{"gene":"YWHAQ","run_date":"2026-06-11T09:02:06","timeline":{"discoveries":[{"year":1994,"finding":"14-3-3 zeta (YWHAQ) and 14-3-3 beta interact with the amino-terminal region of Raf-1, and expression of 14-3-3 proteins in Xenopus oocytes enhanced Raf-1 kinase activity and promoted Raf-1-dependent oocyte maturation; a dominant-negative Raf-1 mutant blocked these effects, establishing 14-3-3 as an activator of Raf-1 signaling.","method":"Yeast two-hybrid, Xenopus oocyte overexpression, dominant-negative epistasis","journal":"Nature","confidence":"High","confidence_rationale":"Tier 2 / Strong — yeast two-hybrid identification combined with in vivo functional rescue and dominant-negative epistasis in Xenopus; foundational result replicated across labs","pmids":["7935795"],"is_preprint":false},{"year":1995,"finding":"14-3-3 epsilon and 14-3-3 beta physically interact with the human CDC25A and CDC25B phosphatases both in vitro and in vivo, as established by yeast two-hybrid and co-immunoprecipitation; however, 14-3-3 does not affect CDC25A phosphatase activity or CDC25A-stimulated Raf-1 kinase activity, suggesting 14-3-3 may scaffold CDC25-Raf-1 association.","method":"Yeast two-hybrid, co-immunoprecipitation, in vitro phosphatase activity assay","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal in vitro and in vivo binding confirmed; functional consequence (no effect on phosphatase activity) explicitly tested; single lab","pmids":["7644510"],"is_preprint":false},{"year":1995,"finding":"The N-terminal 26 amino acids of 14-3-3 are required for dimerization; the N-terminal 12 kDa domain mediates binding to synaptic plasma membranes; intact 14-3-3 potently inhibits protein kinase C (PKC) activity, whereas the isolated N-terminal domain does not, indicating the full-length protein is required for PKC inhibition.","method":"N-terminal deletion mutagenesis, circular dichroism, PKC activity assay, membrane fractionation","journal":"Journal of molecular biology","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — in vitro mutagenesis and enzymatic assay with defined phenotypic readout; single lab with multiple orthogonal methods","pmids":["7837270"],"is_preprint":false},{"year":1998,"finding":"14-3-3 zeta and epsilon isoforms interact with the N-terminal regulatory domain of MEKK3 with ~90 nM affinity, and also bind MEKK1 and MEKK2 but not MEKK4; 14-3-3 does not directly influence MEKK1/2/3 kinase activity but functions as a scaffold for protein-protein interactions; caspase-3 cleavage of MEKK1 releases the kinase domain from the N-terminal 14-3-3-binding region.","method":"Yeast two-hybrid, quantitative binding assay, co-immunoprecipitation, confocal colocalization, in vitro kinase assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal binding confirmed by multiple methods including quantitative affinity measurement and colocalization; functional (kinase activity) tested negatively; single lab with multiple orthogonal methods","pmids":["9452471"],"is_preprint":false},{"year":1998,"finding":"Yeast 14-3-3 homologs BMH1 and BMH2 are multicopy suppressors of rapamycin-induced growth arrest; mutations in the phosphopeptide-binding pocket of 14-3-3 abolish rapamycin resistance, establishing that phosphoserine/threonine binding is required for 14-3-3 participation in TOR/rapamycin-sensitive signaling.","method":"Multicopy suppressor screen, deletion analysis, phosphopeptide-binding pocket mutagenesis, growth assay","journal":"Current biology : CB","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic epistasis with active-site mutagenesis in yeast; phosphobinding requirement directly demonstrated","pmids":["9822578"],"is_preprint":false},{"year":2000,"finding":"Dominant-negative 14-3-3 zeta (R56A/R60A double mutant) inhibits serum-stimulated ERK MAPK activation and increases basal JNK1 and p38 MAPK activation in fibroblasts; targeted expression of dominant-negative 14-3-3 eta in cardiac tissue increases JNK1/p38 activation, causes dilated cardiomyopathy, and massive cardiomyocyte apoptosis, demonstrating that 14-3-3 differentially regulates MAPK cascades and inhibits apoptosis in vivo.","method":"Dominant-negative transfection, transgenic mouse model, MAPK activity assays, histology","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 2 / Strong — dominant-negative mutagenesis validated in both cell culture and transgenic animals with multiple defined phenotypic readouts; independently confirmed across multiple MAPK pathways","pmids":["10654934"],"is_preprint":false},{"year":2001,"finding":"Expression of difopein, a specific inhibitor of 14-3-3/ligand interactions, is sufficient to induce apoptosis in cells and sensitizes cells to cisplatin-induced death, establishing that 14-3-3/ligand interactions are required for cell survival signaling downstream of pro-survival kinases.","method":"Difopein inhibitor expression, cell death assay, drug sensitization assay","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — loss-of-function with specific inhibitor peptide, defined apoptotic phenotype; single lab","pmids":["11577088"],"is_preprint":false},{"year":2002,"finding":"TSC2 (but not TSC1) associates with 14-3-3 in vivo, and phosphorylation of Ser1210 in TSC2 is required for this association; 14-3-3 binding may inhibit TSC2 function and represents a mechanism of Akt-dependent TSC2 regulation.","method":"Co-immunoprecipitation, phosphorylation site mutagenesis","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — co-IP with phospho-site mutagenesis; functional consequence inferred but not directly reconstituted; single lab","pmids":["12364343"],"is_preprint":false},{"year":2003,"finding":"14-3-3 sigma interacts with p53 in response to the DNA-damaging agent adriamycin; 14-3-3 sigma expression stabilizes p53 by blocking Mdm2-mediated p53 ubiquitination and nuclear export, and facilitates p53 oligomerization and enhances p53 transcriptional activity.","method":"Co-immunoprecipitation, ubiquitination assay, nuclear export assay, transcriptional reporter assay","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal biochemical assays (co-IP, ubiquitination, nuclear export, transcription) in a single study; mechanistic cascade defined","pmids":["14517281"],"is_preprint":false},{"year":2009,"finding":"Akt kinase-dependent phosphorylation of Chibby (Cby) enables 14-3-3 binding to Cby, and the resulting 14-3-3/Cby complex facilitates nuclear export of beta-catenin, providing a mechanism for cross-talk between Wnt and Akt signaling pathways in regulating beta-catenin nucleo-cytoplasmic trafficking.","method":"Co-immunoprecipitation, phosphorylation assay, nuclear export assay","journal":"Cell cycle (Georgetown, Tex.)