{"gene":"RPS6KA6","run_date":"2026-06-10T07:46:27","timeline":{"discoveries":[{"year":2005,"finding":"RSK4 is a predominantly cytosolic protein that is constitutively (maximally) activated under serum-starved conditions in all cell types analyzed, unlike RSK1-3 which require growth factor stimulation. Constitutive activation results from constitutive phosphorylation of Ser232, Ser372, and Ser389. Low basal ERK activity in serum-starved cells is sufficient for ~50% of constitutive RSK4 activity. PDK1 gene deletion in mouse embryonic stem cells suggested PDK1 is not required for phosphorylation of the key activation-loop site Ser232, unlike other RSK family members.","method":"In vitro kinase assays, phospho-specific antibodies, subcellular fractionation, PDK1-knockout mouse embryonic stem cells, immunoblotting","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — direct biochemical characterization with mutagenesis-equivalent site mapping, knockout cells, and multiple orthogonal methods in a single rigorous study","pmids":["15632195"],"is_preprint":false},{"year":2004,"finding":"Rsk4 functions as an inhibitor of fibroblast growth factor-RAS-ERK (RTK) signaling. RTK inhibition is specific to RSK4 among RSK family members and depends on a region of RSK4 that is divergent from other RSK members. Rsk4 inhibits transcriptional activation of specific RTK signaling targets as well as ERK activation. Rsk4 expression in extraembryonic tissue is inversely correlated with activated ERK1/2 levels.","method":"Overexpression and domain-deletion experiments in cell lines, transcriptional reporter assays, ERK activation assays, in situ hybridization/immunostaining for expression vs. pERK correlation","journal":"Molecular and cellular biology","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — multiple functional assays in one lab, domain specificity established, but no in vitro reconstitution","pmids":["15121846"],"is_preprint":false},{"year":2009,"finding":"RSK4 overexpression induces cell cycle arrest and senescence features in normal fibroblasts (IMR90) and malignant colon carcinoma cells (HCT116). RSK4 mRNA levels increase during both replicative and stress-induced senescence. RSK4-induced senescence is mediated through p21, but is independent of p16, p38 MAPKs, and reactive oxygen species. Cells with RSK4 shRNA knockdown partially escape senescence and show increased resistance to cisplatin. E1A expression or Rb siRNA confers resistance to RSK4-mediated senescence.","method":"RSK4 cDNA overexpression and shRNA knockdown in IMR90 and HCT116 cells; senescence assays; pharmacological inhibitors of p16, p38, ROS; E1A overexpression; Rb siRNA epistasis","journal":"Clinical cancer research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — epistasis experiments with multiple pathway components, loss- and gain-of-function in multiple cell lines, single lab","pmids":["19584160"],"is_preprint":false},{"year":2011,"finding":"RSK4 inhibition via shRNA allows normal human fibroblasts to bypass stress-induced senescence (oxidative stress, DNA damage) and oncogene-induced senescence (KRAS-V12, BRAF-E600 overexpression). RSK4-induced senescence is mediated through p21 but is independent of p16, p38 MAPKs, and reactive oxygen species induction.","method":"shRNA knockdown of RSK4 in normal human fibroblasts; oncogene overexpression; pharmacological inhibitors; senescence assays; p21 inhibition epistasis","journal":"Carcinogenesis","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — epistasis with multiple pathway inhibitors, replicated in multiple stress contexts, single lab","pmids":["21239520"],"is_preprint":false},{"year":2004,"finding":"RSK4 (and PAK5) are direct transcriptional targets of HNF4alpha. HNF4alpha binds to novel sites in the RSK4 promoter as confirmed by chromatin immunoprecipitation (ChIP) cloning and electrophoretic mobility shift assays (EMSA). RSK4 expression is repressed in streptozotocin-induced diabetic rat kidney and brain coincident with HNF4alpha repression.","method":"ChIP cloning, EMSA with oligonucleotides harboring HNF4alpha binding sites, quantitative RT-PCR in diabetic rat tissue","journal":"Molecular pharmacology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — two orthogonal methods (ChIP + EMSA) establish direct binding, single lab","pmids":["15615695"],"is_preprint":false},{"year":2021,"finding":"RSK4 promotes drug resistance and metastasis in lung and bladder cancer. CRISPR and RNAi silencing of RSK4 sensitized tumor cells to chemotherapy and hindered metastasis in vitro and in vivo. X-ray crystallography identified an allosteric binding site on RSK4 through which floxacin antibiotics (e.g., trovafloxacin) block RSK4 kinase activation by mimicking a kinase autoinhibitory mechanism involving RSK4's hydrophobic motif. Deuterium exchange and Markov transient analyses confirmed the allosteric mechanism.","method":"CRISPR knockdown, RNAi, X-ray crystallography, hydrogen-deuterium exchange, Markov transient analysis, xenograft models, genetically engineered mouse models, bladder tumor explants","journal":"Science translational medicine","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure with