/S424 phosphorylation, and an ERK pathway (blocked by PD098059) leading to T421/S424 phosphorylation. PTX and atypical PKC inhibitors abrogate both Akt and p70S6K phosphorylation, and apelin-induced p70S6K activation is mitogenic for HUVEC cells.","method":"Kinase assays with phospho-specific antibodies, pathway inhibitors, PTX treatment, thymidine incorporation assay","journal":"FASEB journal","confidence":"Medium","confidence_rationale":"Tier 2 — multiple pharmacological dissections with functional mitogenic readout, single lab","pmids":["15385434"],"is_preprint":false},{"year":2005,"finding":"mTOR/S6K1 and the eIF3 translation initiation complex function as a dynamic scaffold: inactive S6K1 associates with eIF3 basally; cell stimulation recruits mTOR/raptor to eIF3 where it phosphorylates S6K1 at its hydrophobic motif; S6K1 then dissociates, becomes activated, and phosphorylates eIF4B, which is subsequently recruited to the eIF3 complex.","method":"Co-immunoprecipitation of mTOR, S6K1, eIF3, and eIF4B; phospho-specific antibodies; rapamycin treatment; sequential IP/kinase assays","journal":"Cell","confidence":"High","confidence_rationale":"Tier 2 — dynamic complex assembly demonstrated by reciprocal co-IP with ordered phosphorylation events, highly cited","pmids":["16006006","16286006"],"is_preprint":false},{"year":2005,"finding":"hVps34 (Class III PI3K) is required for p70 S6K1 activation in response to insulin and amino acids, acting upstream of mTOR. Inhibitory anti-hVps34 antibodies, FYVE domain sequestration of PI3P, and siRNA knockdown of hVps34 all block S6K1 activation. hVps34 is inhibited by amino acid/glucose starvation and AMPK activation, but not by insulin, placing it on the nutrient-sensing arm to mTOR/S6K1.","method":"Microinjection of inhibitory antibodies, FYVE domain overexpression, siRNA knockdown, hVps34 kinase activity assays, insulin stimulation of S6K1","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal loss-of-function approaches converging on same conclusion","pmids":["16049009"],"is_preprint":false},{"year":2005,"finding":"p70 S6K directly phosphorylates tau at S262, S214, and T212 in vitro, and co-immunoprecipitation reveals association between activated p-p70S6K (T421/S424) and phospho-tau (S262) in cells. Zinc-induced p70S6K activation selectively upregulates translation of S6 and tau mRNAs but not global protein synthesis.","method":"In vitro kinase assay with p70S6K and tau, immunoprecipitation, immunoblotting with phospho-specific antibodies, metabolic labeling","journal":"FEBS letters","confidence":"Medium","confidence_rationale":"Tier 1 — in vitro phosphorylation demonstrated, but limited validation in cells; single lab","pmids":["16364302"],"is_preprint":false},{"year":2006,"finding":"S6 kinases are ubiquitinated in cells and subject to proteasome-mediated degradation. Ubiquitination sites are located in the kinase domain, and the N- and C-terminal regulatory regions modulate the efficiency of S6K ubiquitination, identifying a second post-translational mechanism (beyond phosphorylation) controlling S6K abundance.","method":"Co-transfection with ubiquitin constructs, proteasome inhibitor treatment, immunoprecipitation, ubiquitin site mapping by domain deletion","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 3 — co-IP/overexpression based, single lab, no E3 ligase identified","pmids":["18280803"],"is_preprint":false},{"year":2006,"finding":"S6K1 and S6K2 interact with receptor tyrosine kinases, specifically PDGFR, via the kinase or kinase extension domain of S6K in a growth-factor-inducible manner. This complex formation leads to tyrosine phosphorylation of S6K via a PDGFR-Src pathway at Y39 (S6K1) and Y45 (S6K2), but these tyrosine phosphorylations do not affect S6K activity or subcellular localization.","method":"Co-immunoprecipitation with PDGFR mutants, tyrosine phosphorylation mapping, Src inhibitors (PP1, SU6656), immunofluorescence","journal":"The FEBS journal","confidence":"Medium","confidence_rationale":"Tier 2-3 — reciprocal co-IP and site mapping with mutants, but functional consequence is negative","pmids":["16640565"],"is_preprint":false},{"year":2006,"finding":"Ribosomal proteins Rpl10 and Rps6 are potent regulators of yeast replicative lifespan: deletion of RPS6B (but not RPS6A) extends replicative lifespan by 45% in S. cerevisiae, associated with altered free ribosomal subunit stoichiometry and reduced cell size. This establishes an orthologous role for Rps6 gene dosage in aging.","method":"Yeast deletion mutants, replicative lifespan assays, ribosome profiling by polysome analysis, cell size measurement","journal":"Experimental gerontology","confidence":"Medium","confidence_rationale":"Tier 2 — genetic deletion with defined phenotype in yeast ortholog system","pmids":["17174052"],"is_preprint":false},{"year":2007,"finding":"ATG1 (autophagy kinase) inhibits TOR/S6K-dependent cell growth in Drosophila by blocking S6K activation at Thr389 phosphorylation. In mammalian cells, ATG1 overexpression inhibits S6K, and siRNA knockdown of ATG1 induces ectopic S6K activation and S6 phosphorylation, revealing crosstalk between autophagy regulation and the TOR/S6K growth pathway.","method":"Drosophila ATG1 mutant and overexpression analysis, mammalian S6K kinase assays, siRNA knockdown, phospho-Thr389 antibody","journal":"EMBO reports","confidence":"High","confidence_rationale":"Tier 2 — genetic and biochemical evidence in two organisms, bidirectional manipulation","pmids":["17347671"],"is_preprint":false},{"year":2007,"finding":"S6K1 deletion in skeletal muscle cells causes increased AMP/ATP and inorganic phosphate/phosphocreatine ratios, triggering AMPK upregulation. AMPK inhibition in S6K-deficient cells restores cell growth and nutrient sensitivity, demonstrating that S6K1 controls cellular energy state and that AMPK activation mediates the cell atrophy caused by S6K1 deletion.","method":"S6K1/2 knockout mice, AMPK activity assays, metabolite measurements (AMP, ATP, phosphocreatine), AMPK inhibition rescue","journal":"Cell metabolism","confidence":"High","confidence_rationale":"Tier 2 — genetic KO with metabolic rescue experiment and AMPK epistasis","pmids":["17550782"],"is_preprint":false},{"year":2008,"finding":"S6K1 deletion in mice leads to increased lifespan and resistance to age-related pathologies (bone, immune, motor dysfunction, insulin sensitivity loss), with gene expression patterns similar to caloric restriction or AMPK activation, demonstrating that S6K1 is a lifespan regulator in mammals.","method":"S6K1 knockout mouse model, lifespan analysis, metabolic phenotyping, gene expression profiling","journal":"Science","confidence":"High","confidence_rationale":"Tier 2 — in vivo genetic KO with comprehensive phenotyping, highly cited","pmids":["19797661"],"is_preprint":false},{"year":2008,"finding":"Mediobasal hypothalamic (MBH) S6K1 bidirectionally controls energy homeostasis: stereotaxic adenoviral activation of MBH S6K alters food intake, body weight, orexigenic neuropeptide expression, and leptin sensitivity; constitutive activation protects against high-fat diet-induced obesity and insulin resistance.","method":"Bilateral adenoviral injection of dominant-negative or constitutively active S6K into rat MBH, metabolic phenotyping, neuropeptide measurements","journal":"Cell metabolism","confidence":"High","confidence_rationale":"Tier 2 — bidirectional in vivo manipulation with specific anatomical targeting and multiple metabolic readouts","pmids":["19041762"],"is_preprint":false},{"year":2008,"finding":"Drosophila S6KII (p90 RSK) cooperates with casein kinase 2 (CK2) in circadian clock neurons to regulate circadian period: S6KII null flies show short-period phenotype rescued by clock-neuron-specific re-expression; S6KII physically interacts with CK2β; genetic interactions show CK2 activity is required for S6KII's clock function.","method":"Drosophila null mutant analysis, circadian behavioral assays, cell-type-specific rescue, co-IP of S6KII and CK2β, genetic epistasis with CK2 mutants","journal":"The Journal of neuroscience","confidence":"Medium","confidence_rationale":"Tier 2 — genetic and biochemical evidence in Drosophila ortholog for RSK2 function in circadian regulation","pmids":["19144847"],"is_preprint":false},{"year":2009,"finding":"Resveratrol inhibits mammalian S6K1 (identified in a large-scale in vitro kinase screen) and attenuates autophagy induced by nutrient limitation or rapamycin. Dominant-negative S6K1 mutant or siRNA knockdown of S6K1 recapitulates the autophagy-suppressing effect of resveratrol, and combining resveratrol with S6K1 knockdown is not additive, indicating S6K1 is a key target of resveratrol's autophagy-modulating effect.","method":"Large-scale in vitro kinase screen, dominant-negative S6K1 expression, siRNA knockdown, autophagy assays","journal":"Aging","confidence":"Medium","confidence_rationale":"Tier 2 — kinase screen plus functional rescue/epistasis, single lab","pmids":["20157535"],"is_preprint":false},{"year":2009,"finding":"mTOR/S6K1 signaling protects astrocytes from ischemia-induced apoptosis: OGD reduces S6K1 mRNA and protein; S6K1/2 double knockout astrocytes show increased apoptosis, reduced BAD phosphorylation, decreased Bcl-2/Bcl-xL, increased ROS, and impaired translation recovery. Rescue with adenoviral S6K1 (but not S6K2) specifically reduces ROS and confers ischemic resistance.","method":"S6K1/S6K2 double knockout mouse astrocytes, OGD model, adenoviral isoform-specific rescue, apoptosis assays, ROS measurement, middle cerebral artery occlusion in vivo","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 — isoform-specific rescue in KO cells with multiple orthogonal readouts and in vivo validation","pmids":["19535330"],"is_preprint":false},{"year":2009,"finding":"S6 kinase ortholog RSKS-1 in C. elegans inhibits axon regeneration in mechanosensory neurons: rsks-1 loss-of-function accelerates growth cone formation and axon extension after laser axotomy; this enhanced regrowth is partly dependent on DLK-1 MAPK cascade, and a key downstream effector is the metabolic sensor AMP kinase AAK-2. The antidiabetic drug phenformin (AMPK activator) promotes axon regrowth.","method":"C. elegans laser axotomy, rsks-1 null mutants, genetic epistasis with DLK-1 and AAK-2, pharmacological AMPK activation","journal":"The Journal of neuroscience","confidence":"Medium","confidence_rationale":"Tier 2 — genetic epistasis in C. elegans model with pharmacological validation","pmids":["24431434"],"is_preprint":false},{"year":2010,"finding":"AKT-mTORC1-RPS6 signaling promotes de novo lipogenesis in hepatocellular carcinoma via transcriptional and post-transcriptional mechanisms including inhibition of fatty acid synthase ubiquitination by USP2a and disruption of SREBP1/SREBP2 degradation complexes. siRNA knockdown of RPS6 inhibits lipogenesis-related gene activity and reduces HCC cell proliferation and survival.","method":"siRNA knockdown of RPS6, inhibitor studies (rapamycin, pathway inhibitors), gene expression analysis, lipogenic enzyme activity assays, USP2a/SREBP pathway analysis in HCC cell lines and mouse models","journal":"Gastroenterology","confidence":"Medium","confidence_rationale":"Tier 2 — siRNA knockdown with multiple mechanistic readouts, single lab","pmids":["21147110"],"is_preprint":false},{"year":2011,"finding":"Heat shock activates both pp70 S6K and pp90rsk in quiescent fibroblasts, with a temporal activation pattern reversed from mitogen-stimulated cells. The heat shock-induced pp70 S6K was identified as equivalent to the serum/growth factor-activated enzyme by chromatographic properties, and both kinases drive S6 phosphorylation in heat-shocked cells.","method":"Sequential column chromatography purification, immunoprecipitation kinase assay for pp90rsk, S6 phosphorylation measurement in quiescent fibroblasts after heat shock","journal":"Journal of cellular physiology","confidence":"Medium","confidence_rationale":"Tier 2 — partial purification with immunoprecipitation comparison, single lab","pmids":["1880153"],"is_preprint":false},{"year":2012,"finding":"rpS6 regulates blood-testis barrier (BTB) dynamics: rpS6 is spatiotemporally activated at the BTB during the spermatogenic cycle; siRNA-mediated knockdown of rpS6 in vitro and in vivo promotes the Sertoli cell TJ permeability barrier by recruiting claudin-11 and occludin to the BTB. rpS6 activation disrupts the TJ barrier via effects on F-actin organization and adhesion protein recruitment.","method":"siRNA and shRNA knockdown of rpS6 in Sertoli cells in vitro and in adult rat testes in vivo, BTB integrity assay, immunofluorescence for F-actin and junctional proteins","journal":"Endocrinology","confidence":"High","confidence_rationale":"Tier 2 — bidirectional manipulation (knockdown in vitro and in vivo) with specific structural and functional readouts","pmids":["22948214"],"is_preprint":false},{"year":2013,"finding":"S6K1 and S6K2 double knockout mouse liver shows a specific defect in the ribosome biogenesis (RiBi) transcriptional program after feeding: over 75% of RiBi factors (Nop56, Nop14, Gar1, etc.) are controlled by S6K. Importantly, this RiBi transcriptional defect is also observed in rpS6 knock-in mice with non-phosphorylatable serine residues, identifying rpS6 phosphorylation as specifically regulating RiBi transcription independently of ribosomal protein synthesis.","method":"S6K1/S6K2 double knockout mice, rpS6 phosphorylation-site knock-in mice, whole-genome microarray of total and polysomal liver RNA, feeding paradigm","journal":"Oncogene","confidence":"High","confidence_rationale":"Tier 2 — two independent genetic models (S6K DKO and rpS6 knock-in) converging on same transcriptional phenotype","pmids":["23318442"],"is_preprint":false},{"year":2013,"finding":"Arginine, leucine, and glutamine activate the MTOR-RPS6K-RPS6-EIF4EBP1 pathway in porcine trophectoderm cells to stimulate proliferation; increased nuclear localization of pRPS6K and cytoplasmic pRPS6 were detected. siRNA silencing of MTOR, RPTOR, or RICTOR blocked amino acid-induced proliferation, placing RPS6 downstream of both mTORC1 and mTORC2 in trophectoderm proliferation.","method":"Immunofluorescence, immunoblotting of phospho-RPS6K/RPS6/EIF4EBP1, siRNA knockdown of MTOR/RPTOR/RICTOR, proliferation assay in primary porcine trophectoderm cells","journal":"Biology of reproduction","confidence":"Medium","confidence_rationale":"Tier 2 — siRNA epistasis with multiple pathway components and immunolocalization, single lab","pmids":["23486913"],"is_preprint":false},{"year":2014,"finding":"Phosphorylation of rpS6 mediates compensatory renal hypertrophy: rpS6 knock-in mice expressing non-phosphorylatable rpS6 show significantly blunted uninephrectomy-induced renal hypertrophy, with attenuated cyclin D1 increase and reduced cyclin E decrease compared to wild-type. mTORC1 is still activated in knock-in mice (4E-BP1 phosphorylation intact), but rapamycin (which prevents rpS6 phosphorylation) cannot prevent residual hypertrophy in knock-in mice, unequivocally demonstrating rpS6 phosphorylation as the downstream mTORC1-S6K1 effector mediating hypertrophic cell cycle regulation.","method":"rpS6 phosphorylation-site knock-in mice, uninephrectomy model, rapamycin treatment, immunoblotting for cyclins and phospho-proteins","journal":"Kidney international","confidence":"High","confidence_rationale":"Tier 2 — orthogonal genetic and pharmacological approaches with specific mechanistic readout","pmids":["25229342"],"is_preprint":false},{"year":2014,"finding":"RPS6 interacts with components of mTORC2 (co-immunoprecipitation), and siRNA-mediated knockdown of RPS6 attenuates insulin-induced mTORC2 activity and Akt-Ser473 phosphorylation. Conversely, RPS6 overexpression enhances Akt-Ser473 phosphorylation, establishing that RPS6 provides positive feedback to mTORC2/Akt signaling. In mouse hearts, ischemic preconditioning induces RPS6 phosphorylation at Ser235/236 and activates mTORC2.","method":"Co-immunoprecipitation of RPS6 with mTORC2 components, siRNA knockdown, RPS6 overexpression, ischemic preconditioning in perfused mouse hearts, neonatal rat ventricular myocyte assays","journal":"Circulation research","confidence":"High","confidence_rationale":"Tier 2 — reciprocal co-IP plus bidirectional manipulation (KD and OE) with functional consequence in vitro and in vivo","pmids":["24557881"],"is_preprint":false},{"year":2015,"finding":"In yeast, TORC1 promotes rpS6 phosphorylation on Ser-232/Ser-233 via the AGC kinase Ypk3 (not Sch9 as previously proposed). Ypk3-null cells have completely abolished rpS6 phosphorylation; phosphorylation-deficient mutations in Ypk3 regulatory motifs abrogate rpS6 phosphorylation; and complementation of ypk3Δ cells with human S6 kinase restores rpS6 phosphorylation. Cells expressing non-phosphorylatable rpS6 show reduced growth and 40S biogenesis defects not explained by translation changes.","method":"Yeast deletion mutants, phospho-S6-specific antibody, Ypk3 regulatory mutants, human S6K complementation, polysome profiling, ribosome profiling","journal":"PloS one / Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 1-2 — genetic identification of direct kinase with complementation and multiple orthogonal assays; two complementary papers","pmids":["25767889","26582391"],"is_preprint":false},{"year":2015,"finding":"RPS6 phosphorylation is controlled by both TORC1 (via Ypk3 and PP1/Glc7) and TORC2 (via Ypk1) in S. cerevisiae, with TORC2 specifically regulating the N-terminal phosphosite. Ribosome profiling shows that rpS6 phosphorylation does not regulate global translation or translation of individual mRNAs, challenging the longstanding model that S6 phosphorylation broadly promotes translation of 5'TOP mRNAs.","method":"TORC1/TORC2 genetic dissection, ribosome profiling, polysome profiling, non-phosphorylatable rpS6 knock-in cells","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 1 — genome-wide ribosome profiling with genetic knockins; strong negative evidence against translation model","pmids":["26582391"],"is_preprint":false},{"year":2015,"finding":"RPS6 reviews extensive evidence that rpS6 phosphorylation occurs on five conserved serine residues in response to diverse stimuli; genetic manipulations in yeast and mammals using rpS6 knock-in mice and S6K knockout mice have revealed roles in cell size control, glucose homeostasis, ribosome biogenesis, and DNA damage responses. The physiological role of phosphorylation may include fine-tuning responses by counteracting positive signals from rpS6 kinase.","method":"Synthesis of knock-in mouse, S6K KO, and yeast data","journal":"International review of cell and molecular biology","confidence":"Medium","confidence_rationale":"Tier 2 — review synthesizing genetic model organism data from multiple labs","pmids":["26614871"],"is_preprint":false},{"year":2017,"finding":"S6 kinases phosphorylate the nuclear epigenetic factor ZRF1 (DNAJC2/MIDA1) on Ser47 in cultured cells and in mammalian tissues in vivo (identified by chemical genetic screen). ZRF1 knockdown or expression of a phosphorylation mutant blunts S6K-dependent premature senescence in TSC-mutant fibroblasts, associated with altered p16 levels. This identifies ZRF1 as a novel S6K substrate linking mTORC1-S6K to cellular senescence.","method":"Chemical genetic screen for novel S6K substrates, ZRF1 knockdown, phospho-Ser47 antibody, senescence assays (SA-β-gal, p16), TSC-mutant fibroblasts","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 2 — chemical genetic screen for substrate identification plus knockdown rescue and in vivo phosphorylation","pmids":["28242756"],"is_preprint":false},{"year":2017,"finding":"mTORC1/rpS6 signaling promotes BTB disruption in the adult rat testis in vivo: constitutively active phosphomimetic rpS6 mutant (quadruple phosphomimetic) induces BTB leakiness better than wild-type rpS6, causing spermatid polarity loss and germ cell exfoliation. These effects are mediated through changes in actin- and microtubule-based cytoskeletal organization, specifically spatiotemporal expression of actin- and MT-binding proteins.","method":"In vivo transfection of rat testes with rpS6-WT and phosphomimetic rpS6 mutant, BTB integrity assay, immunofluorescence for cytoskeletal proteins, electron microscopy","journal":"American journal of physiology. Endocrinology and metabolism","confidence":"High","confidence_rationale":"Tier 2 — direct in vivo gain-of-function with phosphomimetic mutant and multiple structural readouts","pmids":["29089336"],"is_preprint":false},{"year":2018,"finding":"β-arrestins constitutively assemble a p70S6K/rpS6 complex in HEK293 cells and Sertoli cells; the interaction between β-arrestin 1 and p70S6K is direct (validated by docking). Upon FSH stimulation, G protein (Gαs)-dependent signaling enhances p70S6K activity within the preassembled β-arrestin/p70S6K/rpS6 complex, leading to rpS6 phosphorylation and translation of 5'TOP mRNAs within minutes.","method":"Co-immunoprecipitation, computational docking validation, Gαs depletion, FSHR stimulation, 5'TOP mRNA translation assay","journal":"FASEB journal","confidence":"Medium","confidence_rationale":"Tier 2 — direct interaction validated by docking with functional translation readout, single lab","pmids":["29084767"],"is_preprint":false},{"year":2018,"finding":"The mTOR effectors S6K1 and S6K2 localize to stress granules in human cells and are required for stress granule assembly and maintenance after mild oxidative stress. S6K1 promotes stress granule formation via regulation of eIF2α phosphorylation, while S6K2 is important for stress granule persistence. In C. elegans, the S6K ortholog RSKS-1 also promotes stress granule assembly and its loss sensitizes to stress-induced death.","method":"Immunofluorescence of S6K1/2 in stress granule markers, siRNA knockdown, eIF2α phosphorylation assays, C. elegans rsks-1 loss-of-function, stress-induced death assays","journal":"Cell death and differentiation","confidence":"High","confidence_rationale":"Tier 2 — localisation with functional consequence, validated in two organisms with mechanistic link to eIF2α","pmids":["29523872"],"is_preprint":false},{"year":2019,"finding":"Somatic mosaicism for RPS6 p.R232H mutation in a hemimegalencephaly patient increases cell proliferation in animal models, while MTOR p.S2215F causes neuronal migration delay and cytomegaly; double mutants show synergistic effects (increased proliferation + migration defects + cytomegalic cells with eccentric nuclei/binucleation). This establishes RPS6 p.R232H as an activating mutation that drives cortical dysplasia through increased proliferation downstream of the mTOR pathway.","method":"Whole-exome sequencing, deep targeted sequencing, in utero electroporation of variant constructs in mice, cell proliferation and migration assays, histological analysis","journal":"Human molecular genetics","confidence":"High","confidence_rationale":"Tier 2 — in vivo functional validation of human disease mutations with quantitative phenotyping","pmids":["31411685"],"is_preprint":false},{"year":2021,"finding":"RPS6 phosphorylation promotes translation of mRNAs with short coding sequences (CDSs) more strongly than mRNAs with long CDSs. Selective ribosome footprinting reveals that RPS6 becomes progressively dephosphorylated on ribosomes as they translate longer ORFs, so short-CDS mRNAs retain more p-RPS6 ribosomes. However, 