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP and functional localization assay; single lab, two orthogonal methods","pmids":["19158508"],"is_preprint":false},{"year":2012,"finding":"14-3-3 isoforms (including beta and epsilon) interact with CFTR at PKA-phosphorylated sites in the regulatory region; 14-3-3 binding increases CFTR biogenesis by reducing retrograde ER retrieval (competing with COP1 binding), increasing CFTR synthesis, reducing degradation, and promoting maturation to cell surface.","method":"Co-immunoprecipitation, NMR, pulse-chase, siRNA knockdown, cell surface biotinylation, anion efflux assay","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 1 / Strong — NMR structural validation of binding site, multiple biochemical and cell biological assays, knockdown with defined functional readout; single lab with multiple orthogonal methods","pmids":["22278744"],"is_preprint":false},{"year":2013,"finding":"14-3-3 functions as a molecular adaptor to recruit chaperone-associated misfolded protein cargoes to dynein motors for transport to aggresomes; 14-3-3 simultaneously binds both dynein intermediate chain (DIC) and the Hsp70 co-chaperone BAG3, and functional 14-3-3 dimerization is required for this process in both yeast and mammalian cells.","method":"Co-immunoprecipitation, dimerization-defective mutant analysis, aggresome formation assay, yeast and mammalian cell models","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal co-IP of ternary complex, dimerization mutant functional test, validated in two organisms; multiple orthogonal methods","pmids":["23843611"],"is_preprint":false},{"year":2015,"finding":"14-3-3 proteins act as cytosolic adaptors mediating ER export of SAC1 (a PI4P phosphatase) in COPII-coated vesicles; 14-3-3 directly binds a minimal 7-aa sorting motif (RLSNTSP) in SAC1 and interacts with the COPII sorting subunit Sec24; recombinant 14-3-3 stimulates SAC1 packaging into COPII vesicles in a cell-free budding reaction.","method":"Cell-free COPII vesicle budding assay, recombinant protein, co-immunoprecipitation, homology modeling, sorting motif mutagenesis","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 / Strong — cell-free reconstitution with recombinant protein, motif mutagenesis, and co-IP; multiple orthogonal methods in a single study","pmids":["26056309"],"is_preprint":false},{"year":2015,"finding":"14-3-3 theta (YWHAQ) overexpression reduces LRRK2 kinase activity and reverses mutant LRRK2 (G2019S and R1441G)-induced neurite shortening in primary neurons; this protective effect requires direct physical binding of 14-3-3 theta to LRRK2; pan-14-3-3 inhibitor difopein conversely promotes LRRK2 kinase activity and exacerbates neurite shortening.","method":"Overexpression in BAC transgenic neurons, LRRK2 kinase activity assay, neurite length measurement, difopein inhibition, LRRK2 kinase inhibitor rescue","journal":"Human molecular genetics","confidence":"High","confidence_rationale":"Tier 2 / Strong — gain- and loss-of-function with binding-deficient controls, defined kinase activity readout and morphological phenotype, pharmacological rescue; single lab with multiple orthogonal methods","pmids":["26546614"],"is_preprint":false},{"year":2017,"finding":"Crystal structures of 14-3-3 bound to phosphopeptides from LRRK2 characterize the multivalent interaction interface; LRRK2 possesses multiple phosphorylation sites that each can bind 14-3-3, and many Parkinson's disease-relevant LRRK2 mutations impair 14-3-3 binding.","method":"X-ray crystallography, biochemical binding assays","journal":"The Biochemical journal","confidence":"High","confidence_rationale":"Tier 1 / Moderate — crystal structure with biochemical validation; single lab","pmids":["28202711"],"is_preprint":false},{"year":2018,"finding":"PKA-dependent phosphorylation of SIK1, SIK2, and SIK3 induces 14-3-3 binding and inhibits their catalytic activity; SIK1 and SIK3 have two functional PKA/14-3-3 sites while SIK2 has four; loss of a single site in SIK1/3 abolishes 14-3-3 association and cAMP responsiveness, demonstrating multi-site cooperativity in 14-3-3-mediated kinase inhibition.","method":"In vitro kinase assay, phosphorylation site mutagenesis, co-immunoprecipitation, cAMP stimulation","journal":"The FEBS journal","confidence":"High","confidence_rationale":"Tier 2 / Strong — mutagenesis of multiple binding sites with defined kinase activity readout; cooperativity mechanism established; single lab with multiple orthogonal methods","pmids":["29211348"],"is_preprint":false},{"year":2018,"finding":"BAP1 (BRCA1-associated protein 1) interacts with 14-3-3 protein; this interaction releases the pro-apoptotic protein Bax from 14-3-3, promoting apoptosis through the intrinsic pathway in neuroblastoma cells.","method":"Co-immunoprecipitation, apoptosis assay, Bax release assay, xenograft","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP with functional apoptosis readout and in vivo xenograft validation; single lab","pmids":["29686263"],"is_preprint":false},{"year":2019,"finding":"14-3-3 binds to two pairs of phosphorylation sites in IRSp53 (established by phosphoproteomics and quantitative binding); each subunit of an IRSp53 homodimer independently binds one 14-3-3 dimer; a FRET-sensor assay shows that 14-3-3 binding induces opposite conformational changes in IRSp53 compared to activatory inputs (Cdc42, Eps8); 14-3-3 inhibits IRSp53 binding to membranes and its interaction with Cdc42.","method":"Phosphoproteomics, quantitative binding assay, X-ray crystallography, bicistronic expression of heterodimer, FRET-sensor assay, liposome binding assay","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure combined with FRET conformational readout, quantitative binding, and functional membrane/GTPase inhibition assays; multiple orthogonal methods in one study","pmids":["30696821"],"is_preprint":false},{"year":2020,"finding":"PKA phosphorylates CaMKK2 at Ser100 and Ser511, which mediates recruitment of 14-3-3 adaptor proteins; 14-3-3 binding holds CaMKK2 in an inactivated state by preventing dephosphorylation of phospho-Ser495 (the Ca2+/CaM activation site); crystal structure of 14-3-3 zeta bound to a diphosphorylated CaMKK2 peptide reveals how canonical (Ser511) and non-canonical (Ser100) 14-3-3 consensus sites cooperate to bind 14-3-3.","method":"In vitro kinase assay, phosphosite mutagenesis, X-ray crystallography, co-immunoprecipitation","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure plus in vitro kinase assay and mutagenesis of multiple phosphosites; mechanism of inactivation directly demonstrated","pmids":["32913128"],"is_preprint":false},{"year":2021,"finding":"14-3-3 epsilon was biochemically identified as an intracellular component of TNFR2 receptor complexes in chondrocytes in response to progranulin (PGRN); 14-3-3 epsilon is required for PGRN/TNFR2 signaling through ERK-dependent Elk-1 activation while suppressing NF-κB; chondrocyte-specific deletion of 14-3-3 epsilon abolishes PGRN's therapeutic effects against osteoarthritis in vivo.","method":"Biochemical copurification, proteomics, co-immunoprecipitation, transcription factor activity screen, conditional knockout mouse, OA disease model","journal":"Annals of the rheumatic diseases","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — proteomics-validated co-purification plus conditional KO with defined signaling and disease phenotype; single lab","pmids":["34226187"],"is_preprint":false},{"year":2022,"finding":"Phosphorylation of 14-3-3 zeta at Ser58 by PKC shifts the dimer-monomer equilibrium strongly toward monomers at physiological concentrations; phosphomimicking mutations (S58E/D) incompletely recapitulate this shift; Ser58 phosphorylation changes the subcellular localization of 14-3-3 zeta in HeLa and U251 cells.","method":"Site-specific phosphorylation, analytical ultracentrifugation/biophysics, thermal stability assay, fluorescence microscopy in human cancer cells","journal":"Frontiers in chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct phosphorylation confirmed with multiple biophysical methods and cell imaging; single lab, novel finding for the zeta isoform","pmids":["35321476"],"is_preprint":false}],"current_model":"YWHAQ (14-3-3 zeta/theta) functions as a phosphoserine/phosphothreonine-binding scaffold protein that, upon recognizing specific phosphorylated motifs on client proteins (including Raf-1, LRRK2, CaMKK2, SIKs, CDC25, TSC2, IRSp53, CFTR, and others), allosterically modulates client activity, subcellular localization, stability, or protein-protein interactions through mechanisms including conformational change, steric occlusion of targeting sequences, scaffolding of multi-protein complexes, and competing with retrograde/degradation machinery; it forms homo- and heterodimers whose integrity (regulated by Ser58 phosphorylation) is required for many of these functions, and it mediates essential anti-apoptotic signaling by sequestering pro-apoptotic proteins such as BAD."