5'TOP mRNAs are not preferentially translated despite their short CDSs, suggesting they use a different mechanism.","method":"Selective ribosome footprinting with phospho-RPS6 antibody, RPS6 phosphorylation-site knock-in cells, genome-wide translation analysis","journal":"Nucleic acids research","confidence":"High","confidence_rationale":"Tier 1 — genome-wide ribosome footprinting with genetic removal of phosphorylation sites; novel mechanistic insight","pmids":["34871442"],"is_preprint":false},{"year":2021,"finding":"RPS6 inhibition by siRNA decreases NRF2 expression in HER2-amplified gastric cancer, suggesting RPS6 lies upstream of NRF2 in anti-HER2 drug resistance. PI3K/TORC1/TORC2 inhibition suppresses pRPS6 and NRF2 and reduces tumor growth in resistant models in vitro and in vivo.","method":"siRNA knockdown of RPS6, PI3K/mTOR inhibitor treatment, cell viability assays, in vivo xenograft models","journal":"Clinical cancer research","confidence":"Medium","confidence_rationale":"Tier 2 — siRNA knockdown with functional readout; mechanistic link to NRF2 is indirect","pmids":["30504425"],"is_preprint":false},{"year":2021,"finding":"The lnc-Rps4l-encoded peptide RPS4XL interacts with RPS6 (identified by CoIP and mass spectrometry) and inhibits RPS6 phosphorylation at Ser240/Ser244, thereby inhibiting hypoxia-induced proliferation of pulmonary artery smooth muscle cells.","method":"Co-immunoprecipitation, mass spectrometry, immunofluorescence, MTT/BrdU proliferation assays, phospho-RPS6 immunoblotting","journal":"Molecular therapy","confidence":"Medium","confidence_rationale":"Tier 2-3 — co-IP with MS for interaction, functional knockdown data; single lab","pmids":["33429084"],"is_preprint":false},{"year":2023,"finding":"In mammalian skin wounding, rpS6 is rapidly phosphorylated within minutes of injury, forming a spatially defined 'p-rpS6-zone' surrounding the wound that persists until healing is complete. This zone encompasses proliferation, cellular senescence, angiogenesis, and growth. A mouse model unable to phosphorylate rpS6 shows accelerated initial wound closure but impaired overall healing, demonstrating that p-rpS6 modulates (but does not drive) the healing process.","method":"Immunofluorescence for p-rpS6 in skin wounds, rpS6 non-phosphorylatable knock-in mice, wound healing assays, time-course analysis","journal":"Developmental cell","confidence":"High","confidence_rationale":"Tier 2 — spatial mapping with genetic knock-in model showing in vivo functional consequence","pmids":["37098351"],"is_preprint":false},{"year":1995,"finding":"Human ribosomal protein S6 contains three nuclear localization signals (NLSs) in its C-terminal half. Deletion mutagenesis of S6-β-galactosidase chimeras showed that a single NLS is sufficient for nuclear import, removal of all three NLSs abolishes nuclear entry, and chimeras retaining at least one NLS accumulate specifically in the nucleolus via a distinct domain required but not sufficient for nucleolar targeting.","method":"Chimeric S6-LacZ construct transfection in L cells, deletion mutagenesis, β-galactosidase activity localization assay","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 1-2 — systematic deletion mutagenesis with functional targeting assay defining NLS and nucleolar targeting domain","pmids":["8590812"],"is_preprint":false},{"year":2004,"finding":"Yeast rpS6 (Rps6p) functionally interacts with the 60S subunit protein rpL10/Grc5p in ribosome subunit joining and differential protein expression; Grc5p also interacts with the nuclear export factor Nmd3p to modulate polysome complement, establishing rpS6 as part of a network of ribosomal proteins governing subunit joining fidelity.","method":"Yeast two-hybrid, polysome profiling, genetic interaction analysis in S. cerevisiae","journal":"FEMS yeast research","confidence":"Medium","confidence_rationale":"Tier 3 — yeast two-hybrid with polysome profiling; functional consequence not fully defined","pmids":["15556089"],"is_preprint":false},{"year":2013,"finding":"Human 80S ribosome cryo-EM structure at high resolution reveals the position of RPS6 in the 40S subunit, as part of the metazoan-specific structural architecture with additional inner and outer RNA layers, providing structural context for RPS6's role in ribosome function.","method":"High-resolution cryo-electron microscopy of human and Drosophila 80S ribosomes","journal":"Nature","confidence":"High","confidence_rationale":"Tier 1 — near-atomic cryo-EM structure from highly cited paper; defines RPS6 structural context","pmids":["23636399"],"is_preprint":false},{"year":2015,"finding":"Near-atomic (3.6 Å average, 2.9 Å in stable regions) cryo-EM structure of the human 80S ribosome defines the precise atomic positions of ribosomal proteins including RPS6, revealing amino acid side chain contacts, tRNA binding site interactions, and dynamic subunit interface remodeling during rotation.","method":"Single-particle cryo-EM, atomic model building","journal":"Nature","confidence":"High","confidence_rationale":"Tier 1 — near-atomic resolution structure with functional validation of interface dynamics","pmids":["25901680"],"is_preprint":false}],"current_model":"RPS6 (ribosomal protein S6) is a component of the 40S ribosomal subunit that is phosphorylated on five conserved C-terminal serine residues (Ser235, Ser236, Ser240, Ser244, Ser247) by S6K1/S6K2 (activated downstream of the PI3K–mTORC1–PDK1 axis) and by p90 RSK; phosphorylation is progressively lost as ribosomes translate longer ORFs, selectively promoting translation of short-CDS mRNAs; beyond translation, phospho-rpS6 mediates compensatory organ hypertrophy via cyclin D1/E regulation, controls blood-testis barrier dynamics through F-actin reorganization, provides positive feedback to mTORC2/Akt signaling, regulates the ribosome biogenesis transcriptional program, marks a wound-healing activation zone, and is subject to negative regulation by PP2A and AMPK, while activating mutations (e.g., p.R232H) drive cortical dysplasia through increased cell proliferation."},"narrative":{"teleology":[{"year":1981,"claim":"Early kinetic studies established that rpS6 phosphorylation occurs rapidly after mitogenic stimulation and correlates with mRNA recruitment into polysomes, raising the hypothesis that S6 phosphorylation facilitates translational activation.","evidence":"Polysome profiling and S6 phosphorylation kinetics in serum-stimulated HeLa cells","pmids":["7333277"],"confidence":"Medium","gaps":["Correlation does not establish causation between S6 phosphorylation and mRNA recruitment","No identification of which mRNAs are affected"]},{"year":1984,"claim":"S6 phosphorylation was shown to induce conformational changes in the 40S subunit, altering accessibility of multiple ribosomal proteins, providing a biophysical mechanism by which S6 phosphorylation could modulate ribosome function.","evidence":"Reductive methylation accessibility assay comparing phosphorylated vs. unphosphorylated ribosomes","pmids":["6693389"],"confidence":"Medium","gaps":["Single method; no functional translation consequence demonstrated","No structural resolution of conformational changes"]},{"year":1991,"claim":"Purification and characterization of p70 S6K established that a single mitogen-activated kinase phosphorylates rpS6 on exactly five C-terminal serines (Ser235/236/240/244 plus a fifth site), matching the in vivo pattern, and that the kinase itself is regulated by phosphorylation reversible by PP2A.","evidence":"Biochemical purification from Swiss 3T3 cells, in vitro kinase assay with 40S subunits, tryptic phosphopeptide mapping, PP2A inactivation","pmids":["3257566","1939282"],"confidence":"High","gaps":["Upstream signaling pathway not yet defined","Identity of the fifth low-stoichiometry site uncertain"]},{"year":1995,"claim":"The discovery that rapamycin selectively inhibits p70 S6K and that FRAP/mTOR kinase activity is required for p70 S6K activation placed rpS6 phosphorylation downstream of the rapamycin-sensitive mTOR pathway, distinct from the Ras–MAPK cascade.","evidence":"Selective rapamycin inhibition in multiple cell lines; FRAP kinase-dead mutants failing to activate S6K; dominant-negative Ras/Raf not blocking S6K","pmids":["1380182","7566123","8090223"],"confidence":"High","gaps":["Direct phosphorylation of S6K by mTOR not yet demonstrated","Mechanism of rapamycin resistance in truncation mutants unclear"]},{"year":1995,"claim":"Mapping of three nuclear localization signals in the C-terminal half of rpS6 and identification of a distinct nucleolar-targeting domain established that rpS6 traffics through the nucleus/nucleolus, consistent with ribosome biogenesis roles.","evidence":"Deletion mutagenesis of S6–β-galactosidase chimeras in L cells","pmids":["8590812"],"confidence":"High","gaps":["Functional consequence of nuclear localization for rpS6 not tested","Whether phosphorylation regulates NLS function unknown"]},{"year":1998,"claim":"Direct phosphorylation of S6K1 at Thr389 by mTOR was demonstrated in vitro and shown to be essential for S6K activity, while amino acid withdrawal inactivates S6K through a pathway independent of Akt, establishing amino acid sensing as a distinct input to rpS6 phosphorylation.","evidence":"In vitro kinase assay with immunoprecipitated mTOR, phosphosite mapping; amino acid deprivation in CHO cells with PI3K/mTOR inhibitors","pmids":["9465032","9693128"],"confidence":"High","gaps":["Amino acid sensing mechanism upstream of mTOR not identified","Relative contributions of S6K1 vs. S6K2 to rpS6 phosphorylation unclear"]},{"year":2003,"claim":"The TSC1/2–Rheb axis was identified as the critical link between PI3K/Akt and mTOR/S6K: TSC2 acts as a GAP for Rheb, and loss-of-function TSC mutations constitutively hyperphosphorylate rpS6, explaining the elevated S6K activity in tuberous sclerosis.","evidence":"GTPase assays with purified Rheb, genetic epistasis, TSC2 disease-mutant analysis, S6 phosphorylation in TSC2-null cells with tuberin re-expression","pmids":["12045200","12820960"],"confidence":"High","gaps":["How Rheb directly activates mTOR kinase not resolved","Tissue-specific consequences of TSC-driven rpS6 hyperphosphorylation not fully explored"]},{"year":2005,"claim":"The mTOR/S6K1/eIF3 dynamic scaffold model revealed how S6K1 basally associates with the eIF3 translation initiation complex and, upon mTOR-mediated phosphorylation, dissociates to phosphorylate eIF4B, providing a mechanistic link between rpS6 kinase activation and translation initiation complex remodeling.","evidence":"Sequential co-immunoprecipitation of mTOR, S6K1, eIF3, and eIF4B with rapamycin treatment","pmids":["16006006","16286006"],"confidence":"High","gaps":["Whether rpS6 phosphorylation itself contributes to eIF3 complex dynamics unknown","Stoichiometry and kinetics of scaffold assembly not defined"]},{"year":2008,"claim":"S6K1 knockout mice showed extended lifespan and resistance to age-related pathologies, while hypothalamic S6K manipulation bidirectionally controlled energy homeostasis, establishing the S6K–rpS6 axis as a systemic regulator of aging and metabolism in mammals.","evidence":"S6K1 knockout mouse lifespan and metabolic analysis; bilateral adenoviral injection of S6K variants into rat mediobasal hypothalamus","pmids":["19797661","19041762"],"confidence":"High","gaps":["Relative contribution of rpS6 phosphorylation vs. other S6K substrates to lifespan extension not resolved","Human translatability unknown"]},{"year":2013,"claim":"Two independent genetic models—S6K1/S6K2 double-knockout mice and rpS6 non-phosphorylatable knock-in mice—converged on the discovery that rpS6 phosphorylation specifically controls the ribosome biogenesis transcriptional program, establishing a non-translational function for rpS6 phosphorylation.","evidence":"Whole-genome microarray of total and polysomal liver RNA from S6K DKO and rpS6 knock-in mice after feeding","pmids":["23318442"],"confidence":"High","gaps":["Transcription factor mediating rpS6 phosphorylation-dependent RiBi gene induction not identified","Whether this operates through a ribosome-free rpS6 pool unclear"]},{"year":2014,"claim":"rpS6 phosphorylation was identified as the specific mTORC1-S6K1 effector mediating compensatory renal hypertrophy, acting through cyclin D1/E regulation; additionally, rpS6 was found to provide positive feedback to mTORC2/Akt signaling through direct physical interaction with mTORC2 components.","evidence":"rpS6 phospho-site knock-in mice with uninephrectomy; co-IP of rpS6 with mTORC2 components; bidirectional manipulation of rpS6 affecting Akt-Ser473 phosphorylation","pmids":["25229342","24557881"],"confidence":"High","gaps":["Structural basis of rpS6–mTORC2 interaction unknown","Whether extra-ribosomal rpS6 mediates mTORC2 feedback not tested"]},{"year":2015,"claim":"Genome-wide ribosome profiling in yeast with non-phosphorylatable rpS6 definitively showed that rpS6 phosphorylation does not regulate global translation or preferential translation of individual mRNAs, challenging the decades-old model that S6 phosphorylation broadly controls 5′TOP mRNA translation.","evidence":"Ribosome profiling and polysome profiling in yeast rpS6 phospho-mutant cells; identification of Ypk3 as the direct rpS6 kinase via genetic complementation","pmids":["26582391","25767889"],"confidence":"High","gaps":["Yeast findings may not fully translate to mammals with more complex 5′TOP regulation","Non-translational roles of rpS6 phosphorylation in yeast not fully characterized"]},{"year":2019,"claim":"The somatic RPS6 p.R232H mutation was shown to be an activating mutation causing hemimegalencephaly-associated cortical dysplasia through increased cell proliferation, establishing RPS6 as a disease gene for brain overgrowth syndromes within the mTORopathy spectrum.","evidence":"Whole-exome sequencing of hemimegalencephaly patient; in utero electroporation of RPS6-R232H in mice with proliferation and migration assays","pmids":["31411685"],"confidence":"High","gaps":["Mechanism by which R232H activates mTOR signaling not defined","Frequency of RPS6 mutations in cortical dysplasia cohorts not established"]},{"year":2021,"claim":"Selective ribosome footprinting revealed that rpS6 is progressively dephosphorylated as ribosomes translate longer ORFs, providing a length-dependent mechanism whereby rpS6 phosphorylation selectively promotes translation of short-CDS mRNAs rather than acting through 5′TOP elements.","evidence":"Phospho-RPS6 antibody-based selective ribosome profiling in RPS6 phospho-site knock-in cells","pmids":["34871442"],"confidence":"High","gaps":["Phosphatase responsible for elongation-dependent rpS6 dephosphorylation not identified","Whether this mechanism operates identically in all mammalian tissues unknown"]},{"year":2023,"claim":"In vivo imaging of wounded skin revealed a spatially defined p-rpS6 zone that encompasses proliferation, senescence, and angiogenesis around wound margins; rpS6 non-phosphorylatable knock-in mice showed altered wound healing kinetics, demonstrating a tissue-level organizing role for rpS6 phosphorylation.","evidence":"Immunofluorescence mapping of p-rpS6 in skin wounds; rpS6 knock-in mice wound healing assays","pmids":["37098351"],"confidence":"High","gaps":["Signals initiating wound-edge rpS6 phosphorylation not identified","Whether the p-rpS6 zone acts through translation, RiBi, or other mechanisms unclear"]},{"year":null,"claim":"Key unresolved questions include the identity of the phosphatase responsible for elongation-coupled rpS6 dephosphorylation, the structural basis of rpS6's interaction with mTORC2, whether extra-ribosomal rpS6 mediates nuclear/signaling functions, and the transcription factor through which rpS6 phosphorylation controls the ribosome biogenesis program.","evidence":"","pmids":[],"confidence":"Medium","gaps":["Phosphatase for elongation-dependent rpS6 dephosphorylation unknown","Extra-ribosomal rpS6 pool not characterized","Transcription factor mediating RiBi program downstream of rpS6 not identified"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0005198","term_label":"structural molecule activity","supporting_discovery_ids":[58,59]}],"localization":[{"term_id":"GO:0005840","term_label":"ribosome","supporting_discovery_ids":[58,59,5]},{"term_id":"GO:0005730","term_label":"nucleolus","supporting_discovery_ids":[56]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[56]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[52]}],"pathway":[{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[3,52,58,59]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[7,9,11,43]},{"term_id":"R-HSA-1852241","term_label":"Organelle biogenesis and maintenance","supporting_discovery_ids":[40,44]},{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[42]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[51]}],"complexes":["40S ribosomal subunit","80S ribosome"],"partners":["RPS6KB1","RPS6KB2","MTOR","RPTOR","EIF3A","RPL10","ARRB1"],"other_free_text":[]},"mechanistic_narrative":"RPS6 (eS6) is a structural component of the 40S ribosomal subunit whose five conserved C-terminal serine residues (Ser235, Ser236, Ser240, Ser244, Ser247) are phosphorylated by p70 S6K1/S6K2 downstream of the PI3K–mTORC1–PDK1 axis and by p90 RSK, serving as a central signaling node that couples nutrient and growth-factor sensing to translational control [PMID:3257566, PMID:1939282, PMID:9465032]. Phosphorylation of rpS6 selectively promotes translation of short-CDS mRNAs through progressive dephosphorylation as ribosomes traverse longer open reading frames, while genome-wide ribosome profiling in yeast demonstrates that rpS6 phosphorylation does not broadly regulate 5′TOP mRNA translation [PMID:34871442, PMID:26582391]. Beyond translation, rpS6 phosphorylation drives the ribosome biogenesis transcriptional program, mediates compensatory organ hypertrophy through cyclin D1/E regulation, controls blood–testis barrier dynamics via F-actin reorganization, provides positive feedback to mTORC2/Akt signaling, and defines a wound-healing activation zone in skin [PMID:23318442, PMID:25229342, PMID:22948214, PMID:24557881, PMID:37098351]. Somatic activating mutations in RPS6 (e.g., p.R232H) cause hemimegalencephaly-associated cortical dysplasia through increased cell proliferation [PMID:31411685]."},"prefetch_data":{"uniprot":{"accession":"P62753","full_name":"Small ribosomal subunit protein eS6","aliases":["40S ribosomal protein S6","Phosphoprotein NP33"],"length_aa":249,"mass_kda":28.7,"function":"Component of the 40S small ribosomal subunit (PubMed:23636399, PubMed:8706699). Plays an important role in controlling cell growth and proliferation through the selective translation of particular classes of mRNA (PubMed:17220279). Part of the small subunit (SSU) processome, first precursor of the small eukaryotic ribosomal subunit. During the assembly of the SSU processome in the nucleolus, many ribosome biogenesis factors, an RNA chaperone and ribosomal proteins associate with the nascent pre-rRNA and work in concert to generate RNA folding, modifications, rearrangements and cleavage as well as targeted degradation of pre-ribosomal RNA by the RNA exosome (PubMed:34516797)","subcellular_location":"Cytoplasm; Nucleus, nucleolus","url":"https://www.uniprot.org/uniprotkb/P62753/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":true,"resolved_as":"","url":"https://depmap.org/portal/gene/RPS6","classification":"Common Essential","n_dependent_lines":1208,"n_total_lines":1208,"dependency_fraction":1.0},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"EIF2S3","stoichiometry":10.0},{"gene":"EIF3B","stoichiometry":10.0},{"gene":"RACK1","stoichiometry":10.0},{"gene":"RBM8A","stoichiometry":10.0},{"gene":"RPL11","stoichiometry":10.0},{"gene":"RPL4","stoichiometry":10.0},{"gene":"RPL5","stoichiometry":10.0},{"gene":"RPS16","stoichiometry":10.0},{"gene":"SRP19","stoichiometry":10.0},{"gene":"SRP68","stoichiometry":10.0}],"url":"https://opencell.sf.czbiohub.org/search/RPS6","total_profiled":1310},"omim":[{"mim_id":"621460","title":"NEURODEVELOPMENTAL DISORDER WITH SPASTICITY, THIN CORPUS CALLOSUM, AND DECREASED BRAIN WHITE MATTER; NEDSCW","url":"https://www.omim.org/entry/621460"},{"mim_id":"617620","title":"LEUCINE-RICH REPEAT-CONTAINING PROTEIN 3; LRRC3","url":"https://www.omim.org/entry/617620"},{"mim_id":"617517","title":"RIBOSOMAL PROTEIN S6 KINASE C1; RPS6KC1","url":"https://www.omim.org/entry/617517"},{"mim_id":"616900","title":"HYPOTONIA, INFANTILE, WITH PSYCHOMOTOR RETARDATION AND CHARACTERISTIC FACIES 3; IHPRF3","url":"https://www.omim.org/entry/616900"},{"mim_id":"616899","title":"TBC1 DOMAIN-CONTAINING KINASE; TBCK","url":"https://www.omim.org/entry/616899"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Enhanced","locations":[{"location":"Endoplasmic reticulum","reliability":"Enhanced"},{"location":"Cytosol","reliability":"Enhanced"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/RPS6"},"hgnc":{"alias_symbol":["S6","eS6"],"prev_symbol":[]},"alphafold":{"accession":"P62753","domains":[{"cath_id":"-","chopping":"1-114","consensus_level":"high","plddt":96.1338,"start":1,"end":114},{"cath_id":"-","chopping":"137-171","consensus_level":"medium","plddt":95.0386,"start":137,"end":171}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/P62753","model_url":"https://alphafold.ebi.ac.uk/files/AF-P62753-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-P62753-F1-predicted_aligned_error_v6.png","plddt_mean":94.19},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=RPS6","jax_strain_url":"https://www.jax.org/strain/search?query=RPS6"},"sequence":{"accession":"P62753","fasta_url":"https://rest.uniprot.org/uniprotkb/P62753.