},"narrative":{"mechanistic_narrative":"YWHAQ (14-3-3 theta/zeta) is a phosphoserine/phosphothreonine-binding adaptor that recognizes phosphorylated motifs on diverse clients and thereby controls their activity, localization, stability, and complex assembly across kinase signaling, vesicular transport, and cell-survival pathways [PMID:7935795, PMID:9822578, PMID:32913128]. Sequence-specific recognition depends on an intact phosphopeptide-binding pocket, and many clients are engaged through cooperative multi-site binding in which loss of a single phosphosite abolishes association, as shown for SIK kinases, CaMKK2, LRRK2, and IRSp53 [PMID:9822578, PMID:29211348, PMID:32913128, PMID:28202711, PMID:30696821]. Functionally, 14-3-3 binding can either activate or restrain clients: it stimulates Raf-1 to promote MAPK signaling [PMID:7935795], holds CaMKK2 and SIK kinases in an inactivated state by occluding sites and blocking dephosphorylation [PMID:32913128, PMID:29211348], reduces LRRK2 kinase activity and rescues mutant LRRK2-induced neurite shortening [PMID:26546614], and drives conformational change in IRSp53 that inhibits its membrane and Cdc42 binding [PMID:30696821]. As a molecular adaptor it couples cargoes to trafficking machinery — recruiting misfolded protein cargo and BAG3 to dynein for aggresome delivery, and SAC1 to the COPII subunit Sec24 for ER export — and it modulates CFTR biogenesis by competing with retrograde ER retrieval [PMID:23843611, PMID:26056309, PMID:22278744]. 14-3-3 function requires dimerization, and PKC phosphorylation of Ser58 shifts the equilibrium toward monomers and alters subcellular localization [PMID:23843611, PMID:35321476]. The protein mediates essential anti-apoptotic signaling: loss of 14-3-3/ligand interactions induces apoptosis and dilated cardiomyopathy in vivo, and displacement of pro-apoptotic Bax from 14-3-3 by BAP1 promotes intrinsic-pathway death [PMID:10654934, PMID:11577088, PMID:29686263].","teleology":[{"year":1994,"claim":"Established that 14-3-3 is not merely a passive binder but a positive regulator of a signaling kinase, defining its role as an activator of Raf-1-dependent MAPK signaling.","evidence":"Yeast two-hybrid, Xenopus oocyte overexpression, and dominant-negative Raf-1 epistasis","pmids":["7935795"],"confidence":"High","gaps":["Structural basis of Raf-1 activation not resolved","Did not define the phospho-motif on Raf-1 recognized by 14-3-3"]},{"year":1995,"claim":"Distinguished scaffolding from catalytic modulation by showing 14-3-3 binds CDC25 phosphatases without altering their activity, and mapped dimerization and membrane-binding determinants to the N-terminus while showing full-length protein is needed to inhibit PKC.","evidence":"Yeast two-hybrid, co-IP, in vitro phosphatase assay; N-terminal deletion mutagenesis, circular dichroism, PKC activity assay","pmids":["7644510","7837270"],"confidence":"Medium","gaps":["Functional outcome of CDC25 binding in vivo not defined","Single-lab biochemistry without independent confirmation"]},{"year":1998,"claim":"Demonstrated that 14-3-3 acts as a scaffold for MEKK kinases without altering their catalytic activity, and that phosphopeptide-pocket binding is genetically required for participation in TOR/rapamycin signaling.","evidence":"Quantitative binding and co-IP for MEKK1/2/3; multicopy suppressor screen with pocket mutagenesis in yeast","pmids":["9452471","9822578"],"confidence":"High","gaps":["Direct phospho-clients in the TOR pathway not identified","Selectivity for MEKK1/2/3 over MEKK4 mechanism unexplained"]},{"year":2000,"claim":"Showed in vivo that 14-3-3 differentially tunes MAPK cascades (promoting ERK, restraining JNK/p38) and is required to suppress apoptosis at the organ level.","evidence":"Dominant-negative 14-3-3 transfection and cardiac transgenic mouse with MAPK assays and histology","pmids":["10654934"],"confidence":"High","gaps":["Specific apoptotic clients responsible for the cardiac phenotype not pinpointed","Isoform-specific contributions not separated"]},{"year":2001,"claim":"Established that 14-3-3/ligand interactions are necessary for cell survival, using a generic interaction inhibitor to trigger apoptosis.","evidence":"Difopein inhibitor expression with cell death and drug-sensitization assays","pmids":["11577088"],"confidence":"Medium","gaps":["Does not identify which client displacement drives death","Single-lab loss-of-function"]},{"year":2002,"claim":"Linked 14-3-3 to growth control by showing phospho-dependent binding to TSC2, providing a node for Akt-dependent regulation.","evidence":"Co-IP and Ser1210 phospho-site mutagenesis","pmids":["12364343"],"confidence":"Medium","gaps":["Inhibition of TSC2 function inferred, not reconstituted","Effect on downstream mTOR output not measured here"]},{"year":2003,"claim":"Defined a mechanism by which 14-3-3 stabilizes a client by blocking its ubiquitination and nuclear export, illustrated by p53.","evidence":"Co-IP, ubiquitination assay, nuclear export assay, transcriptional reporter","pmids":["14517281"],"confidence":"High","gaps":["Demonstrated for the sigma isoform; YWHAQ-specific contribution not isolated"]},{"year":2009,"claim":"Showed 14-3-3 controls nucleo-cytoplasmic trafficking of a signaling effector, coupling Akt phosphorylation of Chibby to beta-catenin nuclear export.","evidence":"Co-IP, phosphorylation assay, nuclear export assay","pmids":["19158508"],"confidence":"Medium","gaps":["Direct transport mechanism of the 14-3-3/Cby/beta-catenin complex not resolved","Single-lab"]},{"year":2012,"claim":"Defined 14-3-3 as a regulator of membrane protein biogenesis, increasing CFTR maturation by competing with COPI-mediated ER retrieval.","evidence":"Co-IP, NMR mapping, pulse-chase, siRNA, surface biotinylation, anion efflux","pmids":["22278744"],"confidence":"High","gaps":["Isoform redundancy at the CFTR R-region not dissected","Quantitative contribution of synthesis vs degradation effects not separated"]},{"year":2013,"claim":"Established 14-3-3 as a dimerization-dependent adaptor linking chaperone-bound misfolded cargo to dynein for aggresome transport.","evidence":"Reciprocal co-IP of a DIC/14-3-3/BAG3 ternary complex, dimerization mutants, aggresome assays in yeast and mammalian cells","pmids":["23843611"],"confidence":"High","gaps":["Phospho-recognition basis of cargo selection not defined","Stoichiometry of the dynein-cargo bridge unresolved"]},{"year":2015,"claim":"Demonstrated by reconstitution that 14-3-3 directly drives COPII packaging of cargo (SAC1) via a minimal sorting motif and Sec24 interaction.","evidence":"Cell-free COPII budding with recombinant protein, sorting-motif mutagenesis, co-IP; plus LRRK2 protective gain/loss-of-function in neurons","pmids":["26056309","26546614"],"confidence":"High","gaps":["Generality of 14-3-3-dependent COPII sorting beyond SAC1 untested","LRRK2 binding sites not yet mapped structurally at this stage"]},{"year":2017,"claim":"Provided the structural basis for multivalent 14-3-3/LRRK2 recognition and linked Parkinson's mutations to impaired 14-3-3 binding.","evidence":"X-ray crystallography of 14-3-3/LRRK2 phosphopeptides with binding assays","pmids":["28202711"],"confidence":"High","gaps":["Functional consequence of each individual site in cells not quantified"]},{"year":2018,"claim":"Established cooperative multi-site phospho-recognition as the mechanism by which 14-3-3 inhibits kinase activity (SIK1/2/3) and revealed how 14-3-3 displacement releases pro-apoptotic Bax.","evidence":"In vitro kinase assays with multi-site mutagenesis and cAMP stimulation; co-IP/apoptosis/Bax-release assays and xenograft for BAP1","pmids":["29211348","29686263"],"confidence":"High","gaps":["Whether 14-3-3 sterically occludes the SIK catalytic site or blocks dephosphorylation not fully separated","BAP1 study is single-lab Medium-confidence"]},{"year":2019,"claim":"Showed that 14-3-3 binding allosterically reshapes a client conformation, inhibiting IRSp53 membrane association and Cdc42 binding in opposition to activating inputs.","evidence":"Phosphoproteomics, crystallography, bicistronic heterodimer expression, FRET-sensor and liposome assays","pmids":["30696821"],"confidence":"High","gaps":["In vivo cytoskeletal consequence of IRSp53 conformational switching not addressed"]},{"year":2020,"claim":"Defined how dual PKA-driven canonical and non-canonical sites cooperate to recruit 14-3-3 and lock CaMKK2 inactive by protecting its activation-site phosphorylation from phosphatases.","evidence":"In vitro kinase assay, phosphosite mutagenesis, co-IP, crystal structure of 14-3-3 zeta with a diphospho-CaMKK2 peptide","pmids":["32913128"],"confidence":"High","gaps":["Identity of the phosphatase shielded from phospho-Ser495 not established"]},{"year":2021,"claim":"Placed 14-3-3 inside a transmembrane receptor signaling complex (TNFR2) required for progranulin-driven ERK/Elk-1 output and disease protection.","evidence":"Copurification/proteomics, co-IP, transcription-factor screen, chondrocyte conditional knockout, OA model","pmids":["34226187"],"confidence":"Medium","gaps":["Direct phospho-motif engaged within TNFR2 complex not mapped","Demonstrated for the epsilon isoform"]},{"year":2022,"claim":"Established a regulatory switch for 14-3-3 itself: PKC phosphorylation of Ser58 drives monomerization and relocalization, controlling adaptor availability.","evidence":"Site-specific phosphorylation with analytical ultracentrifugation, thermal stability, and microscopy in human cancer cells","pmids":["35321476"],"confidence":"Medium","gaps":["Functional consequence of monomerization for specific clients not measured","Phosphomimetics incompletely recapitulate the effect"]},{"year":null,"claim":"It remains unresolved how isoform-specific (YWHAQ vs other 14-3-3 paralogs) client engagement and the Ser58 dimer-monomer switch are integrated to set context-dependent activating versus inhibitory outcomes on shared clients.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No systematic isoform-resolved client map","Cellular triggers of Ser58 phosphorylation in physiological settings undefined","How a single adaptor selects activation vs inhibition of a given client not mechanistically unified"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[11,12,18]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[0,13,15,17,18]},{"term_id":"GO:0140313","term_label":"molecular sequestering activity","supporting_discovery_ids":[16,6]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[12,20]},{"term_id":"GO:0005783","term_label":"endoplasmic reticulum","supporting_discovery_ids":[10,12]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[0,5,18,15]},{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[5,6,16]},{"term_id":"R-HSA-5653656","term_label":"Vesicle-mediated transport","supporting_discovery_ids":[11,12,10]}],"complexes":["TNFR2 receptor complex","14-3-3/dynein-DIC/BAG3 transport complex","14-3-3/Sec24 COPII sorting complex"],"partners":["RAF1","LRRK2","CAMKK2","TSC2","CFTR","SAC1","BAG3","BAP1"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"P27348","full_name":"14-3-3 protein theta","aliases":["14-3-3 protein T-cell","14-3-3 protein tau","Protein HS1"],"length_aa":245,"mass_kda":27.8,"function":"Adapter protein implicated in the regulation of a large spectrum of both general and specialized signaling pathways. Binds to a large number of partners, usually by recognition of a phosphoserine or phosphothreonine motif. Binding generally results in the modulation of the activity of the binding partner. Negatively regulates the kinase activity of PDPK1","subcellular_location":"Cytoplasm","url":"https://www.uniprot.org/uniprotkb/P27348/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/YWHAQ","classification":"Not Classified","n_dependent_lines":1,"n_total_lines":1208,"dependency_fraction":0.0008278145695364238},"opencell":{"profiled":true,"resolved_as":"","ensg_id":"ENSG00000134308","cell_line_id":"CID000466","localizations":[{"compartment":"cytoplasmic","grade":3},{"compartment":"centrosome","grade":2},{"compartment":"nucleoplasm","grade":2}],"interactors":[{"gene":"KRAS","stoichiometry":10.0},{"gene":"YWHAG","stoichiometry":10.0},{"gene":"YWHAZ","stoichiometry":10.0},{"gene":"ACTR2","stoichiometry":4.0},{"gene":"ARL1","stoichiometry":4.0},{"gene":"ARL3","stoichiometry":4.0},{"gene":"TRAPPC1","stoichiometry":4.0},{"gene":"YWHAB","stoichiometry":4.0},{"gene":"YWHAE","stoichiometry":4.0},{"gene":"APPL1","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/target/CID000466","total_profiled":1310},"omim":[{"mim_id":"620230","title":"FHF COMPLEX SUBUNIT HOOK-INTERACTING PROTEIN 2B; FHIP2B","url":"https://www.omim.org/entry/620230"},{"mim_id":"618051","title":"INNATE IMMUNITY ACTIVATOR; 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Drosophila.","date":"2016","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/27151460","citation_count":37,"is_preprint":false},{"pmid":"19158508","id":"PMC_19158508","title":"Fine-tuning of nuclear-catenin by Chibby and 14-3-3.","date":"2009","source":"Cell cycle (Georgetown, Tex.)","url":"https://pubmed.ncbi.nlm.nih.gov/19158508","citation_count":37,"is_preprint":false},{"pmid":"26056309","id":"PMC_26056309","title":"Phosphoregulatory protein 14-3-3 facilitates SAC1 transport from the endoplasmic reticulum.","date":"2015","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/26056309","citation_count":36,"is_preprint":false},{"pmid":"19366886","id":"PMC_19366886","title":"14-3-3 and its binding partners are regulators of protein-protein interactions during spermatogenesis.","date":"2009","source":"The Journal of endocrinology","url":"https://pubmed.ncbi.nlm.nih.gov/19366886","citation_count":36,"is_preprint":false},{"pmid":"15949538","id":"PMC_15949538","title":"14-3-3 protein signaling in development and growth factor responses.","date":"2005","source":"Current topics in developmental biology","url":"https://pubmed.ncbi.nlm.nih.gov/15949538","citation_count":34,"is_preprint":false},{"pmid":"32913128","id":"PMC_32913128","title":"CaMKK2 is inactivated by cAMP-PKA signaling and 14-3-3 adaptor proteins.","date":"2020","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/32913128","citation_count":34,"is_preprint":false},{"pmid":"36053201","id":"PMC_36053201","title":"14-3-3 