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/P62753/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/P62753"}},"corpus_meta":[{"pmid":"9465032","id":"PMC_9465032","title":"RAFT1 phosphorylation of the translational regulators p70 S6 kinase and 4E-BP1.","date":"1998","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/9465032","citation_count":948,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"19797661","id":"PMC_19797661","title":"Ribosomal protein S6 kinase 1 signaling regulates mammalian life span.","date":"2009","source":"Science (New York, N.Y.)","url":"https://pubmed.ncbi.nlm.nih.gov/19797661","citation_count":905,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"7566123","id":"PMC_7566123","title":"Control of p70 s6 kinase by kinase activity of FRAP in vivo.","date":"1995","source":"Nature","url":"https://pubmed.ncbi.nlm.nih.gov/7566123","citation_count":626,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"10497130","id":"PMC_10497130","title":"Drosophila S6 kinase: a regulator of cell size.","date":"1999","source":"Science (New York, N.Y.)","url":"https://pubmed.ncbi.nlm.nih.gov/10497130","citation_count":614,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"1380182","id":"PMC_1380182","title":"Rapamycin-induced inhibition of the 70-kilodalton S6 protein kinase.","date":"1992","source":"Science (New York, N.Y.)","url":"https://pubmed.ncbi.nlm.nih.gov/1380182","citation_count":614,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"10579915","id":"PMC_10579915","title":"Ribosomal S6 kinase signaling and the control of translation.","date":"1999","source":"Experimental cell research","url":"https://pubmed.ncbi.nlm.nih.gov/10579915","citation_count":583,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"21147110","id":"PMC_21147110","title":"Increased lipogenesis, induced by AKT-mTORC1-RPS6 signaling, promotes development of human hepatocellular carcinoma.","date":"2010","source":"Gastroenterology","url":"https://pubmed.ncbi.nlm.nih.gov/21147110","citation_count":495,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"16049009","id":"PMC_16049009","title":"hVps34 is a nutrient-regulated lipid kinase required for activation of p70 S6 kinase.","date":"2005","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/16049009","citation_count":430,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"14502240","id":"PMC_14502240","title":"M-CSF, TNFalpha and RANK ligand promote osteoclast survival by signaling through mTOR/S6 kinase.","date":"2003","source":"Cell death and differentiation","url":"https://pubmed.ncbi.nlm.nih.gov/14502240","citation_count":294,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"9693128","id":"PMC_9693128","title":"Amino acid availability regulates p70 S6 kinase and multiple translation factors.","date":"1998","source":"The Biochemical journal","url":"https://pubmed.ncbi.nlm.nih.gov/9693128","citation_count":291,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"20932932","id":"PMC_20932932","title":"Functions and regulation of the 70kDa ribosomal S6 kinases.","date":"2010","source":"The international journal of biochemistry & cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/20932932","citation_count":270,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"20736484","id":"PMC_20736484","title":"Akt-RSK-S6 kinase signaling networks activated by oncogenic receptor tyrosine kinases.","date":"2010","source":"Science signaling","url":"https://pubmed.ncbi.nlm.nih.gov/20736484","citation_count":261,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"26614871","id":"PMC_26614871","title":"Ribosomal Protein S6 Phosphorylation: Four Decades of Research.","date":"2015","source":"International review of cell and molecular biology","url":"https://pubmed.ncbi.nlm.nih.gov/26614871","citation_count":236,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"23318442","id":"PMC_23318442","title":"Ribosomal protein S6 kinase activity controls the ribosome biogenesis transcriptional program.","date":"2013","source":"Oncogene","url":"https://pubmed.ncbi.nlm.nih.gov/23318442","citation_count":228,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"8090223","id":"PMC_8090223","title":"Activation of p70/p85 S6 kinase by a pathway independent of p21ras.","date":"1994","source":"Nature","url":"https://pubmed.ncbi.nlm.nih.gov/8090223","citation_count":212,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"3257566","id":"PMC_3257566","title":"Identification and characterization of a mitogen-activated S6 kinase.","date":"1988","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/3257566","citation_count":203,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"11124802","id":"PMC_11124802","title":"TGF-beta inhibits p70 S6 kinase via protein phosphatase 2A to induce G(1) arrest.","date":"2000","source":"Genes & development","url":"https://pubmed.ncbi.nlm.nih.gov/11124802","citation_count":192,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"18703402","id":"PMC_18703402","title":"Physiological roles of ribosomal protein S6: one of its kind.","date":"2008","source":"International review of cell and molecular biology","url":"https://pubmed.ncbi.nlm.nih.gov/18703402","citation_count":187,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"15385434","id":"PMC_15385434","title":"Apelin (65-77) activates p70 S6 kinase and is mitogenic for umbilical endothelial cells.","date":"2004","source":"FASEB journal : official publication of the Federation of American Societies for Experimental Biology","url":"https://pubmed.ncbi.nlm.nih.gov/15385434","citation_count":172,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"19041762","id":"PMC_19041762","title":"Mediobasal hypothalamic p70 S6 kinase 1 modulates the control of energy homeostasis.","date":"2008","source":"Cell metabolism","url":"https://pubmed.ncbi.nlm.nih.gov/19041762","citation_count":153,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"17550782","id":"PMC_17550782","title":"S6 kinase deletion suppresses muscle growth adaptations to nutrient availability by activating AMP kinase.","date":"2007","source":"Cell metabolism","url":"https://pubmed.ncbi.nlm.nih.gov/17550782","citation_count":149,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"20157535","id":"PMC_20157535","title":"Inhibition of mammalian S6 kinase by resveratrol suppresses autophagy.","date":"2009","source":"Aging","url":"https://pubmed.ncbi.nlm.nih.gov/20157535","citation_count":142,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"17347671","id":"PMC_17347671","title":"ATG1, an autophagy regulator, inhibits cell growth by negatively regulating S6 kinase.","date":"2007","source":"EMBO reports","url":"https://pubmed.ncbi.nlm.nih.gov/17347671","citation_count":132,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"1939282","id":"PMC_1939282","title":"Mitogen-activated 70K S6 kinase. Identification of in vitro 40 S ribosomal S6 phosphorylation sites.","date":"1991","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/1939282","citation_count":127,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"11500301","id":"PMC_11500301","title":"Leucine and insulin activate p70 S6 kinase through different pathways in human skeletal muscle.","date":"2001","source":"American journal of physiology. Endocrinology and metabolism","url":"https://pubmed.ncbi.nlm.nih.gov/11500301","citation_count":119,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"2760046","id":"PMC_2760046","title":"Purification of a hepatic S6 kinase from cycloheximide-treated Rats.","date":"1989","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/2760046","citation_count":111,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"17174052","id":"PMC_17174052","title":"Ribosomal proteins Rpl10 and Rps6 are potent regulators of yeast replicative life span.","date":"2006","source":"Experimental gerontology","url":"https://pubmed.ncbi.nlm.nih.gov/17174052","citation_count":99,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"16364302","id":"PMC_16364302","title":"P70 S6 kinase mediates tau phosphorylation and synthesis.","date":"2005","source":"FEBS letters","url":"https://pubmed.ncbi.nlm.nih.gov/16364302","citation_count":95,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"26045471","id":"PMC_26045471","title":"Glutamine promotes ovarian cancer cell proliferation through the mTOR/S6 pathway.","date":"2015","source":"Endocrine-related cancer","url":"https://pubmed.ncbi.nlm.nih.gov/26045471","citation_count":93,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"30482859","id":"PMC_30482859","title":"TOR and RPS6 transmit light signals to enhance protein translation in deetiolating Arabidopsis seedlings.","date":"2018","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/30482859","citation_count":90,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"23486913","id":"PMC_23486913","title":"Arginine, leucine, and glutamine stimulate proliferation of porcine trophectoderm cells through the MTOR-RPS6K-RPS6-EIF4EBP1 signal transduction pathway.","date":"2013","source":"Biology of reproduction","url":"https://pubmed.ncbi.nlm.nih.gov/23486913","citation_count":88,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"19525323","id":"PMC_19525323","title":"Resistance to the Pseudomonas syringae effector HopA1 is governed by the TIR-NBS-LRR protein RPS6 and is enhanced by mutations in SRFR1.","date":"2009","source":"Plant physiology","url":"https://pubmed.ncbi.nlm.nih.gov/19525323","citation_count":86,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"15064379","id":"PMC_15064379","title":"Phytohormones participate in an S6 kinase signal transduction pathway in Arabidopsis.","date":"2004","source":"Plant physiology","url":"https://pubmed.ncbi.nlm.nih.gov/15064379","citation_count":84,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"29523872","id":"PMC_29523872","title":"The mTOR-S6 kinase pathway promotes stress granule assembly.","date":"2018","source":"Cell death and differentiation","url":"https://pubmed.ncbi.nlm.nih.gov/29523872","citation_count":83,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"12087098","id":"PMC_12087098","title":"Regulation of ribosomal S6 kinase 2 by mammalian target of rapamycin.","date":"2002","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/12087098","citation_count":81,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"25767889","id":"PMC_25767889","title":"TORC1 promotes phosphorylation of ribosomal protein S6 via the AGC kinase Ypk3 in Saccharomyces cerevisiae.","date":"2015","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/25767889","citation_count":80,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"10214966","id":"PMC_10214966","title":"Structural requirement of leucine for activation of p70 S6 kinase.","date":"1999","source":"FEBS letters","url":"https://pubmed.ncbi.nlm.nih.gov/10214966","citation_count":79,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"19535330","id":"PMC_19535330","title":"mTOR/S6 kinase pathway contributes to astrocyte survival during ischemia.","date":"2009","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/19535330","citation_count":75,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"35008473","id":"PMC_35008473","title":"Ribosomal Protein S6: A Potential Therapeutic Target against Cancer?","date":"2021","source":"International journal of molecular sciences","url":"https://pubmed.ncbi.nlm.nih.gov/35008473","citation_count":74,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"26582391","id":"PMC_26582391","title":"TORC1 and TORC2 work together to regulate ribosomal protein S6 phosphorylation in Saccharomyces cerevisiae.","date":"2015","source":"Molecular biology of the cell","url":"https://pubmed.ncbi.nlm.nih.gov/26582391","citation_count":73,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"10455142","id":"PMC_10455142","title":"Osmotic stress inhibits p70/85 S6 kinase through activation of a protein phosphatase.","date":"1999","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/10455142","citation_count":71,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"19474189","id":"PMC_19474189","title":"S6 kinase 1 knockout inhibits uninephrectomy- or diabetes-induced renal hypertrophy.","date":"2009","source":"American journal of physiology. Renal physiology","url":"https://pubmed.ncbi.nlm.nih.gov/19474189","citation_count":70,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"2553707","id":"PMC_2553707","title":"An insulin-stimulated ribosomal protein S6 kinase from rabbit liver.","date":"1989","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/2553707","citation_count":70,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"22948214","id":"PMC_22948214","title":"rpS6 Regulates blood-testis barrier dynamics by affecting F-actin organization and protein recruitment.","date":"2012","source":"Endocrinology","url":"https://pubmed.ncbi.nlm.nih.gov/22948214","citation_count":69,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"25100792","id":"PMC_25100792","title":"p70 Ribosomal protein S6 kinase (Rps6kb1): an update.","date":"2014","source":"Journal of clinical pathology","url":"https://pubmed.ncbi.nlm.nih.gov/25100792","citation_count":68,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"18565837","id":"PMC_18565837","title":"Resistance exercise decreases eIF2Bepsilon phosphorylation and potentiates the feeding-induced stimulation of p70S6K1 and rpS6 in young men.","date":"2008","source":"American journal of physiology. Regulatory, integrative and comparative physiology","url":"https://pubmed.ncbi.nlm.nih.gov/18565837","citation_count":63,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"30504425","id":"PMC_30504425","title":"NRF2 through RPS6 Activation Is Related to Anti-HER2 Drug Resistance in HER2-Amplified Gastric Cancer.","date":"2018","source":"Clinical cancer research : an official journal of the American Association for Cancer Research","url":"https://pubmed.ncbi.nlm.nih.gov/30504425","citation_count":62,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"18688088","id":"PMC_18688088","title":"p70 S6 kinase and tau in Alzheimer's disease.","date":"2008","source":"Journal of Alzheimer's disease : JAD","url":"https://pubmed.ncbi.nlm.nih.gov/18688088","citation_count":59,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"20437080","id":"PMC_20437080","title":"The two Arabidopsis RPS6 genes, encoding for cytoplasmic ribosomal proteins S6, are functionally equivalent.","date":"2010","source":"Plant molecular biology","url":"https://pubmed.ncbi.nlm.nih.gov/20437080","citation_count":58,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"24557881","id":"PMC_24557881","title":"Pivotal role of mTORC2 and involvement of ribosomal protein S6 in cardioprotective signaling.","date":"2014","source":"Circulation research","url":"https://pubmed.ncbi.nlm.nih.gov/24557881","citation_count":58,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"8524322","id":"PMC_8524322","title":"Constitutive activation of S6 kinase by deletion of amino-terminal autoinhibitory and rapamycin sensitivity domains.","date":"1996","source":"Molecular and cellular biology","url":"https://pubmed.ncbi.nlm.nih.gov/8524322","citation_count":58,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"22069735","id":"PMC_22069735","title":"Immunotoxins and other conjugates containing saporin-s6 for cancer therapy.","date":"2011","source":"Toxins","url":"https://pubmed.ncbi.nlm.nih.gov/22069735","citation_count":56,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"8590812","id":"PMC_8590812","title":"Nuclear and nucleolar targeting of human ribosomal protein S6.","date":"1995","source":"Molecular biology of the cell","url":"https://pubmed.ncbi.nlm.nih.gov/8590812","citation_count":55,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"20192830","id":"PMC_20192830","title":"Different domains of Phytophthora sojae effector Avr4/6 are recognized by soybean resistance genes Rps4 and Rps6.","date":"2010","source":"Molecular plant-microbe interactions : MPMI","url":"https://pubmed.ncbi.nlm.nih.gov/20192830","citation_count":55,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"16887816","id":"PMC_16887816","title":"Effect of TRB3 on insulin and nutrient-stimulated hepatic p70 S6 kinase activity.","date":"2006","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/16887816","citation_count":55,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"34871442","id":"PMC_34871442","title":"Phosphorylation of ribosomal protein S6 differentially affects mRNA translation based on ORF length.","date":"2021","source":"Nucleic acids research","url":"https://pubmed.ncbi.nlm.nih.gov/34871442","citation_count":52,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"17483958","id":"PMC_17483958","title":"Co-expression of cyclin D1 and phosphorylated ribosomal S6 proteins in hemimegalencephaly.","date":"2007","source":"Acta neuropathologica","url":"https://pubmed.ncbi.nlm.nih.gov/17483958","citation_count":51,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"1880153","id":"PMC_1880153","title":"Heat shock induces two distinct S6 protein kinase activities in quiescent mammalian fibroblasts.","date":"1991","source":"Journal of cellular physiology","url":"https://pubmed.ncbi.nlm.nih.gov/1880153","citation_count":49,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"15150413","id":"PMC_15150413","title":"Simulation, experiment, and evolution: understanding nucleation in protein S6 folding.","date":"2004","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/15150413","citation_count":49,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"29403507","id":"PMC_29403507","title":"Phosphorylation of Ribosomal Protein RPS6 Integrates Light Signals and Circadian Clock Signals.","date":"2018","source":"Frontiers in plant science","url":"https://pubmed.ncbi.nlm.nih.gov/29403507","citation_count":47,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"21427355","id":"PMC_21427355","title":"S6 kinase 2 promotes breast cancer cell survival via Akt.","date":"2011","source":"Cancer research","url":"https://pubmed.ncbi.nlm.nih.gov/21427355","citation_count":47,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"25043857","id":"PMC_25043857","title":"Palmatine inhibits growth and invasion in prostate cancer cell: Potential role for rpS6/NFκB/FLIP.","date":"2014","source":"Molecular carcinogenesis","url":"https://pubmed.ncbi.nlm.nih.gov/25043857","citation_count":47,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"35176419","id":"PMC_35176419","title":"p70 S6 kinase as a therapeutic target in cancers: More than just an mTOR effector.","date":"2022","source":"Cancer letters","url":"https://pubmed.ncbi.nlm.nih.gov/35176419","citation_count":46,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"31411685","id":"PMC_31411685","title":"Somatic double-hit in MTOR and RPS6 in hemimegalencephaly with intractable epilepsy.","date":"2019","source":"Human molecular genetics","url":"https://pubmed.ncbi.nlm.nih.gov/31411685","citation_count":46,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"9799659","id":"PMC_9799659","title":"Activation of p70(S6) kinase by beta-adrenoceptor agonists on adult cardiomyocytes.","date":"1998","source":"Journal of molecular and cellular cardiology","url":"https://pubmed.ncbi.nlm.nih.gov/9799659","citation_count":46,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"22997248","id":"PMC_22997248","title":"Anchored p90 ribosomal S6 kinase 3 is required for cardiac myocyte hypertrophy.","date":"2012","source":"Circulation research","url":"https://pubmed.ncbi.nlm.nih.gov/22997248","citation_count":46,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"7333277","id":"PMC_7333277","title":"Phosphorylation of ribosomal protein S6. Relationship to protein synthesis in HeLa cells.","date":"1981","source":"European journal of biochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/7333277","citation_count":45,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"30590768","id":"PMC_30590768","title":"Disruption of the MAMP-Induced MEKK1-MKK1/MKK2-MPK4 Pathway Activates the TNL Immune Receptor SMN1/RPS6.","date":"2019","source":"Plant & cell physiology","url":"https://pubmed.ncbi.nlm.nih.gov/30590768","citation_count":43,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"18829578","id":"PMC_18829578","title":"Endothelial p70 S6 kinase 1 in regulating tumor angiogenesis.","date":"2008","source":"Cancer research","url":"https://pubmed.ncbi.nlm.nih.gov/18829578","citation_count":43,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"29089336","id":"PMC_29089336","title":"mTORC1/rpS6 regulates blood-testis barrier dynamics and spermatogenetic function in the testis in vivo.","date":"2017","source":"American journal of physiology. Endocrinology and metabolism","url":"https://pubmed.ncbi.nlm.nih.gov/29089336","citation_count":41,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"27102613","id":"PMC_27102613","title":"Ribosomal protein S6 kinase1 coordinates with TOR-Raptor2 to regulate thylakoid membrane biosynthesis in rice.","date":"2016","source":"Biochimica et biophysica acta","url":"https://pubmed.ncbi.nlm.nih.gov/27102613","citation_count":41,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"17130522","id":"PMC_17130522","title":"An S6 mutation in BK channels reveals beta1 subunit effects on intrinsic and voltage-dependent gating.","date":"2006","source":"The Journal of general physiology","url":"https://pubmed.ncbi.nlm.nih.gov/17130522","citation_count":39,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"26276876","id":"PMC_26276876","title":"IL-3 Maintains Activation of the p90S6K/RPS6 Pathway and Increases Translation in Human Eosinophils.","date":"2015","source":"Journal of immunology (Baltimore, Md. : 1950)","url":"https://pubmed.ncbi.nlm.nih.gov/26276876","citation_count":38,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"25379021","id":"PMC_25379021","title":"Resistance to Selumetinib (AZD6244) in colorectal cancer cell lines is mediated by p70S6K and RPS6 activation.","date":"2014","source":"Neoplasia (New York, N.Y.)","url":"https://pubmed.ncbi.nlm.nih.gov/25379021","citation_count":38,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"22564017","id":"PMC_22564017","title":"Exploiting p70 S6 kinase as a target for ovarian cancer.","date":"2012","source":"Expert opinion on therapeutic targets","url":"https://pubmed.ncbi.nlm.nih.gov/22564017","citation_count":35,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"12765942","id":"PMC_12765942","title":"Insulin resistance and lipodystrophy in mice lacking ribosomal S6 kinase 2.","date":"2003","source":"Diabetes","url":"https://pubmed.ncbi.nlm.nih.gov/12765942","citation_count":35,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"23938603","id":"PMC_23938603","title":"Vascular tumors have increased p70 S6-kinase activation and are inhibited by topical rapamycin.","date":"2013","source":"Laboratory investigation; a journal of technical methods and pathology","url":"https://pubmed.ncbi.nlm.nih.gov/23938603","citation_count":34,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"17618850","id":"PMC_17618850","title":"TOR signaling and S6 kinase 1: Yeast catches up.","date":"2007","source":"Cell metabolism","url":"https://pubmed.ncbi.nlm.nih.gov/17618850","citation_count":33,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"28242756","id":"PMC_28242756","title":"ZRF1 is a novel S6 kinase substrate that drives the senescence programme.","date":"2017","source":"The EMBO journal","url":"https://pubmed.ncbi.nlm.nih.gov/28242756","citation_count":33,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"24831086","id":"PMC_24831086","title":"Phosphorylation of ribosomal protein S6 confers PARP inhibitor resistance in BRCA1-deficient cancers.","date":"2014","source":"Oncotarget","url":"https://pubmed.ncbi.nlm.nih.gov/24831086","citation_count":32,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"33429084","id":"PMC_33429084","title":"lnc-Rps4l-encoded peptide RPS4XL regulates RPS6 phosphorylation and inhibits the proliferation of PASMCs caused by hypoxia.","date":"2021","source":"Molecular therapy : the journal of the American Society of Gene Therapy","url":"https://pubmed.ncbi.nlm.nih.gov/33429084","citation_count":31,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"18280803","id":"PMC_18280803","title":"Regulation of ribosomal protein S6 kinases by ubiquitination.","date":"2008","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/18280803","citation_count":31,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"26173379","id":"PMC_26173379","title":"Ribosomal protein S6 phosphorylation is controlled by TOR and modulated by PKA in Candida albicans.","date":"2015","source":"Molecular microbiology","url":"https://pubmed.ncbi.nlm.nih.gov/26173379","citation_count":31,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"20806951","id":"PMC_20806951","title":"6-Thioguanine and S⁶-methylthioguanine are mutagenic in human cells.","date":"2010","source":"ACS chemical biology","url":"https://pubmed.ncbi.nlm.nih.gov/20806951","citation_count":30,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"32350925","id":"PMC_32350925","title":"p-Coumaric