proteins contribute to autophagy by modulating SINAT-mediated degradation of ATG13.","date":"2022","source":"The Plant cell","url":"https://pubmed.ncbi.nlm.nih.gov/36053201","citation_count":32,"is_preprint":false},{"pmid":"35321476","id":"PMC_35321476","title":"Phosphorylated and Phosphomimicking Variants May Differ-A Case Study of 14-3-3 Protein.","date":"2022","source":"Frontiers in chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/35321476","citation_count":32,"is_preprint":false},{"pmid":"32950519","id":"PMC_32950519","title":"14-3-3 σ: A potential biomolecule for cancer therapy.","date":"2020","source":"Clinica chimica acta; international journal of clinical chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/32950519","citation_count":31,"is_preprint":false},{"pmid":"29218076","id":"PMC_29218076","title":"14-3-3 proteins: an important regulator of autophagy in diseases.","date":"2017","source":"American journal of translational research","url":"https://pubmed.ncbi.nlm.nih.gov/29218076","citation_count":30,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":38367,"output_tokens":5479,"usd":0.098643,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":13817,"output_tokens":5090,"usd":0.098168,"stage2_stop_reason":"end_turn"},"total_usd":0.196811,"stage1_batch_id":"msgbatch_01UT1AahFPCAyn2uVweS7A9z","stage2_batch_id":"msgbatch_01HuKWtKNT4Pqj1bc8eEP5RV","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1994,\n      \"finding\": \"14-3-3 zeta (YWHAQ) and 14-3-3 beta interact with the amino-terminal region of Raf-1, and expression of 14-3-3 proteins in Xenopus oocytes enhanced Raf-1 kinase activity and promoted Raf-1-dependent oocyte maturation; a dominant-negative Raf-1 mutant blocked these effects, establishing 14-3-3 as an activator of Raf-1 signaling.\",\n      \"method\": \"Yeast two-hybrid, Xenopus oocyte overexpression, dominant-negative epistasis\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — yeast two-hybrid identification combined with in vivo functional rescue and dominant-negative epistasis in Xenopus; foundational result replicated across labs\",\n      \"pmids\": [\"7935795\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1995,\n      \"finding\": \"14-3-3 epsilon and 14-3-3 beta physically interact with the human CDC25A and CDC25B phosphatases both in vitro and in vivo, as established by yeast two-hybrid and co-immunoprecipitation; however, 14-3-3 does not affect CDC25A phosphatase activity or CDC25A-stimulated Raf-1 kinase activity, suggesting 14-3-3 may scaffold CDC25-Raf-1 association.\",\n      \"method\": \"Yeast two-hybrid, co-immunoprecipitation, in vitro phosphatase activity assay\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal in vitro and in vivo binding confirmed; functional consequence (no effect on phosphatase activity) explicitly tested; single lab\",\n      \"pmids\": [\"7644510\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1995,\n      \"finding\": \"The N-terminal 26 amino acids of 14-3-3 are required for dimerization; the N-terminal 12 kDa domain mediates binding to synaptic plasma membranes; intact 14-3-3 potently inhibits protein kinase C (PKC) activity, whereas the isolated N-terminal domain does not, indicating the full-length protein is required for PKC inhibition.\",\n      \"method\": \"N-terminal deletion mutagenesis, circular dichroism, PKC activity assay, membrane fractionation\",\n      \"journal\": \"Journal of molecular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro mutagenesis and enzymatic assay with defined phenotypic readout; single lab with multiple orthogonal methods\",\n      \"pmids\": [\"7837270\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"14-3-3 zeta and epsilon isoforms interact with the N-terminal regulatory domain of MEKK3 with ~90 nM affinity, and also bind MEKK1 and MEKK2 but not MEKK4; 14-3-3 does not directly influence MEKK1/2/3 kinase activity but functions as a scaffold for protein-protein interactions; caspase-3 cleavage of MEKK1 releases the kinase domain from the N-terminal 14-3-3-binding region.\",\n      \"method\": \"Yeast two-hybrid, quantitative binding assay, co-immunoprecipitation, confocal colocalization, in vitro kinase assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal binding confirmed by multiple methods including quantitative affinity measurement and colocalization; functional (kinase activity) tested negatively; single lab with multiple orthogonal methods\",\n      \"pmids\": [\"9452471\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"Yeast 14-3-3 homologs BMH1 and BMH2 are multicopy suppressors of rapamycin-induced growth arrest; mutations in the phosphopeptide-binding pocket of 14-3-3 abolish rapamycin resistance, establishing that phosphoserine/threonine binding is required for 14-3-3 participation in TOR/rapamycin-sensitive signaling.\",\n      \"method\": \"Multicopy suppressor screen, deletion analysis, phosphopeptide-binding pocket mutagenesis, growth assay\",\n      \"journal\": \"Current biology : CB\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic epistasis with active-site mutagenesis in yeast; phosphobinding requirement directly demonstrated\",\n      \"pmids\": [\"9822578\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"Dominant-negative 14-3-3 zeta (R56A/R60A double mutant) inhibits serum-stimulated ERK MAPK activation and increases basal JNK1 and p38 MAPK activation in fibroblasts; targeted expression of dominant-negative 14-3-3 eta in cardiac tissue increases JNK1/p38 activation, causes dilated cardiomyopathy, and massive cardiomyocyte apoptosis, demonstrating that 14-3-3 differentially regulates MAPK cascades and inhibits apoptosis in vivo.\",\n      \"method\": \"Dominant-negative transfection, transgenic mouse model, MAPK activity assays, histology\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — dominant-negative mutagenesis validated in both cell culture and transgenic animals with multiple defined phenotypic readouts; independently confirmed across multiple MAPK pathways\",\n      \"pmids\": [\"10654934\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"Expression of difopein, a specific inhibitor of 14-3-3/ligand interactions, is sufficient to induce apoptosis in cells and sensitizes cells to cisplatin-induced death, establishing that 14-3-3/ligand interactions are required for cell survival signaling downstream of pro-survival kinases.\",\n      \"method\": \"Difopein inhibitor expression, cell death assay, drug sensitization assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — loss-of-function with specific inhibitor peptide, defined apoptotic phenotype; single lab\",\n      \"pmids\": [\"11577088\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"TSC2 (but not TSC1) associates with 14-3-3 in vivo, and phosphorylation of Ser1210 in TSC2 is required for this association; 14-3-3 binding may inhibit TSC2 function and represents a mechanism of Akt-dependent TSC2 regulation.\",\n      \"method\": \"Co-immunoprecipitation, phosphorylation site mutagenesis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — co-IP with phospho-site mutagenesis; functional consequence inferred but not directly reconstituted; single lab\",\n      \"pmids\": [\"12364343\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"14-3-3 sigma interacts with p53 in response to the DNA-damaging agent adriamycin; 14-3-3 sigma expression stabilizes p53 by blocking Mdm2-mediated p53 ubiquitination and nuclear export, and facilitates p53 oligomerization and enhances p53 transcriptional activity.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, nuclear export assay, transcriptional reporter assay\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal biochemical assays (co-IP, ubiquitination, nuclear export, transcription) in a single study; mechanistic cascade defined\",\n      \"pmids\": [\"14517281\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Akt kinase-dependent phosphorylation of Chibby (Cby) enables 14-3-3 binding to Cby, and the resulting 14-3-3/Cby complex facilitates nuclear export of beta-catenin, providing a mechanism for cross-talk between Wnt and Akt signaling pathways in regulating beta-catenin nucleo-cytoplasmic trafficking.\",\n      \"method\": \"Co-immunoprecipitation, phosphorylation assay, nuclear export assay\",\n      \"journal\": \"Cell cycle (Georgetown, Tex.)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP and functional localization assay; single lab, two orthogonal methods\",\n      \"pmids\": [\"19158508\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"14-3-3 isoforms (including beta and epsilon) interact with CFTR at PKA-phosphorylated sites in the regulatory region; 14-3-3 binding increases CFTR biogenesis by reducing retrograde ER retrieval (competing with COP1 binding), increasing CFTR synthesis, reducing degradation, and promoting maturation to cell surface.\",\n      \"method\": \"Co-immunoprecipitation, NMR, pulse-chase, siRNA knockdown, cell surface biotinylation, anion efflux assay\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — NMR structural validation of binding site, multiple biochemical and cell biological assays, knockdown with defined functional readout; single lab with multiple orthogonal methods\",\n      \"pmids\": [\"22278744\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"14-3-3 functions as a molecular adaptor to recruit chaperone-associated misfolded protein cargoes to dynein motors for transport to aggresomes; 14-3-3 simultaneously binds both dynein intermediate chain (DIC) and the Hsp70 co-chaperone BAG3, and functional 14-3-3 dimerization is required for this process in both yeast and mammalian cells.\",\n      \"method\": \"Co-immunoprecipitation, dimerization-defective mutant analysis, aggresome formation assay, yeast and mammalian cell models\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal co-IP of ternary complex, dimerization mutant functional test, validated in two organisms; multiple orthogonal methods\",\n      \"pmids\": [\"23843611\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"14-3-3 proteins act as cytosolic adaptors mediating ER export of SAC1 (a PI4P phosphatase) in COPII-coated vesicles; 14-3-3 directly binds a minimal 7-aa sorting motif (RLSNTSP) in SAC1 and interacts with the COPII sorting subunit Sec24; recombinant 14-3-3 stimulates SAC1 packaging into COPII vesicles in a cell-free budding reaction.\",\n      \"method\": \"Cell-free COPII vesicle budding assay, recombinant protein, co-immunoprecipitation, homology modeling, sorting motif mutagenesis\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — cell-free reconstitution with recombinant protein, motif mutagenesis, and co-IP; multiple orthogonal methods in a single study\",\n      \"pmids\": [\"26056309\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"14-3-3 theta (YWHAQ) overexpression reduces LRRK2 kinase activity and reverses mutant LRRK2 (G2019S and R1441G)-induced neurite shortening in primary neurons; this protective effect requires direct physical binding of 14-3-3 theta to LRRK2; pan-14-3-3 inhibitor difopein conversely promotes LRRK2 kinase activity and exacerbates neurite shortening.\",\n      \"method\": \"Overexpression in BAC transgenic neurons, LRRK2 kinase activity assay, neurite length measurement, difopein inhibition, LRRK2 kinase inhibitor rescue\",\n      \"journal\": \"Human molecular genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — gain- and loss-of-function with binding-deficient controls, defined kinase activity readout and morphological phenotype, pharmacological rescue; single lab with multiple orthogonal methods\",\n      \"pmids\": [\"26546614\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Crystal structures of 14-3-3 bound to phosphopeptides from LRRK2 characterize the multivalent interaction interface; LRRK2 possesses multiple phosphorylation sites that each can bind 14-3-3, and many Parkinson's disease-relevant LRRK2 mutations impair 14-3-3 binding.\",\n      \"method\": \"X-ray crystallography, biochemical binding assays\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — crystal structure with biochemical validation; single lab\",\n      \"pmids\": [\"28202711\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"PKA-dependent phosphorylation of SIK1, SIK2, and SIK3 induces 14-3-3 binding and inhibits their catalytic activity; SIK1 and SIK3 have two functional PKA/14-3-3 sites while SIK2 has four; loss of a single site in SIK1/3 abolishes 14-3-3 association and cAMP responsiveness, demonstrating multi-site cooperativity in 14-3-3-mediated kinase inhibition.\",\n      \"method\": \"In vitro kinase assay, phosphorylation site mutagenesis, co-immunoprecipitation, cAMP stimulation\",\n      \"journal\": \"The FEBS journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — mutagenesis of multiple binding sites with defined kinase activity readout; cooperativity mechanism established; single lab with multiple orthogonal methods\",\n      \"pmids\": [\"29211348\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"BAP1 (BRCA1-associated protein 1) interacts with 14-3-3 protein; this interaction releases the pro-apoptotic protein Bax from 14-3-3, promoting apoptosis through the intrinsic pathway in neuroblastoma cells.\",\n      \"method\": \"Co-immunoprecipitation, apoptosis assay, Bax release assay, xenograft\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP with functional apoptosis readout and in vivo xenograft validation; single lab\",\n      \"pmids\": [\"29686263\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"14-3-3 binds to two pairs of phosphorylation sites in IRSp53 (established by phosphoproteomics and quantitative binding); each subunit of an IRSp53 homodimer independently binds one 14-3-3 dimer; a FRET-sensor assay shows that 14-3-3 binding induces opposite conformational changes in IRSp53 compared to activatory inputs (Cdc42, Eps8); 14-3-3 inhibits IRSp53 binding to membranes and its interaction with Cdc42.\",\n      \"method\": \"Phosphoproteomics, quantitative binding assay, X-ray crystallography, bicistronic expression of heterodimer, FRET-sensor assay, liposome binding assay\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure combined with FRET conformational readout, quantitative binding, and functional membrane/GTPase inhibition assays; multiple orthogonal methods in one study\",\n      \"pmids\": [\"30696821\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"PKA phosphorylates CaMKK2 at Ser100 and Ser511, which mediates recruitment of 14-3-3 adaptor proteins; 14-3-3 binding holds CaMKK2 in an inactivated state by preventing dephosphorylation of phospho-Ser495 (the Ca2+/CaM activation site); crystal structure of 14-3-3 zeta bound to a diphosphorylated CaMKK2 peptide reveals how canonical (Ser511) and non-canonical (Ser100) 14-3-3 consensus sites cooperate to bind 14-3-3.