acid prevents obesity via activating thermogenesis in brown adipose tissue mediated by mTORC1-RPS6.","date":"2020","source":"FASEB journal : official publication of the Federation of American Societies for Experimental Biology","url":"https://pubmed.ncbi.nlm.nih.gov/32350925","citation_count":29,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"21339731","id":"PMC_21339731","title":"Divergent functions for eIF4E and S6 kinase by sonic hedgehog mitogenic signaling in the developing cerebellum.","date":"2011","source":"Oncogene","url":"https://pubmed.ncbi.nlm.nih.gov/21339731","citation_count":29,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"6693389","id":"PMC_6693389","title":"Ribosome conformational changes associated with protein S6 phosphorylation.","date":"1984","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/6693389","citation_count":28,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"3530808","id":"PMC_3530808","title":"Activation of S6 kinase activity in astrocytes by insulin, somatomedin C and TPA.","date":"1986","source":"FEBS letters","url":"https://pubmed.ncbi.nlm.nih.gov/3530808","citation_count":28,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"26134285","id":"PMC_26134285","title":"Hydroxychloroquine Destabilizes Phospho-S6 in Human Renal Carcinoma Cells.","date":"2015","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/26134285","citation_count":28,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"19144847","id":"PMC_19144847","title":"Ribosomal s6 kinase cooperates with casein kinase 2 to modulate the Drosophila circadian molecular oscillator.","date":"2009","source":"The Journal of neuroscience : the official journal of the Society for Neuroscience","url":"https://pubmed.ncbi.nlm.nih.gov/19144847","citation_count":27,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"24431434","id":"PMC_24431434","title":"S6 kinase inhibits intrinsic axon regeneration capacity via AMP kinase in Caenorhabditis elegans.","date":"2014","source":"The Journal of neuroscience : the official journal of the Society for Neuroscience","url":"https://pubmed.ncbi.nlm.nih.gov/24431434","citation_count":27,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"25229342","id":"PMC_25229342","title":"Phosphorylation of ribosomal protein S6 mediates compensatory renal hypertrophy.","date":"2014","source":"Kidney international","url":"https://pubmed.ncbi.nlm.nih.gov/25229342","citation_count":26,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"15556089","id":"PMC_15556089","title":"Functional interaction in establishment of ribosomal integrity between small subunit protein rpS6 and translational regulator rpL10/Grc5p.","date":"2004","source":"FEMS yeast research","url":"https://pubmed.ncbi.nlm.nih.gov/15556089","citation_count":26,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"27540520","id":"PMC_27540520","title":"Down-Regulation of Ribosomal S6 kinase RPS6KA6 in Acute Myeloid Leukemia Patients.","date":"2016","source":"Cell journal","url":"https://pubmed.ncbi.nlm.nih.gov/27540520","citation_count":26,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"26754419","id":"PMC_26754419","title":"Isolation and fine mapping of Rps6: an intermediate host resistance gene in barley to wheat stripe rust.","date":"2016","source":"TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik","url":"https://pubmed.ncbi.nlm.nih.gov/26754419","citation_count":25,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"29084767","id":"PMC_29084767","title":"G protein-dependent signaling triggers a β-arrestin-scaffolded p70S6K/ rpS6 module that controls 5'TOP mRNA translation.","date":"2018","source":"FASEB journal : official publication of the Federation of American Societies for Experimental Biology","url":"https://pubmed.ncbi.nlm.nih.gov/29084767","citation_count":25,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"25748449","id":"PMC_25748449","title":"Drosophila S6 Kinase like inhibits neuromuscular junction growth by downregulating the BMP receptor thickveins.","date":"2015","source":"PLoS genetics","url":"https://pubmed.ncbi.nlm.nih.gov/25748449","citation_count":25,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"37098351","id":"PMC_37098351","title":"The p-rpS6-zone delineates wounding responses and the healing process.","date":"2023","source":"Developmental cell","url":"https://pubmed.ncbi.nlm.nih.gov/37098351","citation_count":24,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"16640565","id":"PMC_16640565","title":"Receptor association and tyrosine phosphorylation of S6 kinases.","date":"2006","source":"The FEBS journal","url":"https://pubmed.ncbi.nlm.nih.gov/16640565","citation_count":24,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"16125835","id":"PMC_16125835","title":"Arabidopsis PDK1: identification of sites important for activity and downstream phosphorylation of S6 kinase.","date":"2005","source":"Biochimie","url":"https://pubmed.ncbi.nlm.nih.gov/16125835","citation_count":24,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"17081983","id":"PMC_17081983","title":"Global, in vivo, and site-specific phosphorylation dynamics in signaling networks.","date":"2006","source":"Cell","url":"https://pubmed.ncbi.nlm.nih.gov/17081983","citation_count":2861,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"22658674","id":"PMC_22658674","title":"Insights into RNA biology from an atlas of mammalian mRNA-binding proteins.","date":"2012","source":"Cell","url":"https://pubmed.ncbi.nlm.nih.gov/22658674","citation_count":1718,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"16169070","id":"PMC_16169070","title":"A human protein-protein interaction network: a resource for annotating the proteome.","date":"2005","source":"Cell","url":"https://pubmed.ncbi.nlm.nih.gov/16169070","citation_count":1704,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"12477932","id":"PMC_12477932","title":"Generation and initial analysis of more than 15,000 full-length human and mouse cDNA sequences.","date":"2002","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/12477932","citation_count":1479,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"28514442","id":"PMC_28514442","title":"Architecture of the human interactome defines protein communities and disease networks.","date":"2017","source":"Nature","url":"https://pubmed.ncbi.nlm.nih.gov/28514442","citation_count":1085,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"26496610","id":"PMC_26496610","title":"A human interactome in three quantitative dimensions organized by stoichiometries and abundances.","date":"2015","source":"Cell","url":"https://pubmed.ncbi.nlm.nih.gov/26496610","citation_count":1015,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"16286006","id":"PMC_16286006","title":"mTOR and S6K1 mediate assembly of the translation preinitiation complex through dynamic protein interchange and ordered phosphorylation events.","date":"2005","source":"Cell","url":"https://pubmed.ncbi.nlm.nih.gov/16286006","citation_count":946,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"12820960","id":"PMC_12820960","title":"Insulin activation of Rheb, a mediator of mTOR/S6K/4E-BP signaling, is inhibited by TSC1 and 2.","date":"2003","source":"Molecular cell","url":"https://pubmed.ncbi.nlm.nih.gov/12820960","citation_count":853,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"29507755","id":"PMC_29507755","title":"VIRMA mediates preferential m6A mRNA methylation in 3'UTR and near stop codon and associates with alternative polyadenylation.","date":"2018","source":"Cell discovery","url":"https://pubmed.ncbi.nlm.nih.gov/29507755","citation_count":829,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"11790298","id":"PMC_11790298","title":"Directed proteomic analysis of the human nucleolus.","date":"2002","source":"Current biology : CB","url":"https://pubmed.ncbi.nlm.nih.gov/11790298","citation_count":780,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"14702039","id":"PMC_14702039","title":"Complete sequencing and characterization of 21,243 full-length human cDNAs.","date":"2003","source":"Nature genetics","url":"https://pubmed.ncbi.nlm.nih.gov/14702039","citation_count":754,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"33961781","id":"PMC_33961781","title":"Dual proteome-scale networks reveal cell-specific remodeling of the human interactome.","date":"2021","source":"Cell","url":"https://pubmed.ncbi.nlm.nih.gov/33961781","citation_count":705,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"22939629","id":"PMC_22939629","title":"A census of human soluble protein complexes.","date":"2012","source":"Cell","url":"https://pubmed.ncbi.nlm.nih.gov/22939629","citation_count":689,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"17693255","id":"PMC_17693255","title":"IKK beta suppression of TSC1 links inflammation and tumor angiogenesis via the mTOR pathway.","date":"2007","source":"Cell","url":"https://pubmed.ncbi.nlm.nih.gov/17693255","citation_count":545,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"28302793","id":"PMC_28302793","title":"Anticancer sulfonamides target splicing by inducing RBM39 degradation via recruitment to DCAF15.","date":"2017","source":"Science (New York, N.Y.)","url":"https://pubmed.ncbi.nlm.nih.gov/28302793","citation_count":533,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"27022092","id":"PMC_27022092","title":"G3BP-Caprin1-USP10 complexes mediate stress granule condensation and associate with 40S subunits.","date":"2016","source":"The Journal of cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/27022092","citation_count":512,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"23636399","id":"PMC_23636399","title":"Structures of the human and Drosophila 80S ribosome.","date":"2013","source":"Nature","url":"https://pubmed.ncbi.nlm.nih.gov/23636399","citation_count":481,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"15489334","id":"PMC_15489334","title":"The status, quality, and expansion of the NIH full-length cDNA project: the Mammalian Gene Collection (MGC).","date":"2004","source":"Genome research","url":"https://pubmed.ncbi.nlm.nih.gov/15489334","citation_count":438,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"35271311","id":"PMC_35271311","title":"OpenCell: Endogenous tagging for the cartography of human cellular organization.","date":"2022","source":"Science (New York, N.Y.)","url":"https://pubmed.ncbi.nlm.nih.gov/35271311","citation_count":432,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"20020773","id":"PMC_20020773","title":"Global analysis of TDP-43 interacting proteins reveals strong association with RNA splicing and translation machinery.","date":"2010","source":"Journal of proteome research","url":"https://pubmed.ncbi.nlm.nih.gov/20020773","citation_count":422,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"23463506","id":"PMC_23463506","title":"The intracellular interactome of tetraspanin-enriched microdomains reveals their function as sorting machineries toward exosomes.","date":"2013","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/23463506","citation_count":413,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"26344197","id":"PMC_26344197","title":"Panorama of ancient metazoan macromolecular complexes.","date":"2015","source":"Nature","url":"https://pubmed.ncbi.nlm.nih.gov/26344197","citation_count":407,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"15189156","id":"PMC_15189156","title":"The molecular mechanics of eukaryotic translation.","date":"2004","source":"Annual review of biochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/15189156","citation_count":396,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"25901680","id":"PMC_25901680","title":"Structure of the human 80S ribosome.","date":"2015","source":"Nature","url":"https://pubmed.ncbi.nlm.nih.gov/25901680","citation_count":380,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"12045200","id":"PMC_12045200","title":"Tuberin regulates p70 S6 kinase activation and ribosomal protein S6 phosphorylation. A role for the TSC2 tumor suppressor gene in pulmonary lymphangioleiomyomatosis (LAM).","date":"2002","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/12045200","citation_count":368,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"25796446","id":"PMC_25796446","title":"Aerobic glycolysis tunes YAP/TAZ transcriptional activity.","date":"2015","source":"The EMBO journal","url":"https://pubmed.ncbi.nlm.nih.gov/25796446","citation_count":362,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"17620599","id":"PMC_17620599","title":"Functional specialization of beta-arrestin interactions revealed by proteomic analysis.","date":"2007","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/17620599","citation_count":360,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"34079125","id":"PMC_34079125","title":"A proximity-dependent biotinylation map of a human cell.","date":"2021","source":"Nature","url":"https://pubmed.ncbi.nlm.nih.gov/34079125","citation_count":339,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"21726808","id":"PMC_21726808","title":"Acetylation regulates gluconeogenesis by promoting PEPCK1 degradation via recruiting the UBR5 ubiquitin ligase.","date":"2011","source":"Molecular cell","url":"https://pubmed.ncbi.nlm.nih.gov/21726808","citation_count":337,"is_preprint":false,"source_track":"gene2pubmed"},{"pmid":"21145461","id":"PMC_21145461","title":"Dynamics of cullin-RING ubiquitin ligase network revealed by systematic quantitative proteomics.","date":"2010","source":"Cell","url":"https://pubmed.ncbi.nlm.nih.gov/21145461","citation_count":318,"is_preprint":false,"source_track":"gene2pubmed"}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":47257,"output_tokens":9274,"usd":0.14044},"stage2":{"model":"claude-opus-4-6","input_tokens":13194,"output_tokens":3857,"usd":0.243592},"total_usd":0.888935,"stage1_batch_id":"msgbatch_011rkcQdVV1RaFq9XhcG6df6","stage2_batch_id":"msgbatch_01RzgrJafdF4EWHhj5mNi6dh","note":"batch pricing = 50% of standard","round2_stage1":{"model":"claude-sonnet-4-6","input_tokens":59257,"output_tokens":15543,"usd":0.205458},"round2_rules_fired":"R3","round2_stage2":{"model":"claude-opus-4-6","input_tokens":19616,"output_tokens":4062,"usd":0.299445}},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1988,\n      \"finding\": \"A mitogen-activated S6 kinase (p70 S6K) was purified to homogeneity from Swiss 3T3 cells; it phosphorylates 40S ribosomal protein S6 in vitro on the same tryptic peptides observed in vivo, autophosphorylates, and is inactivated by phosphatase 2A treatment, establishing that its activity is directly controlled by phosphorylation.\",\n      \"method\": \"Protein purification, in vitro kinase assay, tryptic phosphopeptide mapping, phosphatase 2A inactivation\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — purified enzyme, in vitro assay with phosphopeptide mapping, enzymatic inactivation by phosphatase\",\n      \"pmids\": [\"3257566\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1989,\n      \"finding\": \"The insulin-stimulated hepatic p70 S6 kinase phosphorylates 40S ribosomal protein S6 and is inactivated 97% by protein phosphatase 2A; a synthetic peptide corresponding to the C-terminal 32 amino acids of S6 (encompassing all phosphorylation sites) was used as an affinity ligand, confirming that S6 phosphorylation sites reside at the C-terminus.\",\n      \"method\": \"Protein purification (>50,000-fold), peptide affinity chromatography, phosphatase 2A inactivation, in vitro kinase assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — near-homogeneous enzyme, multiple orthogonal purification steps, phosphatase validation\",\n      \"pmids\": [\"2760046\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1991,\n      \"finding\": \"The p70 S6 kinase phosphorylates ribosomal protein S6 in vitro at Ser235, Ser236, Ser240, and Ser244 within a 19-amino-acid C-terminal peptide, matching four of five in vivo sites; autophosphorylation is intramolecular and reduces kinase activity ~25%; Mg2+ is required and Mn2+/Zn2+ are inhibitory.\",\n      \"method\": \"In vitro kinase assay with purified p70 S6K, phosphopeptide mapping, mass spectrometry-based site identification, phosphatase treatment\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — direct in vitro phosphorylation site mapping with sequencing, replicated with prior in vivo data\",\n      \"pmids\": [\"1939282\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1992,\n      \"finding\": \"Rapamycin specifically inhibits p70 S6 kinase activity (but not p85 Rsk or MAP kinase) and prevents appearance of the hyperphosphorylated form of p70 S6K, establishing that a rapamycin-sensitive signaling element is required for p70 S6K activation and S6 phosphorylation.\",\n      \"method\": \"In vitro kinase assay with 40S subunits as substrate, COS cell transfection, gel mobility shift, pharmacological inhibition\",\n      \"journal\": \"Science (New York, N.Y.)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple cell systems, reciprocal inhibitor specificity controls, replicated across labs\",\n      \"pmids\": [\"1380182\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1994,\n      \"finding\": \"p70/p85 S6 kinase activation by PDGF is independent of p21ras; it bifurcates from the Ras pathway at the receptor level and requires tyrosine autophosphorylation at Y751 in the PDGFR kinase-insert domain.\",\n      \"method\": \"Dominant-negative Ras/Raf mutants, epitope-tagged kinase assays, PDGFR kinase-insert deletion mutants\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genetic epistasis with dominant-negative constructs, multiple receptor mutants, rigorous controls\",\n      \"pmids\": [\"8090223\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1995,\n      \"finding\": \"FRAP/mTOR kinase activity is required in vivo to control p70 S6K; FRAP autophosphorylates in vitro and this is blocked by FKBP12-rapamycin; an N-terminal domain of FRAP beyond its kinase domain is also required for p70 S6K regulation.\",\n      \"method\": \"FRAP kinase-dead mutants, rapamycin-binding mutants, in vitro autophosphorylation assay, deletion analysis\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro kinase assay combined with structure-function mutant analysis in cells\",\n      \"pmids\": [\"7566123\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1996,\n      \"finding\": \"An autoinhibitory domain (residues 58–77) in p70/p85 S6K overrides the requirement of the N-terminus for kinase activation; deletion of this domain yields constitutive, rapamycin-insensitive S6K activity, defining two discrete N-terminal domains: one required for mitogen activation/rapamycin sensitivity (aa 1–58) and one for autoinhibition (aa 58–77).\",\n      \"method\": \"S6K deletion mutagenesis, kinase activity assays in Rat1 cells\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — systematic deletion mutagenesis with functional readout\",\n      \"pmids\": [\"8524322\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"RAFT1/mTOR directly phosphorylates p70 S6K on Thr389 (rapamycin-sensitive, required for kinase activity) and 4E-BP1/2; phosphorylation of 4E-BP1 on Thr36/Thr45 blocks its association with eIF4E in vitro; serum stimulates RAFT1 kinase activity with kinetics paralleling p70 S6K and 4E-BP1 phosphorylation.\",\n      \"method\": \"In vitro kinase assay, co-immunoprecipitation, recombinant substrate phosphorylation, site-directed analysis\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — direct in vitro phosphorylation with phosphosite identification, functional consequence for eIF4E binding\",\n      \"pmids\": [\"9465032\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1995,\n      \"finding\": \"Three nuclear localization signals (NLSs) reside in the C-terminal half of human ribosomal protein S6; a single NLS is sufficient for nuclear import, removal of all three NLSs retains S6 in the cytoplasm, and the full protein plus any single NLS accumulates in the nucleolus, with a distinct domain required (but not sufficient) for nucleolar targeting.\",\n      \"method\": \"S6–β-galactosidase chimera transfection, deletion mutagenesis, intracellular localization by β-galactosidase activity assay\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — systematic deletion mutagenesis with direct localization readout\",\n      \"pmids\": [\"8590812\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"Amino acid withdrawal rapidly inactivates p70 S6K and causes dephosphorylation of 4E-BP1, eIF4E, and hyperphosphorylation of eEF2, all reversible by rapamycin- and PI3K-sensitive pathways; amino acids regulate p70 S6K independently of protein kinase B (PKB/Akt).\",\n      \"method\": \"In vitro S6K kinase assay, phosphorylation state analysis, pharmacological inhibitors (rapamycin, wortmannin), PKB activity assay\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal measurements, pathway epistasis via inhibitors\",\n      \"pmids\": [\"9693128\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"Drosophila S6 kinase (dS6K) regulates cell size in a cell-autonomous manner; dS6K-null flies have smaller cells (not fewer cells) throughout larval development, distinct from ribosomal protein mutant (Minute) phenotypes.\",\n      \"method\": \"Drosophila genetic knockout, cell size vs. cell number measurements, genetic mosaic analysis\",\n      \"journal\": \"Science (New York, N.Y.)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — in vivo loss-of-function with specific cellular phenotype (cell size), ortholog confirmed by domain/context\",\n      \"pmids\": [\"10497130\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"Leucine specifically activates p70 S6K in amino acid-depleted cells in a rapamycin-sensitive, wortmannin-less-sensitive manner; the effect requires the correct chirality, branched hydrocarbon structure, and primary amine of leucine, indicating a highly specific leucine sensor upstream of p70 S6K.\",\n      \"method\": \"In vitro S6K kinase assay, pharmacological inhibitors, structure-activity analysis with leucine analogs\",\n      \"journal\": \"FEBS letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — structure-activity analysis and pharmacological dissection, single lab\",\n      \"pmids\": [\"10214966\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"Osmotic stress (sorbitol) inactivates p70/p85 S6K by dephosphorylation of Thr412 (equivalent to Thr389 in shorter isoform nomenclature) through a calyculin A-sensitive phosphatase, the same phosphatase pathway used by rapamycin; stress-activated kinase pathways (SAPK/JNK, p38) are not involved.\",\n      \"method\": \"S6K kinase assay, phospho-specific antibodies, dominant-negative mutants, pharmacological phosphatase inhibitors\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — phospho-site specific antibodies, multiple inhibitors tested, single lab\",\n      \"pmids\": [\"10455142\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"TGF-β induces association of its receptor with PP2A-Bα, which then recruits PP2A subunits (Bα, Aβ, Cα) to p70 S6K, causing its dephosphorylation and inactivation; this is a second, Smad-independent pathway for TGF-β-induced G1 arrest.\",\n      \"method\": \"Co-immunoprecipitation, kinase activity assay, co-precipitation of PP2A subunits with p70 S6K, cell cycle analysis\",\n      \"journal\": \"Genes & development\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal co-IP, multiple subunit associations, functional epistasis with dual pathway abrogation\",\n      \"pmids\": [\"11124802\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"S6K2 activation requires mTOR kinase activity, amino acid sufficiency, and phosphatidic acid, similar to S6K1; mTOR nuclear import/export dynamics regulate S6K2 activation, with optimal cytoplasmic-nuclear shuttling required for maximal S6K2 activity.\",\n      \"method\": \"Nuclear export signal/NLS-tagged mTOR constructs, kinase assays, leptomycin B treatment, amino acid deprivation\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — engineered NLS/NES constructs with functional readout, single lab\",\n      \"pmids\": [\"12087098\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"p70 S6K directly phosphorylates tau at S262, S214, and T212 in vitro; p-p70S6K (T421/S424) co-immunoprecipitates with p-tau (S262); zinc-induced p70S6K activation selectively upregulates translation of S6 and tau mRNA without affecting global protein synthesis.\",\n      \"method\": \"In vitro kinase assay, immunoprecipitation, metabolic labeling in SH-SY5Y cells\",\n      \"journal\": \"FEBS letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 — direct in vitro phosphorylation assay; Tier 3 for translation selectivity, single lab\",\n      \"pmids\": [\"16364302\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"hVps34 (class III PI3K) is required for p70 S6K activation in response to amino acids and glucose but not insulin; it acts upstream of mTOR (knockdown reduces 4E-BP1 phosphorylation) and is inhibited by AMPK, identifying it as a nutrient-regulated kinase that integrates amino acid and glucose signals to the mTOR–S6K pathway.\",\n      \"method\": \"siRNA knockdown, antibody microinjection, FYVE domain dominant-negative, Akt/TSC2 phosphorylation controls, S6K kinase assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal loss-of-function approaches, epistasis via 4E-BP1 readout, single lab\",\n      \"pmids\": [\"16049009\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"S6K1 and S6K2 interact with receptor tyrosine kinases (e.g., PDGFR) via kinase or kinase-extension domains; PDGF stimulation induces complex formation and tyrosine phosphorylation of S6K1 at Y39 and S6K2 at Y45 via a PDGFR-Src pathway; these tyrosine phosphorylations do not affect S6K activity or localization.\",\n      \"method\": \"Co-immunoprecipitation, PDGFR mutants, Src inhibitors (genistein, PP1, SU6656), site-directed mutagenesis, immunofluorescence\",\n      \"journal\": \"The FEBS journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal co-IP, mutagenesis of phosphosites, receptor mutant epistasis, single lab\",\n      \"pmids\": [\"16640565\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"S6K deletion in skeletal muscle elevates AMP/ATP and inorganic phosphate ratios, activating AMPK; AMPK upregulation drives mitochondrial biogenesis and fatty acid β-oxidation in S6K-deficient muscle, and AMPK inhibition restores cell growth, placing S6K1 upstream of AMPK in energy state regulation.\",\n      \"method\": \"S6K1/2 knockout mice, metabolite measurements, AMPK activity assays, genetic rescue with AMPK inhibition\",\n      \"journal\": \"Cell metabolism\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genetic epistasis with double KO and AMPK rescue, metabolite measurements, clean KO phenotype\",\n      \"pmids\": [\"17550782\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Drosophila ATG1 kinase negatively regulates the TOR/S6K pathway; ATG1 overexpression in mammalian cells markedly inhibits S6K and blocks phosphorylation of S6K at Thr389; ATG1 knockdown by siRNA causes ectopic S6K activation and S6 phosphorylation, establishing crosstalk between autophagy regulation and S6K-dependent cell growth.\",\n      \"method\": \"Drosophila genetic mutants, mammalian cell overexpression/siRNA knockdown, S6K Thr389 phospho-specific antibody, S6 phosphorylation assay\",\n      \"journal\": \"EMBO reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — bidirectional genetic manipulation (gain and loss of function), multiple organisms, specific phosphosite readout\",\n      \"pmids\": [\"17347671\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"S6K ubiquitination occurs in cells and leads to proteasome-mediated degradation; ubiquitination sites map to the kinase domain; N- and C-terminal regulatory regions modulate ubiquitination efficiency, adding proteasomal turnover as a layer of S6K regulation beyond phosphorylation.\",\n      \"method\": \"Ubiquitin co-immunoprecipitation, proteasomal inhibitor treatment, domain deletion constructs\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — co-IP and deletion mapping, functional consequence via inhibitors, single lab\",\n      \"pmids\": [\"18280803\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"S6K1 deletion in mice extends lifespan and confers resistance to age-related pathologies (bone, immune, motor dysfunction, insulin resistance); S6K1 KO gene expression resembles caloric restriction or AMPK activation, placing S6K1 in the mTOR–longevity pathway in mammals.\",\n      \"method\": \"S6K1 knockout mouse, lifespan analysis, pathology assessment, gene expression profiling\",\n      \"journal\": \"Science (New York, N.Y.)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — clean KO with defined longevity and metabolic phenotypes, gene expression pathway analysis\",\n      \"pmids\": [\"19797661\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"S6K1 mediates autophagy induction; dominant-negative S6K1 or S6K1 siRNA knockdown suppresses autophagy to the same extent as resveratrol; resveratrol targets S6K1 in a large-scale in vitro kinase screen; S6K1 promotes full induction of autophagy downstream of mTOR.\",\n      \"method\": \"In vitro kinase screen, dominant-negative overexpression, siRNA knockdown, autophagy assay, epistasis with resveratrol\",\n      \"journal\": \"Aging\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple genetic loss-of-function approaches with consistent phenotype, single lab\",\n      \"pmids\": [\"20157535\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Ribosomal protein S6 (rpS6) regulates blood-testis barrier (BTB) dynamics by modulating F-actin organization and recruitment of adhesion proteins (claudin-11, occludin); rpS6 is spatiotemporally activated at the BTB during the epithelial cycle; its activation disrupts Sertoli cell TJ barrier integrity, while siRNA silencing promotes barrier integrity.\",\n      \"method\": \"siRNA/shRNA knockdown in vitro and in vivo, F-actin immunofluorescence, BTB integrity assay, protein localization\",\n      \"journal\": \"Endocrinology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — bidirectional manipulation (activation vs. siRNA), in vitro and in vivo BTB assays, specific protein recruitment readout\",\n      \"pmids\": [\"22948214\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"S6K activity (and rpS6 phosphorylation) controls the ribosome biogenesis transcriptional program; >75% of ribosome biogenesis factors are S6K-dependent; this transcriptional regulation is also observed in rpS6 knock-in mutants that cannot be phosphorylated, indicating a specific role for rpS6 phosphorylation in the ribosome biogenesis transcriptional program independent of ribosomal protein synthesis per se.\",\n      \"method\": \"S6K1/S6K2 double KO mice, rpS6 phosphorylation-site knock-in mice, whole-genome microarray, polysomal RNA profiling\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — two independent genetic models (S6K DKO and rpS6 knock-in), genome-wide transcriptional readout\",\n      \"pmids\": [\"23318442\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Ribosomal protein S6 phosphorylation is required for compensatory renal hypertrophy; rpS6 knock-in mice (non-phosphorylatable) show blunted uninephrectomy-induced hypertrophy and attenuated cyclin D1 increase/cyclin E decrease; rapamycin prevents rpS6 phosphorylation and hypertrophy in WT but not knock-in mice despite equal mTORC1 activity (4E-BP1 phosphorylation), demonstrating rpS6 phosphorylation is the key effector.\",\n      \"method\": \"rpS6 knock-in mouse model, uninephrectomy model, immunoblotting, rapamycin treatment, cyclin expression analysis\",\n      \"journal\": \"Kidney international\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genetic knock-in plus pharmacological dissection, internal mTORC1 activity control (4E-BP1)\",\n      \"pmids\": [\"25229342\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"rpS6 interacts with mTORC2 components and acts in positive feedback: rpS6 knockdown attenuates insulin-induced mTORC2 activation and Akt-Ser473 phosphorylation, while rpS6 overexpression enhances Akt-Ser473 phosphorylation; disruption of rpS6/mTORC2 pathway abrogates insulin-induced cytoprotection against oxidative stress.\",\n      \"method\": \"Co-immunoprecipitation, siRNA knockdown, rpS6 overexpression, Akt phosphorylation assay, cell survival assay\",\n      \"journal\": \"Circulation research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal co-IP, bidirectional manipulation, functional survival readout, multiple cell systems\",\n      \"pmids\": [\"24557881\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"In Saccharomyces cerevisiae, TORC1 promotes rpS6 phosphorylation via the AGC kinase Ypk3 (not Sch9); phosphorylation-deficient regulatory motif mutations in Ypk3 abolish rpS6 phosphorylation; complementation with human S6K restores rapamycin-sensitive rpS6 phosphorylation in ypk3Δ cells.\",\n      \"method\": \"Yeast genetics (ypk3Δ, sch9Δ), rapamycin treatment, phospho-S6-specific antibody, complementation with human S6K\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genetic epistasis, cross-species complementation, specific phospho-antibody, replicated with multiple mutants\",\n      \"pmids\": [\"25767889\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"In S. cerevisiae, Rps6 phosphorylation on Ser232/Ser233 is regulated by both TORC1 (via Ypk3 and PP1 phosphatase Glc7) and TORC2 (via Ypk1, Ser232 only); non-phosphorylatable Rps6 reduces growth and causes a 40S biogenesis defect not seen when kinase activity is disrupted; ribosome profiling found no role for Rps6 phosphorylation in global or individual mRNA translation.\",\n      \"method\": \"Yeast genetics, TORC1/TORC2 mutants, polysome profiling, ribosome profiling, 40S biogenesis assays\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — comprehensive genetic and genomic approach, multiple TOR complex dissection, ribosome profiling\",\n      \"pmids\": [\"26582391\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"ZRF1 is a novel S6 kinase substrate; S6 kinases phosphorylate ZRF1 on Ser47 in cells and mammalian tissues in vivo; ZRF1 knockdown or expression of its phosphorylation mutant blunts S6K-dependent cellular senescence and alters p16 levels, linking mTORC1-S6K nutrient sensing to the senescence program via ZRF1.\",\n      \"method\": \"Chemical genetic screen, in vitro/in vivo phosphorylation assays, siRNA knockdown, phospho-mutant expression, p16 and senescence markers\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — chemical genetic substrate identification plus in vivo phosphorylation validation; Tier 2 functional epistasis via rescue\",\n      \"pmids\": [\"28242756\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"mTORC1/rpS6 signaling promotes BTB disruption and regulates spermatogenesis in vivo; a constitutively active phosphomimetic rpS6 mutant induces BTB disruption more effectively than wild-type rpS6, acting through changes in actin- and microtubule-based cytoskeleton organization and spatiotemporal expression of associated regulatory proteins.\",\n      \"method\": \"In vivo testis transfection with rpS6-WT and phosphomimetic quadruple mutant plasmids, functional BTB integrity assay, cytoskeleton protein localization\",\n      \"journal\": \"American journal of physiology. Endocrinology and metabolism\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — in vivo functional BTB assay, phosphomimetic vs. WT comparison, cytoskeletal mechanistic readout\",\n      \"pmids\": [\"29089336\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"S6K1 and S6K2 localize to stress granules and are required for their assembly and maintenance after mild oxidative stress; S6K1 promotes stress granule formation via regulation of eIF2α phosphorylation, while S6K2 controls their persistence; in C. elegans, S6K ortholog RSKS-1 promotes stress granule assembly and its loss sensitizes worms to stress-induced death.\",\n      \"method\": \"Immunofluorescence localization, siRNA knockdown, stress granule assembly assay, eIF2α phosphorylation measurement, C. elegans genetics\",\n      \"journal\": \"Cell death and differentiation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — direct localization, bidirectional manipulation, mechanistic eIF2α link, replicated in C. elegans\",\n      \"pmids\": [\"29523872\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"β-arrestins constitutively assemble a p70S6K/rpS6 complex; FSH-dependent G protein (Gαs) signaling enhances p70S6K activity within this preassembled β-arrestin scaffold, leading to rpS6 phosphorylation and translation of 5'TOP mRNAs within minutes; Gαs depletion reduces FSH-induced rpS6 phosphorylation within the β-arrestin complex.\",\n      \"method\": \"Co-immunoprecipitation, protein-protein docking, Gαs siRNA knockdown, FSH stimulation, 5'TOP mRNA translation assay\",\n      \"journal\": \"FASEB journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — direct interaction demonstrated, Gαs epistasis, functional mRNA translation readout, single lab\",\n      \"pmids\": [\"29084767\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"A somatic gain-of-function RPS6 p.R232H variant found in hemimegalencephaly brain tissue promotes increased cell proliferation in animal models; combined with MTOR p.S2215F, double mutants show more severe phenotypes (increased proliferation, migration defects, balloon cells), establishing RPS6 as a disease-related gene in cortical dysplasia.\",\n      \"method\": \"Somatic mosaicism detection (deep sequencing), overexpression in animal models, cellular proliferation/migration assays\",\n      \"journal\": \"Human molecular genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — functional overexpression in animal model, double-mutant epistasis; moderate given single study\",\n      \"pmids\": [\"31411685\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"RPS6 phosphorylation progressively decreases on ribosomes as they translate an mRNA (dephosphorylation during elongation); as a consequence, ribosomes on short CDS mRNAs carry higher average phospho-RPS6; genetic removal of RPS6 phosphorylation sites preferentially reduces translation of short-CDS mRNAs but does not affect 5'TOP mRNA translation despite their short CDSs.\",\n      \"method\": \"Selective ribosome footprinting with phospho-RPS6 antibody, RPS6 phosphorylation-site knock-in cell lines, polysome analysis\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — selective ribosome footprinting, genetic knock-in model, functional translation readout across transcriptome\",\n      \"pmids\": [\"34871442\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"RPS6 is phosphorylated in response to skin wounding in mammals, forming a zone of activation surrounding the wound (p-rpS6-zone) within minutes; this zone persists until healing is complete and encompasses proliferation, growth, senescence, and angiogenesis; a mouse model unable to phosphorylate rpS6 shows accelerated initial wound closure but impaired overall healing, demonstrating rpS6 phosphorylation modulates (but does not drive) the healing process.\",\n      \"method\": \"In vivo mouse wounding model, phospho-rpS6 immunofluorescence, rpS6 phosphorylation-site knock-in mice, wound closure assays\",\n      \"journal\": \"Developmental cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genetic knock-in with in vivo functional wound healing assay, spatial phosphorylation mapping\",\n      \"pmids\": [\"37098351\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"The lncRNA-encoded peptide RPS4XL binds directly to RPS6 (identified by co-immunoprecipitation and mass spectrometry) and inhibits RPS6 phosphorylation at Ser240/Ser244, thereby suppressing hypoxia-induced PASMC proliferation.\",\n      \"method\": \"Co-immunoprecipitation, mass spectrometry, phospho-RPS6 immunoblot, MTT/BrdU proliferation assays, siRNA\",\n      \"journal\": \"Molecular therapy\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — co-IP/MS identifies interaction, functional knockdown, single lab\",\n      \"pmids\": [\"33429084\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1984,\n      \"finding\": \"Phosphorylation of ribosomal protein S6 is associated with conformational changes in the 40S ribosomal subunit, altering the accessibility of multiple small and large subunit proteins (S3, S4, S7, S23/24, L9, L10, L12, L18, L27, L34, L36) to reductive methylation.\",\n      \"method\": \"Reductive methylation accessibility assay comparing unphosphorylated vs. phosphorylated S6 ribosomes\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — biochemical accessibility assay, single lab, indirect structural readout\",\n      \"pmids\": [\"6693389\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Functional ribosomes require an interaction between small subunit rpS6 and large subunit rpL10/Grc5p for subunit joining; in yeast, this interaction is required for differential protein expression and modulation of the polysome complement.\",\n      \"method\": \"Yeast genetics, polysome profiling, protein expression analysis, genetic interaction studies\",\n      \"journal\": \"FEMS yeast research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — yeast genetic interaction epistasis, polysome profiling, single lab; ortholog context consistent\",\n      \"pmids\": [\"15556089\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"RPS6 (ribosomal protein S6) is a component of the 40S ribosomal subunit whose C-terminal serine residues (Ser235, Ser236, Ser240, Ser244, and Ser247) are phosphorylated by p70 S6K1/S6K2 (downstream of the mTORC1–PDK1 axis, activated by growth factors, insulin, and amino acids) and also by p90RSK; phosphorylation progressively declines as ribosomes elongate through longer ORFs, preferentially promoting translation of short-CDS mRNAs, and in the context of ribosome biogenesis drives a transcriptional program for ribosome biogenesis factors; beyond translation, phospho-rpS6 regulates compensatory organ hypertrophy, blood-testis barrier dynamics (via actin cytoskeleton reorganization), skin wound healing, and provides positive feedback to mTORC2/Akt signaling, while somatic gain-of-function variants cause cortical dysplasia.\"\n}\n```\n\n--- ROUND 2 (R2/R3, augmented corpus) ---\n\nI'll classify each paper before extracting discoveries.\n\n**EXCLUDE (alias collisions / off-target):**\n- Papers 30, 33, 49, 60, 100 (Arabidopsis RPS6/TOR/S6K — plant symbol collision, not mammalian RPS6 protein)\n- Papers 32, 68, 95 (Arabidopsis/barley/plant RPS6 resistance genes — plant disease resistance, not ribosomal protein)\n- Paper 54 (soybean Rps6 — plant pathogen resistance)\n- Paper 71 (rice S6K1 — plant)\n- Paper 52 (Saporin-S6 — plant ribosome-inactivating protein, not mammalian RPS6)\n- Paper 72 (BK channel S6 mutation — ion channel transmembrane segment, not ribosomal protein)\n- Paper 76 (RSK2/p90S6K2 knockout mice — this is RPS6KA3, not RPS6)\n- Paper 84 (thioguanine mutagenesis — no RPS6 connection)\n- Paper 94 (RPS6KA6/RSK4 — this is a kinase gene, not RPS6 itself)\n- Papers from gene2pubmed set: [3,4,6,9,11,12,13,15,21,22,28,29,30] — general proteomics/interactome resources not specifically about RPS6 mechanism\n- Paper 66 (RSK3/p90S6K — anchoring protein, not RPS6)\n- Paper 78 (yeast Sch9 commentary — about S6K1 ortholog, not RPS6 itself)\n- Paper 59 (protein folding simulation of S6 — ribosomal protein S6 folding study, but purely computational/biophysical with no cellular function)\n\n**KEEP:** Papers about mammalian/model organism RPS6 (ribosomal protein S6) function, phosphorylation, or its kinases acting on RPS6 as substrate.\n\n```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1988,\n      \"finding\": \"A mitogen-activated S6 kinase of Mr 70,000 (p70 S6K) was purified to homogeneity from Swiss 3T3 cells stimulated by EGF, serum, or orthovanadate. The purified kinase phosphorylates ribosomal protein S6 of the 40S subunit in vitro, exhibits autophosphorylation activity, and is inactivated by phosphatase 2A treatment, establishing that the kinase itself is regulated by phosphorylation.\",\n      \"method\": \"Biochemical purification (Mono Q/Mono S chromatography), in vitro kinase assay with 40S subunit, phosphatase 2A inactivation\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — purification to homogeneity with in vitro reconstitution and phosphatase inactivation\",\n      \"pmids\": [\"3257566\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1989,\n      \"finding\": \"A hepatic 70 kDa S6 kinase purified from cycloheximide-treated rats phosphorylates ribosomal protein S6 and additional substrates including histone H2b, glycogen synthase, and ATP citrate lyase (the latter on the same serine phosphorylated by insulin in intact cells), demonstrating broad substrate specificity beyond S6.\",\n      \"method\": \"50,000-fold purification using peptide affinity column (C-terminal S6 peptide), in vitro kinase assays with multiple substrates\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — near-homogeneous purification with defined substrate mapping\",\n      \"pmids\": [\"2760046\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1989,\n      \"finding\": \"An insulin-stimulated S6 kinase purified from rabbit liver has a molecular weight of ~70,000 and its activity is abolished by phosphatase 2A treatment, confirming phosphorylation-dependent activation. The inactivated enzyme can be re-activated ~5-fold by preincubation with partially purified MAP-2 kinase, placing a MAP kinase-like activity upstream.\",\n      \"method\": \"Multi-step chromatographic purification, phosphatase 2A inactivation, reactivation by MAP-2 kinase\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 methods but single lab, upstream kinase identification partial\",\n      \"pmids\": [\"2553707\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1991,\n      \"finding\": \"The purified 70K S6 kinase phosphorylates S6 exclusively on five sites (Ser235, Ser236, Ser240, Ser244, and a fifth low-stoichiometry site) within a 19-amino acid peptide at the carboxyl terminus of S6, matching the in vivo phosphorylation pattern. Mn²⁺ and Zn²⁺ inhibit the kinase with IC₅₀ ~2 µM for Zn²⁺, and autophosphorylation (~1.2 mol/mol) reduces activity ~25%.\",\n      \"method\": \"In vitro kinase assay with purified 70K S6K and 40S subunit, tryptic phosphopeptide mapping, site identification by sequencing, metal ion inhibition studies\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro reconstitution with direct site identification matching in vivo data\",\n      \"pmids\": [\"1939282\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1981,\n      \"finding\": \"S6 phosphorylation in HeLa cells peaks rapidly (~30 min) after serum stimulation, preceding the slower increase in translation elongation/initiation rates, and correlates temporally with mRNA recruitment into polysomes, suggesting rpS6 phosphorylation may facilitate mRNA recruitment rather than globally controlling elongation.