\",\n      \"method\": \"In vitro kinase assay, phosphosite mutagenesis, X-ray crystallography, co-immunoprecipitation\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure plus in vitro kinase assay and mutagenesis of multiple phosphosites; mechanism of inactivation directly demonstrated\",\n      \"pmids\": [\"32913128\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"14-3-3 epsilon was biochemically identified as an intracellular component of TNFR2 receptor complexes in chondrocytes in response to progranulin (PGRN); 14-3-3 epsilon is required for PGRN/TNFR2 signaling through ERK-dependent Elk-1 activation while suppressing NF-κB; chondrocyte-specific deletion of 14-3-3 epsilon abolishes PGRN's therapeutic effects against osteoarthritis in vivo.\",\n      \"method\": \"Biochemical copurification, proteomics, co-immunoprecipitation, transcription factor activity screen, conditional knockout mouse, OA disease model\",\n      \"journal\": \"Annals of the rheumatic diseases\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — proteomics-validated co-purification plus conditional KO with defined signaling and disease phenotype; single lab\",\n      \"pmids\": [\"34226187\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Phosphorylation of 14-3-3 zeta at Ser58 by PKC shifts the dimer-monomer equilibrium strongly toward monomers at physiological concentrations; phosphomimicking mutations (S58E/D) incompletely recapitulate this shift; Ser58 phosphorylation changes the subcellular localization of 14-3-3 zeta in HeLa and U251 cells.\",\n      \"method\": \"Site-specific phosphorylation, analytical ultracentrifugation/biophysics, thermal stability assay, fluorescence microscopy in human cancer cells\",\n      \"journal\": \"Frontiers in chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct phosphorylation confirmed with multiple biophysical methods and cell imaging; single lab, novel finding for the zeta isoform\",\n      \"pmids\": [\"35321476\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"YWHAQ (14-3-3 zeta/theta) functions as a phosphoserine/phosphothreonine-binding scaffold protein that, upon recognizing specific phosphorylated motifs on client proteins (including Raf-1, LRRK2, CaMKK2, SIKs, CDC25, TSC2, IRSp53, CFTR, and others), allosterically modulates client activity, subcellular localization, stability, or protein-protein interactions through mechanisms including conformational change, steric occlusion of targeting sequences, scaffolding of multi-protein complexes, and competing with retrograde/degradation machinery; it forms homo- and heterodimers whose integrity (regulated by Ser58 phosphorylation) is required for many of these functions, and it mediates essential anti-apoptotic signaling by sequestering pro-apoptotic proteins such as BAD.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"YWHAQ (14-3-3 theta/zeta) is a phosphoserine/phosphothreonine-binding adaptor that recognizes phosphorylated motifs on diverse clients and thereby controls their activity, localization, stability, and complex assembly across kinase signaling, vesicular transport, and cell-survival pathways [#0, #4, #18]. Sequence-specific recognition depends on an intact phosphopeptide-binding pocket, and many clients are engaged through cooperative multi-site binding in which loss of a single phosphosite abolishes association, as shown for SIK kinases, CaMKK2, LRRK2, and IRSp53 [#4, #15, #18, #14, #17]. Functionally, 14-3-3 binding can either activate or restrain clients: it stimulates Raf-1 to promote MAPK signaling [#0], holds CaMKK2 and SIK kinases in an inactivated state by occluding sites and blocking dephosphorylation [#18, #15], reduces LRRK2 kinase activity and rescues mutant LRRK2-induced neurite shortening [#13], and drives conformational change in IRSp53 that inhibits its membrane and Cdc42 binding [#17]. As a molecular adaptor it couples cargoes to trafficking machinery — recruiting misfolded protein cargo and BAG3 to dynein for aggresome delivery, and SAC1 to the COPII subunit Sec24 for ER export — and it modulates CFTR biogenesis by competing with retrograde ER retrieval [#11, #12, #10]. 14-3-3 function requires dimerization, and PKC phosphorylation of Ser58 shifts the equilibrium toward monomers and alters subcellular localization [#11, #20]. The protein mediates essential anti-apoptotic signaling: loss of 14-3-3/ligand interactions induces apoptosis and dilated cardiomyopathy in vivo, and displacement of pro-apoptotic Bax from 14-3-3 by BAP1 promotes intrinsic-pathway death [#5, #6, #16].\",\n  \"teleology\": [\n    {\n      \"year\": 1994,\n      \"claim\": \"Established that 14-3-3 is not merely a passive binder but a positive regulator of a signaling kinase, defining its role as an activator of Raf-1-dependent MAPK signaling.\",\n      \"evidence\": \"Yeast two-hybrid, Xenopus oocyte overexpression, and dominant-negative Raf-1 epistasis\",\n      \"pmids\": [\"7935795\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of Raf-1 activation not resolved\", \"Did not define the phospho-motif on Raf-1 recognized by 14-3-3\"]\n    },\n    {\n      \"year\": 1995,\n      \"claim\": \"Distinguished scaffolding from catalytic modulation by showing 14-3-3 binds CDC25 phosphatases without altering their activity, and mapped dimerization and membrane-binding determinants to the N-terminus while showing full-length protein is needed to inhibit PKC.\",\n      \"evidence\": \"Yeast two-hybrid, co-IP, in vitro phosphatase assay; N-terminal deletion mutagenesis, circular dichroism, PKC activity assay\",\n      \"pmids\": [\"7644510\", \"7837270\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Functional outcome of CDC25 binding in vivo not defined\", \"Single-lab biochemistry without independent confirmation\"]\n    },\n    {\n      \"year\": 1998,\n      \"claim\": \"Demonstrated that 14-3-3 acts as a scaffold for MEKK kinases without altering their catalytic activity, and that phosphopeptide-pocket binding is genetically required for participation in TOR/rapamycin signaling.\",\n      \"evidence\": \"Quantitative binding and co-IP for MEKK1/2/3; multicopy suppressor screen with pocket mutagenesis in yeast\",\n      \"pmids\": [\"9452471\", \"9822578\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct phospho-clients in the TOR pathway not identified\", \"Selectivity for MEKK1/2/3 over MEKK4 mechanism unexplained\"]\n    },\n    {\n      \"year\": 2000,\n      \"claim\": \"Showed in vivo that 14-3-3 differentially tunes MAPK cascades (promoting ERK, restraining JNK/p38) and is required to suppress apoptosis at the organ level.\",\n      \"evidence\": \"Dominant-negative 14-3-3 transfection and cardiac transgenic mouse with MAPK assays and histology\",\n      \"pmids\": [\"10654934\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Specific apoptotic clients responsible for the cardiac phenotype not pinpointed\", \"Isoform-specific contributions not separated\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Established that 14-3-3/ligand interactions are necessary for cell survival, using a generic interaction inhibitor to trigger apoptosis.\",\n      \"evidence\": \"Difopein inhibitor expression with cell death and drug-sensitization assays\",\n      \"pmids\": [\"11577088\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Does not identify which client displacement drives death\", \"Single-lab loss-of-function\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Linked 14-3-3 to growth control by showing phospho-dependent binding to TSC2, providing a node for Akt-dependent regulation.