\",\n      \"method\": \"Polysome profiling, kinetic analysis of S6 phosphorylation vs. protein synthesis rates in suspension culture\",\n      \"journal\": \"European journal of biochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — kinetic correlation study in intact cells, single lab\",\n      \"pmids\": [\"7333277\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1984,\n      \"finding\": \"Extensive phosphorylation of ribosomal protein S6 is associated with conformational changes in the 40S ribosomal subunit, specifically altered accessibility of proteins S3, S4, S7, S23/24 (small subunit) and L9, L10, L12, L18, L27, L34, L36 (large subunit) to reductive methylation.\",\n      \"method\": \"Reductive methylation accessibility assay comparing ribosomes with unphosphorylated vs. phosphorylated S6\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — direct biochemical comparison, single lab, single method\",\n      \"pmids\": [\"6693389\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1986,\n      \"finding\": \"Insulin, IGF-1, and TPA activate a cytosolic S6 kinase in rat astrocytes, demonstrating that S6 kinase responds to tyrosine kinase receptors and protein kinase C activation in neural cells. The effect is insensitive to cycloheximide, indicating activation occurs through post-translational modification of a pre-existing kinase.\",\n      \"method\": \"S6 kinase activity assay in cytosolic fractions of cultured astrocytes after hormone treatment; cycloheximide control\",\n      \"journal\": \"FEBS letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — direct kinase activity measurement, multiple stimuli tested\",\n      \"pmids\": [\"3530808\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1992,\n      \"finding\": \"Rapamycin selectively inhibits p70 S6 kinase activity and blocks the appearance of the hyperphosphorylated form of p70 S6K in COS cells, but does not affect p85 Rsk S6 kinase or MAP kinase, demonstrating that a rapamycin-sensitive signaling element is specifically required for p70 S6K activation.\",\n      \"method\": \"Kinase assays with 40S subunits, SDS-PAGE mobility shift, selective inhibitor treatment in hepatoma cells and transfected COS cells\",\n      \"journal\": \"Science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — replicated across cell lines with selective pharmacological dissection\",\n      \"pmids\": [\"1380182\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1994,\n      \"finding\": \"The p70/p85 S6 kinase signaling pathway is independent of p21ras: dominant-negative p74raf and p21ras block p44MAPK but not p70S6K activation. Furthermore, activation of p70S6K by PDGFR requires autophosphorylation at tyrosine 751 within the kinase-insert domain of the receptor, bifurcating the signaling from the ras-MAPK pathway at the receptor level.\",\n      \"method\": \"Dominant-negative Raf and Ras mutant expression, epitope-tagged kinase assays, PDGFR kinase-insert domain mutants\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genetic epistasis with multiple mutants, replicated in multiple cell lines\",\n      \"pmids\": [\"8090223\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1995,\n      \"finding\": \"FRAP/mTOR kinase activity is required in vivo for activation of p70 S6K: kinase-dead FRAP variants fail to activate p70S6K, and FRAP autophosphorylates in vitro; autophosphorylation is inhibited by the FKBP12-rapamycin complex. An N-terminal domain of FRAP beyond its kinase domain is also required for p70S6K regulation.\",\n      \"method\": \"FRAP variant overexpression, in vitro autophosphorylation, rapamycin-FKBP12 inhibition of autophosphorylation, kinase-dead mutant analysis\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro kinase assay combined with genetic epistasis, replicated\",\n      \"pmids\": [\"7566123\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1996,\n      \"finding\": \"S6 kinase contains an autoinhibitory domain (codons 58–77) that overrides mitogen-dependence; deletion of the first 77 amino acids produces a constitutively active, rapamycin-insensitive S6K. A separate N-terminal domain (codons 1–58) is required for mitogen-induced activation and rapamycin sensitivity, defining two functionally distinct N-terminal regulatory regions.\",\n      \"method\": \"Deletion mutagenesis of S6K, expression in Rat1 cells, kinase activity assays, morphological phenotype analysis\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — systematic deletion analysis with reconstitution and functional readout\",\n      \"pmids\": [\"8524322\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"RAFT1/mTOR directly phosphorylates p70 S6K on Thr-389 (a rapamycin-sensitive site essential for S6K activity) in vitro and in vivo, and also phosphorylates 4E-BP1 and 4E-BP2. Thr-389 phosphorylation is necessary for S6K activity, and RAFT1 phosphorylates p70S6K much more efficiently than 4E-BP1.\",\n      \"method\": \"In vitro kinase assay with immunoprecipitated RAFT1/mTOR, phospho-specific site mapping, rapamycin treatment, serum stimulation kinetics\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — direct in vitro phosphorylation with site mapping and functional validation\",\n      \"pmids\": [\"9465032\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"Amino acid withdrawal rapidly inactivates p70 S6K and promotes 4E-BP1 dephosphorylation in CHO cells through a pathway sensitive to rapamycin and PI3K inhibitors but independent of protein kinase B (PKB/Akt), demonstrating that amino acids regulate p70 S6K via a distinct upstream pathway from insulin/PKB.\",\n      \"method\": \"Amino acid deprivation/repletion in CHO cells, kinase activity assays, PI3K and mTOR inhibitor treatments, PKB activity measurements\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple inhibitors dissecting pathway, replicated by multiple groups\",\n      \"pmids\": [\"9693128\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"Drosophila S6 kinase (dS6K) regulates cell size in a cell-autonomous manner: dS6K null flies display extreme developmental delay, severe body size reduction due to smaller cells (not fewer cells), a phenotype distinct from ribosomal protein mutations (Minutes). This genetically establishes S6K as a cell-size regulator.\",\n      \"method\": \"Drosophila dS6K null mutant analysis, cell counting, cell size measurement, mosaic analysis\",\n      \"journal\": \"Science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — clean genetic loss-of-function with specific cellular phenotype, highly cited\",\n      \"pmids\": [\"10497130\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"Leucine activation of p70S6K in amino acid-depleted cells requires strict structural features: the correct chirality, the four-branched hydrocarbon structure, and a primary amine. The effect is rapamycin-sensitive but less sensitive to wortmannin, indicating that leucine acts via mTOR rather than PI3K to activate p70S6K.\",\n      \"method\": \"p70S6K activity assays with leucine analogues in H4IIE cells, pharmacological inhibition with rapamycin and wortmannin\",\n      \"journal\": \"FEBS letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — structure-activity relationship with pharmacological dissection, single lab\",\n      \"pmids\": [\"10214966\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"Osmotic stress (sorbitol) inactivates p70/p85 S6K and causes dephosphorylation of Thr412 in the COOH-terminus via a calyculin A-sensitive phosphatase, similar to rapamycin. Rapamycin-resistant truncation mutants of S6K are also resistant to osmotic stress-induced inactivation, demonstrating that both rapamycin and osmotic stress converge on a common calyculin A-sensitive phosphatase-dependent mechanism.\",\n      \"method\": \"S6K kinase assays, phospho-specific antibodies, pharmacological inhibitors of phosphatases, rapamycin-resistant S6K truncation mutants\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — genetic (truncation mutants) and pharmacological epistasis converging on same mechanism\",\n      \"pmids\": [\"10455142\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"TGF-β induces association of its receptor with PP2A-Bα subunit, leading to assembly of a PP2A complex (Bα, Aβ, Cα subunits) with p70S6K, dephosphorylation and inactivation of p70S6K, and G1 arrest. This is a second pathway for TGF-β-induced cell cycle arrest parallel to Smad2/3 activation.\",\n      \"method\": \"Co-immunoprecipitation of PP2A subunits with TGF-β receptor and p70S6K, kinase activity assays, G1 arrest rescue experiments\",\n      \"journal\": \"Genes & development\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal co-IP plus functional epistasis with two independent pathways dissected\",\n      \"pmids\": [\"11124802\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"S6K1 and S6K2 activation requires sequential phosphorylation: PI3K-mediated phosphorylation of C-terminal sites permits PDK1 to phosphorylate the activation loop residue, and mTOR acts as a checkpoint for amino acid availability. At least eight phosphorylation sites mediate S6K1 activation in a hierarchical fashion.\",\n      \"method\": \"Review synthesizing phosphorylation site mutant data, PI3K/mTOR pathway dissection, PDK1 phosphorylation of activation loop\",\n      \"journal\": \"Experimental cell research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — synthesis of multiple labs' mutagenesis and epistasis data\",\n      \"pmids\": [\"10579915\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"S6K2 activation requires mTOR kinase activity, amino acid sufficiency, and phosphatidic acid, similar to S6K1. Manipulation of mTOR nuclear/cytoplasmic distribution (using NLS/NES tags) showed that optimal mTOR shuttling is required for maximal S6K2 activation, with S6K2 being a predominantly nuclear protein.\",\n      \"method\": \"mTOR NLS/NES tagging constructs, kinase activity assays, subcellular fractionation, leptomycin B treatment\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — direct manipulation of localization with functional consequence, single lab\",\n      \"pmids\": [\"12087098\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"Loss of TSC2/tuberin leads to constitutive hyperphosphorylation of ribosomal protein S6 and constitutive activation of p70S6K in LAM patient-derived smooth muscle cells and TSC2-/- cells. Re-introduction of wild-type tuberin abolishes S6 phosphorylation and inhibits p70S6K, demonstrating that TSC2 negatively regulates the p70S6K–rpS6 axis.\",\n      \"method\": \"Immunoblotting of S6 phosphorylation, p70S6K kinase assays, tuberin re-expression in TSC2-null cells, rapamycin treatment\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genetic rescue experiment in patient-derived and TSC2-null cells with multiple readouts\",\n      \"pmids\": [\"12045200\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"TSC1/2 acts as a GAP for the small GTPase Rheb, and insulin-activated Rheb (via PI3K) drives S6K1 phosphorylation; loss-of-function TSC1/2 mutations phenocopy Rheb overexpression in activating S6K1. A disease-associated GAP-domain point mutation in TSC2 fails to stimulate Rheb GTPase or block Rheb-induced S6K1 activation.\",\n      \"method\": \"GTPase assay with purified Rheb, epistasis by co-expression, TSC2 disease mutant analysis, S6K1 phosphorylation assays\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — biochemical GAP assay plus genetic epistasis with disease mutant validation\",\n      \"pmids\": [\"12820960\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"M-CSF, RANKL, and TNFα promote osteoclast survival through convergent mTOR/S6K signaling; rapamycin blocks cytokine-dependent osteoclast survival and induces apoptosis. Inhibitors of PI3K, Akt, ERK, and geranylgeranylation suppress cytokine-induced S6K activation and osteoclast survival, and inhibition of translation by other mechanisms also induces apoptosis, demonstrating that mTOR/S6K-dependent protein synthesis is essential for osteoclast survival.\",\n      \"method\": \"Rapamycin and signaling inhibitor treatment, S6K activity assays, apoptosis assays, in vitro bone resorption\",\n      \"journal\": \"Cell death and differentiation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — pharmacological pathway dissection with functional cell death readout\",\n      \"pmids\": [\"14502240\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Apelin (65-77) activates p70 S6K in CHO cells and endothelial cells via dual pathways: a PI3K→Akt pathway (blocked by wortmannin) leading to T389 and T421/S424 phosphorylation, and an ERK pathway (blocked by PD098059) leading to T421/S424 phosphorylation. PTX and atypical PKC inhibitors abrogate both Akt and p70S6K phosphorylation, and apelin-induced p70S6K activation is mitogenic for HUVEC cells.\",\n      \"method\": \"Kinase assays with phospho-specific antibodies, pathway inhibitors, PTX treatment, thymidine incorporation assay\",\n      \"journal\": \"FASEB journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple pharmacological dissections with functional mitogenic readout, single lab\",\n      \"pmids\": [\"15385434\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"mTOR/S6K1 and the eIF3 translation initiation complex function as a dynamic scaffold: inactive S6K1 associates with eIF3 basally; cell stimulation recruits mTOR/raptor to eIF3 where it phosphorylates S6K1 at its hydrophobic motif; S6K1 then dissociates, becomes activated, and phosphorylates eIF4B, which is subsequently recruited to the eIF3 complex.\",\n      \"method\": \"Co-immunoprecipitation of mTOR, S6K1, eIF3, and eIF4B; phospho-specific antibodies; rapamycin treatment; sequential IP/kinase assays\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — dynamic complex assembly demonstrated by reciprocal co-IP with ordered phosphorylation events, highly cited\",\n      \"pmids\": [\"16006006\", \"16286006\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"hVps34 (Class III PI3K) is required for p70 S6K1 activation in response to insulin and amino acids, acting upstream of mTOR. Inhibitory anti-hVps34 antibodies, FYVE domain sequestration of PI3P, and siRNA knockdown of hVps34 all block S6K1 activation. hVps34 is inhibited by amino acid/glucose starvation and AMPK activation, but not by insulin, placing it on the nutrient-sensing arm to mTOR/S6K1.\",\n      \"method\": \"Microinjection of inhibitory antibodies, FYVE domain overexpression, siRNA knockdown, hVps34 kinase activity assays, insulin stimulation of S6K1\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal loss-of-function approaches converging on same conclusion\",\n      \"pmids\": [\"16049009\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"p70 S6K directly phosphorylates tau at S262, S214, and T212 in vitro, and co-immunoprecipitation reveals association between activated p-p70S6K (T421/S424) and phospho-tau (S262) in cells. Zinc-induced p70S6K activation selectively upregulates translation of S6 and tau mRNAs but not global protein synthesis.\",\n      \"method\": \"In vitro kinase assay with p70S6K and tau, immunoprecipitation, immunoblotting with phospho-specific antibodies, metabolic labeling\",\n      \"journal\": \"FEBS letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 — in vitro phosphorylation demonstrated, but limited validation in cells; single lab\",\n      \"pmids\": [\"16364302\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"S6 kinases are ubiquitinated in cells and subject to proteasome-mediated degradation. Ubiquitination sites are located in the kinase domain, and the N- and C-terminal regulatory regions modulate the efficiency of S6K ubiquitination, identifying a second post-translational mechanism (beyond phosphorylation) controlling S6K abundance.\",\n      \"method\": \"Co-transfection with ubiquitin constructs, proteasome inhibitor treatment, immunoprecipitation, ubiquitin site mapping by domain deletion\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — co-IP/overexpression based, single lab, no E3 ligase identified\",\n      \"pmids\": [\"18280803\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"S6K1 and S6K2 interact with receptor tyrosine kinases, specifically PDGFR, via the kinase or kinase extension domain of S6K in a growth-factor-inducible manner. This complex formation leads to tyrosine phosphorylation of S6K via a PDGFR-Src pathway at Y39 (S6K1) and Y45 (S6K2), but these tyrosine phosphorylations do not affect S6K activity or subcellular localization.\",\n      \"method\": \"Co-immunoprecipitation with PDGFR mutants, tyrosine phosphorylation mapping, Src inhibitors (PP1, SU6656), immunofluorescence\",\n      \"journal\": \"The FEBS journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — reciprocal co-IP and site mapping with mutants, but functional consequence is negative\",\n      \"pmids\": [\"16640565\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"Ribosomal proteins Rpl10 and Rps6 are potent regulators of yeast replicative lifespan: deletion of RPS6B (but not RPS6A) extends replicative lifespan by 45% in S. cerevisiae, associated with altered free ribosomal subunit stoichiometry and reduced cell size. This establishes an orthologous role for Rps6 gene dosage in aging.\",\n      \"method\": \"Yeast deletion mutants, replicative lifespan assays, ribosome profiling by polysome analysis, cell size measurement\",\n      \"journal\": \"Experimental gerontology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — genetic deletion with defined phenotype in yeast ortholog system\",\n      \"pmids\": [\"17174052\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"ATG1 (autophagy kinase) inhibits TOR/S6K-dependent cell growth in Drosophila by blocking S6K activation at Thr389 phosphorylation. In mammalian cells, ATG1 overexpression inhibits S6K, and siRNA knockdown of ATG1 induces ectopic S6K activation and S6 phosphorylation, revealing crosstalk between autophagy regulation and the TOR/S6K growth pathway.\",\n      \"method\": \"Drosophila ATG1 mutant and overexpression analysis, mammalian S6K kinase assays, siRNA knockdown, phospho-Thr389 antibody\",\n      \"journal\": \"EMBO reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genetic and biochemical evidence in two organisms, bidirectional manipulation\",\n      \"pmids\": [\"17347671\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"S6K1 deletion in skeletal muscle cells causes increased AMP/ATP and inorganic phosphate/phosphocreatine ratios, triggering AMPK upregulation. AMPK inhibition in S6K-deficient cells restores cell growth and nutrient sensitivity, demonstrating that S6K1 controls cellular energy state and that AMPK activation mediates the cell atrophy caused by S6K1 deletion.\",\n      \"method\": \"S6K1/2 knockout mice, AMPK activity assays, metabolite measurements (AMP, ATP, phosphocreatine), AMPK inhibition rescue\",\n      \"journal\": \"Cell metabolism\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genetic KO with metabolic rescue experiment and AMPK epistasis\",\n      \"pmids\": [\"17550782\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"S6K1 deletion in mice leads to increased lifespan and resistance to age-related pathologies (bone, immune, motor dysfunction, insulin sensitivity loss), with gene expression patterns similar to caloric restriction or AMPK activation, demonstrating that S6K1 is a lifespan regulator in mammals.\",\n      \"method\": \"S6K1 knockout mouse model, lifespan analysis, metabolic phenotyping, gene expression profiling\",\n      \"journal\": \"Science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — in vivo genetic KO with comprehensive phenotyping, highly cited\",\n      \"pmids\": [\"19797661\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Mediobasal hypothalamic (MBH) S6K1 bidirectionally controls energy homeostasis: stereotaxic adenoviral activation of MBH S6K alters food intake, body weight, orexigenic neuropeptide expression, and leptin sensitivity; constitutive activation protects against high-fat diet-induced obesity and insulin resistance.\",\n      \"method\": \"Bilateral adenoviral injection of dominant-negative or constitutively active S6K into rat MBH, metabolic phenotyping, neuropeptide measurements\",\n      \"journal\": \"Cell metabolism\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — bidirectional in vivo manipulation with specific anatomical targeting and multiple metabolic readouts\",\n      \"pmids\": [\"19041762\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Drosophila S6KII (p90 RSK) cooperates with casein kinase 2 (CK2) in circadian clock neurons to regulate circadian period: S6KII null flies show short-period phenotype rescued by clock-neuron-specific re-expression; S6KII physically interacts with CK2β; genetic interactions show CK2 activity is required for S6KII's clock function.\",\n      \"method\": \"Drosophila null mutant analysis, circadian behavioral assays, cell-type-specific rescue, co-IP of S6KII and CK2β, genetic epistasis with CK2 mutants\",\n      \"journal\": \"The Journal of neuroscience\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — genetic and biochemical evidence in Drosophila ortholog for RSK2 function in circadian regulation\",\n      \"pmids\": [\"19144847\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Resveratrol inhibits mammalian S6K1 (identified in a large-scale in vitro kinase screen) and attenuates autophagy induced by nutrient limitation or rapamycin. Dominant-negative S6K1 mutant or siRNA knockdown of S6K1 recapitulates the autophagy-suppressing effect of resveratrol, and combining resveratrol with S6K1 knockdown is not additive, indicating S6K1 is a key target of resveratrol's autophagy-modulating effect.\",\n      \"method\": \"Large-scale in vitro kinase screen, dominant-negative S6K1 expression, siRNA knockdown, autophagy assays\",\n      \"journal\": \"Aging\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — kinase screen plus functional rescue/epistasis, single lab\",\n      \"pmids\": [\"20157535\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"mTOR/S6K1 signaling protects astrocytes from ischemia-induced apoptosis: OGD reduces S6K1 mRNA and protein; S6K1/2 double knockout astrocytes show increased apoptosis, reduced BAD phosphorylation, decreased Bcl-2/Bcl-xL, increased ROS, and impaired translation recovery. Rescue with adenoviral S6K1 (but not S6K2) specifically reduces ROS and confers ischemic resistance.\",\n      \"method\": \"S6K1/S6K2 double knockout mouse astrocytes, OGD model, adenoviral isoform-specific rescue, apoptosis assays, ROS measurement, middle cerebral artery occlusion in vivo\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — isoform-specific rescue in KO cells with multiple orthogonal readouts and in vivo validation\",\n      \"pmids\": [\"19535330\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"S6 kinase ortholog RSKS-1 in C. elegans inhibits axon regeneration in mechanosensory neurons: rsks-1 loss-of-function accelerates growth cone formation and axon extension after laser axotomy; this enhanced regrowth is partly dependent on DLK-1 MAPK cascade, and a key downstream effector is the metabolic sensor AMP kinase AAK-2. The antidiabetic drug phenformin (AMPK activator) promotes axon regrowth.\",\n      \"method\": \"C. elegans laser axotomy, rsks-1 null mutants, genetic epistasis with DLK-1 and AAK-2, pharmacological AMPK activation\",\n      \"journal\": \"The Journal of neuroscience\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — genetic epistasis in C. elegans model with pharmacological validation\",\n      \"pmids\": [\"24431434\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"AKT-mTORC1-RPS6 signaling promotes de novo lipogenesis in hepatocellular carcinoma via transcriptional and post-transcriptional mechanisms including inhibition of fatty acid synthase ubiquitination by USP2a and disruption of SREBP1/SREBP2 degradation complexes. siRNA knockdown of RPS6 inhibits lipogenesis-related gene activity and reduces HCC cell proliferation and survival.\",\n      \"method\": \"siRNA knockdown of RPS6, inhibitor studies (rapamycin, pathway inhibitors), gene expression analysis, lipogenic enzyme activity assays, USP2a/SREBP pathway analysis in HCC cell lines and mouse models\",\n      \"journal\": \"Gastroenterology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — siRNA knockdown with multiple mechanistic readouts, single lab\",\n      \"pmids\": [\"21147110\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Heat shock activates both pp70 S6K and pp90rsk in quiescent fibroblasts, with a temporal activation pattern reversed from mitogen-stimulated cells. The heat shock-induced pp70 S6K was identified as equivalent to the serum/growth factor-activated enzyme by chromatographic properties, and both kinases drive S6 phosphorylation in heat-shocked cells.\",\n      \"method\": \"Sequential column chromatography purification, immunoprecipitation kinase assay for pp90rsk, S6 phosphorylation measurement in quiescent fibroblasts after heat shock\",\n      \"journal\": \"Journal of cellular physiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — partial purification with immunoprecipitation comparison, single lab\",\n      \"pmids\": [\"1880153\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"rpS6 regulates blood-testis barrier (BTB) dynamics: rpS6 is spatiotemporally activated at the BTB during the spermatogenic cycle; siRNA-mediated knockdown of rpS6 in vitro and in vivo promotes the Sertoli cell TJ permeability barrier by recruiting claudin-11 and occludin to the BTB. rpS6 activation disrupts the TJ barrier via effects on F-actin organization and adhesion protein recruitment.\",\n      \"method\": \"siRNA and shRNA knockdown of rpS6 in Sertoli cells in vitro and in adult rat testes in vivo, BTB integrity assay, immunofluorescence for F-actin and junctional proteins\",\n      \"journal\": \"Endocrinology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — bidirectional manipulation (knockdown in vitro and in vivo) with specific structural and functional readouts\",\n      \"pmids\": [\"22948214\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"S6K1 and S6K2 double knockout mouse liver shows a specific defect in the ribosome biogenesis (RiBi) transcriptional program after feeding: over 75% of RiBi factors (Nop56, Nop14, Gar1, etc.) are controlled by S6K. Importantly, this RiBi transcriptional defect is also observed in rpS6 knock-in mice with non-phosphorylatable serine residues, identifying rpS6 phosphorylation as specifically regulating RiBi transcription independently of ribosomal protein synthesis.\",\n      \"method\": \"S6K1/S6K2 double knockout mice, rpS6 phosphorylation-site knock-in mice, whole-genome microarray of total and polysomal liver RNA, feeding paradigm\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — two independent genetic models (S6K DKO and rpS6 knock-in) converging on same transcriptional phenotype\",\n      \"pmids\": [\"23318442\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Arginine, leucine, and glutamine activate the MTOR-RPS6K-RPS6-EIF4EBP1 pathway in porcine trophectoderm cells to stimulate proliferation; increased nuclear localization of pRPS6K and cytoplasmic pRPS6 were detected. siRNA silencing of MTOR, RPTOR, or RICTOR blocked amino acid-induced proliferation, placing RPS6 downstream of both mTORC1 and mTORC2 in trophectoderm proliferation.\",\n      \"method\": \"Immunofluorescence, immunoblotting of phospho-RPS6K/RPS6/EIF4EBP1, siRNA knockdown of MTOR/RPTOR/RICTOR, proliferation assay in primary porcine trophectoderm cells\",\n      \"journal\": \"Biology of reproduction\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — siRNA epistasis with multiple pathway components and immunolocalization, single lab\",\n      \"pmids\": [\"23486913\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Phosphorylation of rpS6 mediates compensatory renal hypertrophy: rpS6 knock-in mice expressing non-phosphorylatable rpS6 show significantly blunted uninephrectomy-induced renal hypertrophy, with attenuated cyclin D1 increase and reduced cyclin E decrease compared to wild-type. mTORC1 is still activated in knock-in mice (4E-BP1 phosphorylation intact), but rapamycin (which prevents rpS6 phosphorylation) cannot prevent residual hypertrophy in knock-in mice, unequivocally demonstrating rpS6 phosphorylation as the downstream mTORC1-S6K1 effector mediating hypertrophic cell cycle regulation.\",\n      \"method\": \"rpS6 phosphorylation-site knock-in mice, uninephrectomy model, rapamycin treatment, immunoblotting for cyclins and phospho-proteins\",\n      \"journal\": \"Kidney international\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — orthogonal genetic and pharmacological approaches with specific mechanistic readout\",\n      \"pmids\": [\"25229342\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"RPS6 interacts with components of mTORC2 (co-immunoprecipitation), and siRNA-mediated knockdown of RPS6 attenuates insulin-induced mTORC2 activity and Akt-Ser473 phosphorylation. Conversely, RPS6 overexpression enhances Akt-Ser473 phosphorylation, establishing that RPS6 provides positive feedback to mTORC2/Akt signaling. In mouse hearts, ischemic preconditioning induces RPS6 phosphorylation at Ser235/236 and activates mTORC2.\",\n      \"method\": \"Co-immunoprecipitation of RPS6 with mTORC2 components, siRNA knockdown, RPS6 overexpression, ischemic preconditioning in perfused mouse hearts, neonatal rat ventricular myocyte assays\",\n      \"journal\": \"Circulation research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal co-IP plus bidirectional manipulation (KD and OE) with functional consequence in vitro and in vivo\",\n      \"pmids\": [\"24557881\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"In yeast, TORC1 promotes rpS6 phosphorylation on Ser-232/Ser-233 via the AGC kinase Ypk3 (not Sch9 as previously proposed). Ypk3-null cells have completely abolished rpS6 phosphorylation; phosphorylation-deficient mutations in Ypk3 regulatory motifs abrogate rpS6 phosphorylation; and complementation of ypk3Δ cells with human S6 kinase restores rpS6 phosphorylation. Cells expressing non-phosphorylatable rpS6 show reduced growth and 40S biogenesis defects not explained by translation changes.\",\n      \"method\": \"Yeast deletion mutants, phospho-S6-specific antibody, Ypk3 regulatory mutants, human S6K complementation, polysome profiling, ribosome profiling\",\n      \"journal\": \"PloS one / Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — genetic identification of direct kinase with complementation and multiple orthogonal assays; two complementary papers\",\n      \"pmids\": [\"25767889\", \"26582391\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"RPS6 phosphorylation is controlled by both TORC1 (via Ypk3 and PP1/Glc7) and TORC2 (via Ypk1) in S. cerevisiae, with TORC2 specifically regulating the N-terminal phosphosite. Ribosome profiling shows that rpS6 phosphorylation does not regulate global translation or translation of individual mRNAs, challenging the longstanding model that S6 phosphorylation broadly promotes translation of 5'TOP mRNAs.\",\n      \"method\": \"TORC1/TORC2 genetic dissection, ribosome profiling, polysome profiling, non-phosphorylatable rpS6 knock-in cells\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — genome-wide ribosome profiling with genetic knockins; strong negative evidence against translation model\",\n      \"pmids\": [\"26582391\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"RPS6 reviews extensive evidence that rpS6 phosphorylation occurs on five conserved serine residues in response to diverse stimuli; genetic manipulations in yeast and mammals using rpS6 knock-in mice and S6K knockout mice have revealed roles in cell size control, glucose homeostasis, ribosome biogenesis, and DNA damage responses. The physiological role of phosphorylation may include fine-tuning responses by counteracting positive signals from rpS6 kinase.\",\n      \"method\": \"Synthesis of knock-in mouse, S6K KO, and yeast data\",\n      \"journal\": \"International review of cell and molecular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — review synthesizing genetic model organism data from multiple labs\",\n      \"pmids\": [\"26614871\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"S6 kinases phosphorylate the nuclear epigenetic factor ZRF1 (DNAJC2/MIDA1) on Ser47 in cultured cells and in mammalian tissues in vivo (identified by chemical genetic screen). ZRF1 knockdown or expression of a phosphorylation mutant blunts S6K-dependent premature senescence in TSC-mutant fibroblasts, associated with altered p16 levels. This identifies ZRF1 as a novel S6K substrate linking mTORC1-S6K to cellular senescence.\",\n      \"method\": \"Chemical genetic screen for novel S6K substrates, ZRF1 knockdown, phospho-Ser47 antibody, senescence assays (SA-β-gal, p16), TSC-mutant fibroblasts\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — chemical genetic screen for substrate identification plus knockdown rescue and in vivo phosphorylation\",\n      \"pmids\": [\"28242756\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"mTORC1/rpS6 signaling promotes BTB disruption in the adult rat testis in vivo: constitutively active phosphomimetic rpS6 mutant (quadruple phosphomimetic) induces BTB leakiness better than wild-type rpS6, causing spermatid polarity loss and germ cell exfoliation. These effects are mediated through changes in actin- and microtubule-based cytoskeletal organization, specifically spatiotemporal expression of actin- and MT-binding proteins.\",\n      \"method\": \"In vivo transfection of rat testes with rpS6-WT and phosphomimetic rpS6 mutant, BTB integrity assay, immunofluorescence for cytoskeletal proteins, electron microscopy\",\n      \"journal\": \"American journal of physiology. Endocrinology and metabolism\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — direct in vivo gain-of-function with phosphomimetic mutant and multiple structural readouts\",\n      \"pmids\": [\"29089336\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"β-arrestins constitutively assemble a p70S6K/rpS6 complex in HEK293 cells and Sertoli cells; the interaction between β-arrestin 1 and p70S6K is direct (validated by docking). Upon FSH stimulation, G protein (Gαs)-dependent signaling enhances p70S6K activity within the preassembled β-arrestin/p70S6K/rpS6 complex, leading to rpS6 phosphorylation and translation of 5'TOP mRNAs within minutes.\",\n      \"method\": \"Co-immunoprecipitation, computational docking validation, Gαs depletion, FSHR stimulation, 5'TOP mRNA translation assay\",\n      \"journal\": \"FASEB journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — direct interaction validated by docking with functional translation readout, single lab\",\n      \"pmids\": [\"29084767\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"The mTOR effectors S6K1 and S6K2 localize to stress granules in human cells and are required for stress granule assembly and maintenance after mild oxidative stress. S6K1 promotes stress granule formation via regulation of eIF2α phosphorylation, while S6K2 is important for stress granule persistence. In C. elegans, the S6K ortholog RSKS-1 also promotes stress granule assembly and its loss sensitizes to stress-induced death.\",\n      \"method\": \"Immunofluorescence of S6K1/2 in stress granule markers, siRNA knockdown, eIF2α phosphorylation assays, C. elegans rsks-1 loss-of-function, stress-induced death assays\",\n      \"journal\": \"Cell death and differentiation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — localisation with functional consequence, validated in two organisms with mechanistic link to eIF2α\",\n      \"pmids\": [\"29523872\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Somatic mosaicism for RPS6 p.R232H mutation in a hemimegalencephaly patient increases cell proliferation in animal models, while MTOR p.S2215F causes neuronal migration delay and cytomegaly; double mutants show synergistic effects (increased proliferation + migration defects + cytomegalic cells with eccentric nuclei/binucleation). This establishes RPS6 p.R232H as an activating mutation that drives cortical dysplasia through increased proliferation downstream of the mTOR pathway.\",\n      \"method\": \"Whole-exome sequencing, deep targeted sequencing, in utero electroporation of variant constructs in mice, cell proliferation and migration assays, histological analysis\",\n      \"journal\": \"Human molecular genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — in vivo functional validation of human disease mutations with quantitative phenotyping\",\n      \"pmids\": [\"31411685\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"RPS6 phosphorylation promotes translation of mRNAs with short coding sequences (CDSs) more strongly than mRNAs with long CDSs. Selective ribosome footprinting reveals that RPS6 becomes progressively dephosphorylated on ribosomes as they translate longer ORFs, so short-CDS mRNAs retain more p-RPS6 ribosomes. However, 5'TOP mRNAs are not preferentially translated despite their short CDSs, suggesting they use a different mechanism.\",\n      \"method\": \"Selective ribosome footprinting with phospho-RPS6 antibody, RPS6 phosphorylation-site knock-in cells, genome-wide translation analysis\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — genome-wide ribosome footprinting with genetic removal of phosphorylation sites; novel mechanistic insight\",\n      \"pmids\": [\"34871442\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"RPS6 inhibition by siRNA decreases NRF2 expression in HER2-amplified gastric cancer, suggesting RPS6 lies upstream of NRF2 in anti-HER2 drug resistance. PI3K/TORC1/TORC2 inhibition suppresses pRPS6 and NRF2 and reduces tumor growth in resistant models in vitro and in vivo.\",\n      \"method\": \"siRNA knockdown of RPS6, PI3K/mTOR inhibitor treatment, cell viability assays, in vivo xenograft models\",\n      \"journal\": \"Clinical cancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — siRNA knockdown with functional readout; mechanistic link to NRF2 is indirect\",\n      \"pmids\": [\"30504425\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"The lnc-Rps4l-encoded peptide RPS4XL interacts with RPS6 (identified by CoIP and mass spectrometry) and inhibits RPS6 phosphorylation at Ser240/Ser244, thereby inhibiting hypoxia-induced proliferation of pulmonary artery smooth muscle cells.\",\n      \"method\": \"Co-immunoprecipitation, mass spectrometry, immunofluorescence, MTT/BrdU proliferation assays, phospho-RPS6 immunoblotting\",\n      \"journal\": \"Molecular therapy\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — co-IP with MS for interaction, functional knockdown data; single lab\",\n      \"pmids\": [\"33429084\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"In mammalian skin wounding, rpS6 is rapidly phosphorylated within minutes of injury, forming a spatially defined 'p-rpS6-zone' surrounding the wound that persists until healing is complete. This zone encompasses proliferation, cellular senescence, angiogenesis, and growth. A mouse model unable to phosphorylate rpS6 shows accelerated initial wound closure but impaired overall healing, demonstrating that p-rpS6 modulates (but does not drive) the healing process.\",\n      \"method\": \"Immunofluorescence for p-rpS6 in skin wounds, rpS6 non-phosphorylatable knock-in mice, wound healing assays, time-course analysis\",\n      \"journal\": \"Developmental cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — spatial mapping with genetic knock-in model showing in vivo functional consequence\",\n      \"pmids\": [\"37098351\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1995,\n      \"finding\": \"Human ribosomal protein S6 contains three nuclear localization signals (NLSs) in its C-terminal half. Deletion mutagenesis of S6-β-galactosidase chimeras showed that a single NLS is sufficient for nuclear import, removal of all three NLSs abolishes nuclear entry, and chimeras retaining at least one NLS accumulate specifically in the nucleolus via a distinct domain required but not sufficient for nucleolar targeting.\",\n      \"method\": \"Chimeric S6-LacZ construct transfection in L cells, deletion mutagenesis, β-galactosidase activity localization assay\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — systematic deletion mutagenesis with functional targeting assay defining NLS and nucleolar targeting domain\",\n      \"pmids\": [\"8590812\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Yeast rpS6 (Rps6p) functionally interacts with the 60S subunit protein rpL10/Grc5p in ribosome subunit joining and differential protein expression; Grc5p also interacts with the nuclear export factor Nmd3p to modulate polysome complement, establishing rpS6 as part of a network of ribosomal proteins governing subunit joining fidelity.\",\n      \"method\": \"Yeast two-hybrid, polysome profiling, genetic interaction analysis in S. cerevisiae\",\n      \"journal\": \"FEMS yeast research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — yeast two-hybrid with polysome profiling; functional consequence not fully defined\",\n      \"pmids\": [\"15556089\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Human 80S ribosome cryo-EM structure at high resolution reveals the position of RPS6 in the 40S subunit, as part of the metazoan-specific structural architecture with additional inner and outer RNA layers, providing structural context for RPS6's role in ribosome function.\",\n      \"method\": \"High-resolution cryo-electron microscopy of human and Drosophila 80S ribosomes\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — near-atomic cryo-EM structure from highly cited paper; defines RPS6 structural context\",\n      \"pmids\": [\"23636399\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Near-atomic (3.6 Å average, 2.9 Å in stable regions) cryo-EM structure of the human 80S ribosome defines the precise atomic positions of ribosomal proteins including RPS6, revealing amino acid side chain contacts, tRNA binding site interactions, and dynamic subunit interface remodeling during rotation.\",\n      \"method\": \"Single-particle cryo-EM, atomic model building\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — near-atomic resolution structure with functional validation of interface dynamics\",\n      \"pmids\": [\"25901680\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"RPS6 (ribosomal protein S6) is a component of the 40S ribosomal subunit that is phosphorylated on five conserved C-terminal serine residues (Ser235, Ser236, Ser240, Ser244, Ser247) by S6K1/S6K2 (activated downstream of the PI3K–mTORC1–PDK1 axis) and by p90 RSK; phosphorylation is progressively lost as ribosomes translate longer ORFs, selectively promoting translation of short-CDS mRNAs; beyond translation, phospho-rpS6 mediates compensatory organ hypertrophy via cyclin D1/E regulation, controls blood-testis barrier dynamics through F-actin reorganization, provides positive feedback to mTORC2/Akt signaling, regulates the ribosome biogenesis transcriptional program, marks a wound-healing activation zone, and is subject to negative regulation by PP2A and AMPK, while activating mutations (e.g., p.R232H) drive cortical dysplasia through increased cell proliferation.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"RPS6 is a core component of the 40S ribosomal subunit whose phosphorylation at C-terminal serines (Ser235, Ser236, Ser240, Ser244, Ser247) by p70 S6K1/S6K2 — downstream of the mTORC1–PI3K axis activated by growth factors, insulin, and amino acids (especially leucine) — serves as a central node coupling nutrient sensing to translational control, ribosome biogenesis, and cell growth [PMID:1939282, PMID:9693128, PMID:10214966]. RPS6 phosphorylation induces conformational changes in the 40S subunit, progressively declines during elongation, and thereby preferentially promotes translation of short-CDS mRNAs rather than broadly regulating 5′TOP mRNA translation [PMID:6693389, PMID:34871442]. Beyond translation, phospho-rpS6 drives a transcriptional program for ribosome biogenesis factors, is required for compensatory organ hypertrophy and skin wound healing, regulates blood–testis barrier dynamics through actin cytoskeleton reorganization, and provides positive feedback to mTORC2/Akt signaling [PMID:23318442, PMID:25229342, PMID:37098351, PMID:22948214, PMID:24557881]. Somatic gain-of-function RPS6 variants cause cortical dysplasia including hemimegalencephaly [PMID:31411685].\",\n  \"teleology\": [\n    {\n      \"year\": 1984,\n      \"claim\": \"Before the upstream kinase was known, phosphorylation of S6 was shown to alter 40S subunit conformation, establishing that S6 phosphorylation has structural consequences for the ribosome rather than merely being a passive mark.\",\n      \"evidence\": \"Reductive methylation accessibility assay on phosphorylated vs. unphosphorylated 40S ribosomes\",\n      \"pmids\": [\"6693389\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Indirect structural readout; no high-resolution structural data\", \"Functional consequence for translation not tested\"]\n    },\n    {\n      \"year\": 1991,\n      \"claim\": \"Identification and purification of p70 S6K as the mitogen-activated kinase that phosphorylates RPS6 at defined C-terminal sites (Ser235, Ser236, Ser240, Ser244) established the direct kinase–substrate relationship and mapped the phosphorylation sites.\",\n      \"evidence\": \"Purification of p70 S6K from Swiss 3T3 and hepatic cells, in vitro kinase assays with tryptic phosphopeptide mapping and mass spectrometry\",\n      \"pmids\": [\"3257566\", \"2760046\", \"1939282\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Fifth in vivo site (Ser247) not phosphorylated by p70 S6K in vitro\", \"Identity of additional kinases (e.g., p90RSK) not yet addressed\"]\n    },\n    {\n      \"year\": 1995,\n      \"claim\": \"Rapamycin specifically blocks p70 S6K activation while sparing parallel MAPK pathways, and FRAP/mTOR kinase activity is required for S6K regulation, placing mTOR as the rapamycin-sensitive kinase upstream of S6K–RPS6 phosphorylation.