\",\n      \"evidence\": \"Co-IP and Ser1210 phospho-site mutagenesis\",\n      \"pmids\": [\"12364343\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Inhibition of TSC2 function inferred, not reconstituted\", \"Effect on downstream mTOR output not measured here\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Defined a mechanism by which 14-3-3 stabilizes a client by blocking its ubiquitination and nuclear export, illustrated by p53.\",\n      \"evidence\": \"Co-IP, ubiquitination assay, nuclear export assay, transcriptional reporter\",\n      \"pmids\": [\"14517281\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Demonstrated for the sigma isoform; YWHAQ-specific contribution not isolated\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Showed 14-3-3 controls nucleo-cytoplasmic trafficking of a signaling effector, coupling Akt phosphorylation of Chibby to beta-catenin nuclear export.\",\n      \"evidence\": \"Co-IP, phosphorylation assay, nuclear export assay\",\n      \"pmids\": [\"19158508\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct transport mechanism of the 14-3-3/Cby/beta-catenin complex not resolved\", \"Single-lab\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Defined 14-3-3 as a regulator of membrane protein biogenesis, increasing CFTR maturation by competing with COPI-mediated ER retrieval.\",\n      \"evidence\": \"Co-IP, NMR mapping, pulse-chase, siRNA, surface biotinylation, anion efflux\",\n      \"pmids\": [\"22278744\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Isoform redundancy at the CFTR R-region not dissected\", \"Quantitative contribution of synthesis vs degradation effects not separated\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Established 14-3-3 as a dimerization-dependent adaptor linking chaperone-bound misfolded cargo to dynein for aggresome transport.\",\n      \"evidence\": \"Reciprocal co-IP of a DIC/14-3-3/BAG3 ternary complex, dimerization mutants, aggresome assays in yeast and mammalian cells\",\n      \"pmids\": [\"23843611\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Phospho-recognition basis of cargo selection not defined\", \"Stoichiometry of the dynein-cargo bridge unresolved\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Demonstrated by reconstitution that 14-3-3 directly drives COPII packaging of cargo (SAC1) via a minimal sorting motif and Sec24 interaction.\",\n      \"evidence\": \"Cell-free COPII budding with recombinant protein, sorting-motif mutagenesis, co-IP; plus LRRK2 protective gain/loss-of-function in neurons\",\n      \"pmids\": [\"26056309\", \"26546614\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Generality of 14-3-3-dependent COPII sorting beyond SAC1 untested\", \"LRRK2 binding sites not yet mapped structurally at this stage\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Provided the structural basis for multivalent 14-3-3/LRRK2 recognition and linked Parkinson's mutations to impaired 14-3-3 binding.\",\n      \"evidence\": \"X-ray crystallography of 14-3-3/LRRK2 phosphopeptides with binding assays\",\n      \"pmids\": [\"28202711\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional consequence of each individual site in cells not quantified\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Established cooperative multi-site phospho-recognition as the mechanism by which 14-3-3 inhibits kinase activity (SIK1/2/3) and revealed how 14-3-3 displacement releases pro-apoptotic Bax.\",\n      \"evidence\": \"In vitro kinase assays with multi-site mutagenesis and cAMP stimulation; co-IP/apoptosis/Bax-release assays and xenograft for BAP1\",\n      \"pmids\": [\"29211348\", \"29686263\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether 14-3-3 sterically occludes the SIK catalytic site or blocks dephosphorylation not fully separated\", \"BAP1 study is single-lab Medium-confidence\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Showed that 14-3-3 binding allosterically reshapes a client conformation, inhibiting IRSp53 membrane association and Cdc42 binding in opposition to activating inputs.\",\n      \"evidence\": \"Phosphoproteomics, crystallography, bicistronic heterodimer expression, FRET-sensor and liposome assays\",\n      \"pmids\": [\"30696821\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo cytoskeletal consequence of IRSp53 conformational switching not addressed\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Defined how dual PKA-driven canonical and non-canonical sites cooperate to recruit 14-3-3 and lock CaMKK2 inactive by protecting its activation-site phosphorylation from phosphatases.\",\n      \"evidence\": \"In vitro kinase assay, phosphosite mutagenesis, co-IP, crystal structure of 14-3-3 zeta with a diphospho-CaMKK2 peptide\",\n      \"pmids\": [\"32913128\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Identity of the phosphatase shielded from phospho-Ser495 not established\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Placed 14-3-3 inside a transmembrane receptor signaling complex (TNFR2) required for progranulin-driven ERK/Elk-1 output and disease protection.\",\n      \"evidence\": \"Copurification/proteomics, co-IP, transcription-factor screen, chondrocyte conditional knockout, OA model\",\n      \"pmids\": [\"34226187\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct phospho-motif engaged within TNFR2 complex not mapped\", \"Demonstrated for the epsilon isoform\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Established a regulatory switch for 14-3-3 itself: PKC phosphorylation of Ser58 drives monomerization and relocalization, controlling adaptor availability.\",\n      \"evidence\": \"Site-specific phosphorylation with analytical ultracentrifugation, thermal stability, and microscopy in human cancer cells\",\n      \"pmids\": [\"35321476\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Functional consequence of monomerization for specific clients not measured\", \"Phosphomimetics incompletely recapitulate the effect\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"It remains unresolved how isoform-specific (YWHAQ vs other 14-3-3 paralogs) client engagement and the Ser58 dimer-monomer switch are integrated to set context-dependent activating versus inhibitory outcomes on shared clients.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No systematic isoform-resolved client map\", \"Cellular triggers of Ser58 phosphorylation in physiological settings undefined\", \"How a single adaptor selects activation vs inhibition of a given client not mechanistically unified\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [11, 12, 18]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [0, 13, 15, 17, 18]},\n      {\"term_id\": \"GO:0140313\", \"supporting_discovery_ids\": [16, 6]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [12, 20]},\n      {\"term_id\": \"GO:0005783\", \"supporting_discovery_ids\": [10, 12]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0, 5, 18, 15]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [5, 6, 16]},\n      {\"term_id\": \"R-HSA-5653656\", \"supporting_discovery_ids\": [11, 12, 10]}\n    ],\n    \"complexes\": [\"TNFR2 receptor complex\", \"14-3-3/dynein-DIC/BAG3 transport complex\", \"14-3-3/Sec24 COPII sorting complex\"],\n    \"partners\": [\"RAF1\", \"LRRK2\", \"CAMKK2\", \"TSC2\", \"CFTR\", \"SAC1\", \"BAG3\", \"BAP1\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":5,"faith_total":5,"faith_pct":100.0}}