\",\n      \"evidence\": \"Pharmacological inhibition in multiple cell types, FRAP kinase-dead and rapamycin-binding mutants, in vitro autophosphorylation\",\n      \"pmids\": [\"1380182\", \"7566123\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct mTOR phosphorylation of S6K Thr389 not yet shown\", \"Ras-independence of the pathway only shown for PDGFR context\"]\n    },\n    {\n      \"year\": 1995,\n      \"claim\": \"RPS6 contains three nuclear localization signals in its C-terminal half, any one of which is sufficient for nuclear import; full-length S6 accumulates in the nucleolus, revealing that RPS6 traffics through the nucleus for ribosome assembly.\",\n      \"evidence\": \"S6–β-galactosidase chimera transfection with systematic NLS deletion mutagenesis\",\n      \"pmids\": [\"8590812\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Nucleolar targeting domain identified but not shown to be sufficient alone\", \"Whether phosphorylation status affects nuclear import not tested\"]\n    },\n    {\n      \"year\": 1999,\n      \"claim\": \"Amino acids — particularly leucine — activate S6K via a rapamycin-sensitive, Akt-independent pathway, establishing nutrient sensing as a distinct input to S6K–RPS6 phosphorylation separate from growth factor signaling.\",\n      \"evidence\": \"Amino acid deprivation/repletion, structure–activity analysis of leucine analogs, pharmacological inhibitor epistasis\",\n      \"pmids\": [\"9693128\", \"10214966\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Identity of the leucine sensor unknown at this stage\", \"Mechanism of amino acid signal transduction to mTOR not resolved\"]\n    },\n    {\n      \"year\": 1999,\n      \"claim\": \"Drosophila S6K loss-of-function showed that S6K controls cell size (not cell number) in a cell-autonomous manner, establishing the biological output of the pathway as growth regulation rather than proliferation control.\",\n      \"evidence\": \"dS6K-null Drosophila, cell size and number quantification, mosaic analysis\",\n      \"pmids\": [\"10497130\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether cell-size control is mediated entirely through rpS6 phosphorylation or other S6K substrates not distinguished\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"hVps34 (class III PI3K) was identified as an upstream nutrient-responsive activator of the mTOR–S6K pathway for amino acid and glucose signals but not insulin, adding a lipid-signaling layer to nutrient sensing.\",\n      \"evidence\": \"siRNA knockdown, antibody microinjection, FYVE dominant-negative, 4E-BP1 and S6K readouts\",\n      \"pmids\": [\"16049009\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism by which hVps34 activates mTOR not defined\", \"Relative contributions of hVps34 vs. Rag GTPases not delineated\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"S6K1 knockout mice showed extended lifespan and metabolic reprogramming resembling caloric restriction, with elevated AMPK and mitochondrial biogenesis, linking S6K activity to organismal aging and energy homeostasis.\",\n      \"evidence\": \"S6K1 KO and S6K1/S6K2 DKO mice, lifespan analysis, AMPK activity, metabolite measurements, gene expression profiling\",\n      \"pmids\": [\"19797661\", \"17550782\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Relative contribution of rpS6 phosphorylation vs. other S6K substrates to longevity not resolved\", \"Sex-specific effects noted but mechanism unclear\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"S6K activity and rpS6 phosphorylation were shown to drive a transcriptional program for ribosome biogenesis, with >75% of ribosome biogenesis factors being S6K-dependent; phosphorylation-deficient rpS6 knock-in mice confirmed that rpS6 phosphorylation itself is required.\",\n      \"evidence\": \"S6K1/S6K2 DKO mice and rpS6 phosphorylation-site knock-in mice, whole-genome microarray, polysomal RNA profiling\",\n      \"pmids\": [\"23318442\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Transcription factor(s) mediating the ribosome biogenesis program downstream of phospho-rpS6 not identified\", \"Whether this is a translational or transcriptional readout of rpS6 remains debated\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"rpS6 phosphorylation was established as the key mTORC1 effector for compensatory renal hypertrophy, with non-phosphorylatable knock-in mice showing blunted hypertrophy despite intact 4E-BP1 phosphorylation, separating rpS6 function from other mTORC1 outputs.\",\n      \"evidence\": \"rpS6 knock-in mouse, uninephrectomy model, rapamycin treatment, 4E-BP1 internal control\",\n      \"pmids\": [\"25229342\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism linking phospho-rpS6 to cyclin D1 upregulation not defined\", \"Whether the effect is translation- or transcription-mediated not established\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"rpS6 was found to interact with mTORC2 components and positively regulate Akt-Ser473 phosphorylation, revealing an unexpected feedback loop from a ribosomal protein to a distinct mTOR complex.\",\n      \"evidence\": \"Co-immunoprecipitation, rpS6 siRNA and overexpression, Akt-Ser473 phosphorylation, oxidative stress survival assay\",\n      \"pmids\": [\"24557881\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct binding interface between rpS6 and mTORC2 not mapped\", \"Whether this occurs on or off the ribosome is unknown\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"mTORC1/rpS6 signaling was shown to regulate blood–testis barrier dynamics through actin and microtubule cytoskeleton reorganization, with phosphomimetic rpS6 promoting BTB disruption in vivo.\",\n      \"evidence\": \"In vivo testis transfection of WT and phosphomimetic rpS6 mutants, BTB integrity assay, cytoskeletal protein localization\",\n      \"pmids\": [\"22948214\", \"29089336\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct molecular targets of phospho-rpS6 at the BTB not identified\", \"Whether this function is ribosome-dependent or extra-ribosomal is unknown\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"A somatic gain-of-function RPS6 p.R232H variant was identified in hemimegalencephaly brain tissue and shown to promote proliferation in animal models, establishing RPS6 as a cortical dysplasia gene.\",\n      \"evidence\": \"Deep sequencing of somatic mosaicism, overexpression in animal models, double-mutant epistasis with MTOR p.S2215F\",\n      \"pmids\": [\"31411685\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single study; not independently replicated\", \"Mechanism by which R232H alters RPS6 function not determined\", \"Whether the variant affects phosphorylation or ribosome assembly not tested\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Selective ribosome footprinting revealed that RPS6 phosphorylation progressively declines during elongation, causing ribosomes on short-CDS mRNAs to carry higher phospho-RPS6 and thereby preferentially promoting translation of short-CDS transcripts — independent of 5′TOP status.\",\n      \"evidence\": \"Phospho-RPS6 selective ribosome footprinting, RPS6 phosphorylation-site knock-in cell lines, transcriptome-wide translation analysis\",\n      \"pmids\": [\"34871442\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism of progressive dephosphorylation during elongation not identified\", \"Identity of the ribosome-associated phosphatase unknown\", \"Whether this selectivity drives specific physiological outcomes not tested\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"rpS6 phosphorylation was shown to form a rapid spatiotemporal activation zone around skin wounds that persists until healing is complete; non-phosphorylatable rpS6 mice have impaired overall wound healing, extending rpS6 function to tissue repair.\",\n      \"evidence\": \"In vivo mouse wounding model, phospho-rpS6 immunofluorescence mapping, rpS6 phosphorylation-site knock-in mice, wound closure assays\",\n      \"pmids\": [\"37098351\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Cell types responsible for the phospho-rpS6 zone not resolved\", \"Whether rpS6 phosphorylation acts via translation or signaling in this context is unknown\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Key open questions include: the identity of the ribosome-associated phosphatase that dephosphorylates rpS6 during elongation; whether rpS6 functions at the BTB and in wound healing are ribosome-dependent or extra-ribosomal; the structural basis of how rpS6 phosphorylation alters 40S conformation at atomic resolution; and the mechanism by which the R232H variant causes cortical dysplasia.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Ribosome-associated rpS6 phosphatase identity unknown\", \"Extra-ribosomal vs. ribosome-dependent functions not dissected\", \"No high-resolution structure of phosphorylated vs. unphosphorylated human 40S showing rpS6 conformational changes\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0005198\", \"supporting_discovery_ids\": [37, 38]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [26, 34]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005840\", \"supporting_discovery_ids\": [0, 1, 2, 37, 38]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [34]},\n      {\"term_id\": \"GO:0005730\", \"supporting_discovery_ids\": [8]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [8]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [34, 37, 38]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [3, 5, 7, 9, 26]},\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [25, 33]},\n      {\"term_id\": \"R-HSA-1852241\", \"supporting_discovery_ids\": [24, 28]}\n    ],\n    \"complexes\": [\n      \"40S ribosomal subunit\",\n      \"80S ribosome\"\n    ],\n    \"partners\": [\n      \"RPS6KB1\",\n      \"RPS6KB2\",\n      \"MTOR\",\n      \"RICTOR\",\n      \"RPL10\",\n      \"ARRB1\",\n      \"ZRF1\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```\n\n--- ROUND 2 (R2/R3, augmented corpus) ---\n\n```json\n{\n  \"mechanistic_narrative\": \"RPS6 (eS6) is a structural component of the 40S ribosomal subunit whose five conserved C-terminal serine residues (Ser235, Ser236, Ser240, Ser244, Ser247) are phosphorylated by p70 S6K1/S6K2 downstream of the PI3K–mTORC1–PDK1 axis and by p90 RSK, serving as a central signaling node that couples nutrient and growth-factor sensing to translational control [PMID:3257566, PMID:1939282, PMID:9465032]. Phosphorylation of rpS6 selectively promotes translation of short-CDS mRNAs through progressive dephosphorylation as ribosomes traverse longer open reading frames, while genome-wide ribosome profiling in yeast demonstrates that rpS6 phosphorylation does not broadly regulate 5′TOP mRNA translation [PMID:34871442, PMID:26582391]. Beyond translation, rpS6 phosphorylation drives the ribosome biogenesis transcriptional program, mediates compensatory organ hypertrophy through cyclin D1/E regulation, controls blood–testis barrier dynamics via F-actin reorganization, provides positive feedback to mTORC2/Akt signaling, and defines a wound-healing activation zone in skin [PMID:23318442, PMID:25229342, PMID:22948214, PMID:24557881, PMID:37098351]. Somatic activating mutations in RPS6 (e.g., p.R232H) cause hemimegalencephaly-associated cortical dysplasia through increased cell proliferation [PMID:31411685].\",\n  \"teleology\": [\n    {\n      \"year\": 1981,\n      \"claim\": \"Early kinetic studies established that rpS6 phosphorylation occurs rapidly after mitogenic stimulation and correlates with mRNA recruitment into polysomes, raising the hypothesis that S6 phosphorylation facilitates translational activation.\",\n      \"evidence\": \"Polysome profiling and S6 phosphorylation kinetics in serum-stimulated HeLa cells\",\n      \"pmids\": [\"7333277\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Correlation does not establish causation between S6 phosphorylation and mRNA recruitment\", \"No identification of which mRNAs are affected\"]\n    },\n    {\n      \"year\": 1984,\n      \"claim\": \"S6 phosphorylation was shown to induce conformational changes in the 40S subunit, altering accessibility of multiple ribosomal proteins, providing a biophysical mechanism by which S6 phosphorylation could modulate ribosome function.\",\n      \"evidence\": \"Reductive methylation accessibility assay comparing phosphorylated vs. unphosphorylated ribosomes\",\n      \"pmids\": [\"6693389\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single method; no functional translation consequence demonstrated\", \"No structural resolution of conformational changes\"]\n    },\n    {\n      \"year\": 1991,\n      \"claim\": \"Purification and characterization of p70 S6K established that a single mitogen-activated kinase phosphorylates rpS6 on exactly five C-terminal serines (Ser235/236/240/244 plus a fifth site), matching the in vivo pattern, and that the kinase itself is regulated by phosphorylation reversible by PP2A.\",\n      \"evidence\": \"Biochemical purification from Swiss 3T3 cells, in vitro kinase assay with 40S subunits, tryptic phosphopeptide mapping, PP2A inactivation\",\n      \"pmids\": [\"3257566\", \"1939282\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Upstream signaling pathway not yet defined\", \"Identity of the fifth low-stoichiometry site uncertain\"]\n    },\n    {\n      \"year\": 1995,\n      \"claim\": \"The discovery that rapamycin selectively inhibits p70 S6K and that FRAP/mTOR kinase activity is required for p70 S6K activation placed rpS6 phosphorylation downstream of the rapamycin-sensitive mTOR pathway, distinct from the Ras–MAPK cascade.\",\n      \"evidence\": \"Selective rapamycin inhibition in multiple cell lines; FRAP kinase-dead mutants failing to activate S6K; dominant-negative Ras/Raf not blocking S6K\",\n      \"pmids\": [\"1380182\", \"7566123\", \"8090223\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct phosphorylation of S6K by mTOR not yet demonstrated\", \"Mechanism of rapamycin resistance in truncation mutants unclear\"]\n    },\n    {\n      \"year\": 1995,\n      \"claim\": \"Mapping of three nuclear localization signals in the C-terminal half of rpS6 and identification of a distinct nucleolar-targeting domain established that rpS6 traffics through the nucleus/nucleolus, consistent with ribosome biogenesis roles.\",\n      \"evidence\": \"Deletion mutagenesis of S6–β-galactosidase chimeras in L cells\",\n      \"pmids\": [\"8590812\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional consequence of nuclear localization for rpS6 not tested\", \"Whether phosphorylation regulates NLS function unknown\"]\n    },\n    {\n      \"year\": 1998,\n      \"claim\": \"Direct phosphorylation of S6K1 at Thr389 by mTOR was demonstrated in vitro and shown to be essential for S6K activity, while amino acid withdrawal inactivates S6K through a pathway independent of Akt, establishing amino acid sensing as a distinct input to rpS6 phosphorylation.\",\n      \"evidence\": \"In vitro kinase assay with immunoprecipitated mTOR, phosphosite mapping; amino acid deprivation in CHO cells with PI3K/mTOR inhibitors\",\n      \"pmids\": [\"9465032\", \"9693128\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Amino acid sensing mechanism upstream of mTOR not identified\", \"Relative contributions of S6K1 vs. S6K2 to rpS6 phosphorylation unclear\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"The TSC1/2–Rheb axis was identified as the critical link between PI3K/Akt and mTOR/S6K: TSC2 acts as a GAP for Rheb, and loss-of-function TSC mutations constitutively hyperphosphorylate rpS6, explaining the elevated S6K activity in tuberous sclerosis.\",\n      \"evidence\": \"GTPase assays with purified Rheb, genetic epistasis, TSC2 disease-mutant analysis, S6 phosphorylation in TSC2-null cells with tuberin re-expression\",\n      \"pmids\": [\"12045200\", \"12820960\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How Rheb directly activates mTOR kinase not resolved\", \"Tissue-specific consequences of TSC-driven rpS6 hyperphosphorylation not fully explored\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"The mTOR/S6K1/eIF3 dynamic scaffold model revealed how S6K1 basally associates with the eIF3 translation initiation complex and, upon mTOR-mediated phosphorylation, dissociates to phosphorylate eIF4B, providing a mechanistic link between rpS6 kinase activation and translation initiation complex remodeling.\",\n      \"evidence\": \"Sequential co-immunoprecipitation of mTOR, S6K1, eIF3, and eIF4B with rapamycin treatment\",\n      \"pmids\": [\"16006006\", \"16286006\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether rpS6 phosphorylation itself contributes to eIF3 complex dynamics unknown\", \"Stoichiometry and kinetics of scaffold assembly not defined\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"S6K1 knockout mice showed extended lifespan and resistance to age-related pathologies, while hypothalamic S6K manipulation bidirectionally controlled energy homeostasis, establishing the S6K–rpS6 axis as a systemic regulator of aging and metabolism in mammals.\",\n      \"evidence\": \"S6K1 knockout mouse lifespan and metabolic analysis; bilateral adenoviral injection of S6K variants into rat mediobasal hypothalamus\",\n      \"pmids\": [\"19797661\", \"19041762\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Relative contribution of rpS6 phosphorylation vs. other S6K substrates to lifespan extension not resolved\", \"Human translatability unknown\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Two independent genetic models—S6K1/S6K2 double-knockout mice and rpS6 non-phosphorylatable knock-in mice—converged on the discovery that rpS6 phosphorylation specifically controls the ribosome biogenesis transcriptional program, establishing a non-translational function for rpS6 phosphorylation.\",\n      \"evidence\": \"Whole-genome microarray of total and polysomal liver RNA from S6K DKO and rpS6 knock-in mice after feeding\",\n      \"pmids\": [\"23318442\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Transcription factor mediating rpS6 phosphorylation-dependent RiBi gene induction not identified\", \"Whether this operates through a ribosome-free rpS6 pool unclear\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"rpS6 phosphorylation was identified as the specific mTORC1-S6K1 effector mediating compensatory renal hypertrophy, acting through cyclin D1/E regulation; additionally, rpS6 was found to provide positive feedback to mTORC2/Akt signaling through direct physical interaction with mTORC2 components.\",\n      \"evidence\": \"rpS6 phospho-site knock-in mice with uninephrectomy; co-IP of rpS6 with mTORC2 components; bidirectional manipulation of rpS6 affecting Akt-Ser473 phosphorylation\",\n      \"pmids\": [\"25229342\", \"24557881\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of rpS6–mTORC2 interaction unknown\", \"Whether extra-ribosomal rpS6 mediates mTORC2 feedback not tested\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Genome-wide ribosome profiling in yeast with non-phosphorylatable rpS6 definitively showed that rpS6 phosphorylation does not regulate global translation or preferential translation of individual mRNAs, challenging the decades-old model that S6 phosphorylation broadly controls 5′TOP mRNA translation.\",\n      \"evidence\": \"Ribosome profiling and polysome profiling in yeast rpS6 phospho-mutant cells; identification of Ypk3 as the direct rpS6 kinase via genetic complementation\",\n      \"pmids\": [\"26582391\", \"25767889\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Yeast findings may not fully translate to mammals with more complex 5′TOP regulation\", \"Non-translational roles of rpS6 phosphorylation in yeast not fully characterized\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"The somatic RPS6 p.R232H mutation was shown to be an activating mutation causing hemimegalencephaly-associated cortical dysplasia through increased cell proliferation, establishing RPS6 as a disease gene for brain overgrowth syndromes within the mTORopathy spectrum.\",\n      \"evidence\": \"Whole-exome sequencing of hemimegalencephaly patient; in utero electroporation of RPS6-R232H in mice with proliferation and migration assays\",\n      \"pmids\": [\"31411685\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism by which R232H activates mTOR signaling not defined\", \"Frequency of RPS6 mutations in cortical dysplasia cohorts not established\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Selective ribosome footprinting revealed that rpS6 is progressively dephosphorylated as ribosomes translate longer ORFs, providing a length-dependent mechanism whereby rpS6 phosphorylation selectively promotes translation of short-CDS mRNAs rather than acting through 5′TOP elements.\",\n      \"evidence\": \"Phospho-RPS6 antibody-based selective ribosome profiling in RPS6 phospho-site knock-in cells\",\n      \"pmids\": [\"34871442\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Phosphatase responsible for elongation-dependent rpS6 dephosphorylation not identified\", \"Whether this mechanism operates identically in all mammalian tissues unknown\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"In vivo imaging of wounded skin revealed a spatially defined p-rpS6 zone that encompasses proliferation, senescence, and angiogenesis around wound margins; rpS6 non-phosphorylatable knock-in mice showed altered wound healing kinetics, demonstrating a tissue-level organizing role for rpS6 phosphorylation.\",\n      \"evidence\": \"Immunofluorescence mapping of p-rpS6 in skin wounds; rpS6 knock-in mice wound healing assays\",\n      \"pmids\": [\"37098351\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Signals initiating wound-edge rpS6 phosphorylation not identified\", \"Whether the p-rpS6 zone acts through translation, RiBi, or other mechanisms unclear\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Key unresolved questions include the identity of the phosphatase responsible for elongation-coupled rpS6 dephosphorylation, the structural basis of rpS6's interaction with mTORC2, whether extra-ribosomal rpS6 mediates nuclear/signaling functions, and the transcription factor through which rpS6 phosphorylation controls the ribosome biogenesis program.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Phosphatase for elongation-dependent rpS6 dephosphorylation unknown\", \"Extra-ribosomal rpS6 pool not characterized\", \"Transcription factor mediating RiBi program downstream of rpS6 not identified\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0005198\", \"supporting_discovery_ids\": [58, 59]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005840\", \"supporting_discovery_ids\": [58, 59, 5]},\n      {\"term_id\": \"GO:0005730\", \"supporting_discovery_ids\": [56]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [56]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [52]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [3, 52, 58, 59]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [7, 9, 11, 43]},\n      {\"term_id\": \"R-HSA-1852241\", \"supporting_discovery_ids\": [40, 44]},\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [42]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [51]}\n    ],\n    \"complexes\": [\n      \"40S ribosomal subunit\",\n      \"80S ribosome\"\n    ],\n    \"partners\": [\n      \"RPS6KB1\",\n      \"RPS6KB2\",\n      \"MTOR\",\n      \"RPTOR\",\n      \"EIF3A\",\n      \"RPL10\",\n      \"ARRB1\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}