allosteric mechanism validated by HDX and functional assays; multiple model systems","pmids":["34261798"],"is_preprint":false},{"year":2016,"finding":"Tandem affinity purification coupled with nano LC-MS/MS identified 82 RSK4-associated proteins and 137 RSK4m (alternative splice variant)-associated proteins in MDA-MB-231 cells. Bioinformatics analysis indicated enrichment in cell senescence pathways. RSK4 and RSK4m have distinct interactomes, with RSK4m associated with more molecular functions.","method":"SF-TAP (tandem affinity purification with STREP II + Flag tags) coupled to nano LC-MS/MS, bioinformatics (GO, IPA)","journal":"International journal of biological macromolecules","confidence":"Low","confidence_rationale":"Tier 3 / Weak — interactome screen without functional validation of individual interactions; single lab, no reciprocal confirmation","pmids":["27986634"],"is_preprint":false},{"year":2022,"finding":"FOXP2 transcription factor directly binds to the RSK4 (RPS6KA6) promoter and transcriptionally activates its expression. Knockdown of RSK4 reverses the anti-proliferative and pro-apoptotic effects of FOXP2 overexpression in thyroid cancer cells, placing RSK4 downstream of FOXP2. FOXP2/RSK4 regulation is associated with the PI3K/AKT pathway.","method":"Dual-luciferase reporter assay, chromatin immunoprecipitation (ChIP), JASPAR binding site prediction, rescue/knockdown experiments in TPC-1 cells","journal":"Experimental and therapeutic medicine","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct promoter binding confirmed by ChIP and luciferase reporter; epistasis by rescue experiment; single lab","pmids":["35607372"],"is_preprint":false},{"year":2019,"finding":"17β-estradiol (E2) reduces RSK4 expression in ER+ breast cancer cells by promoting DNMT3B-mediated methylation of the RSK4 promoter, thereby decreasing RSK4 transcriptional activity. E2-induced RSK4 silencing is associated with increased cell proliferation, clone formation, and invasion.","method":"Bisulfite genomic sequencing for methylation, luciferase reporter assay for transcriptional activity, Western blotting for DNMT1/3A/3B, MTT/Transwell/flow cytometry functional assays, RT-PCR","journal":"OncoTargets and therapy","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — methylation mechanism identified with specific DNMT isoform, luciferase confirms transcriptional regulation, single lab","pmids":["31413588"],"is_preprint":false},{"year":2023,"finding":"TRAF4 negatively regulates RSK4; TRAF4 knockdown leads to increased RSK4 expression in ovarian cancer cells. USP7 deubiquitinates and stabilizes TRAF4 via co-immunoprecipitation-confirmed interaction, and TRAF4 destabilization leads to RSK4 upregulation and suppression of the PI3K/AKT pathway. This USP7/TRAF4/RSK4/PI3K/AKT axis was confirmed in a xenograft mouse model.","method":"Co-immunoprecipitation for TRAF4 ubiquitination, siRNA knockdown of USP7 and TRAF4, RSK4 overexpression, Western blotting, xenograft mouse model","journal":"Journal of cancer research and therapeutics","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — Co-IP for ubiquitination, multiple knockdowns, in vivo confirmation; single lab","pmids":["37006049"],"is_preprint":false},{"year":2023,"finding":"RSK4 promotes macrophage recruitment and M2 polarization in esophageal squamous cell carcinoma (ESCC) by enhancing secretion of soluble ICAM-1 (sICAM-1) via direct and indirect STAT3 phosphorylation. RSK4-conditioned macrophages then secrete CCL22 to enhance tumor proliferation, migration, and invasion.","method":"In vitro co-culture experiments, ELISA for sICAM-1 and CCL22, phospho-STAT3 detection, RSK4 overexpression/knockdown in ESCC cells, xenograft mouse model, IHC in human ESCC tissues","journal":"Biochimica et biophysica acta. Molecular basis of disease","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — mechanistic pathway (RSK4→STAT3→sICAM-1→macrophage M2→CCL22) established with multiple assays; single lab","pmids":["38142759"],"is_preprint":false},{"year":2026,"finding":"RSK4 stability is regulated by antagonistic post-translational modifications: GSK3β phosphorylates RSK4 at Thr402/Ser406, promoting its proteasomal degradation via the FBXW7-dependent ubiquitin-proteasome pathway. OGT-mediated O-GlcNAcylation of RSK4 at Thr405 antagonizes GSK3β phosphorylation, thereby stabilizing RSK4 and enhancing cancer stem cell properties and radioresistance in ESCC. Inhibition of O-GlcNAcylation with OSMI-4 destabilizes RSK4 and sensitizes ESCC to radiotherapy.","method":"Site-directed mutagenesis at Thr402/Ser406/Thr405, co-immunoprecipitation with GSK3β and FBXW7, O-GlcNAc detection, proteasome inhibitor experiments, OSMI-4 treatment, patient-derived xenograft and organoid models","journal":"International journal of biological sciences","confidence":"High","confidence_rationale":"Tier 1 / Moderate — site-specific mutagenesis identifies exact PTM residues, writer (OGT, GSK3β) and reader/eraser (FBXW7) identified, multiple orthogonal methods, in vivo validation; single lab","pmids":["41800247"],"is_preprint":false},{"year":2024,"finding":"Deletion of exon 7 of Rps6ka6 (creating a severely truncated RSK4 protein) in H2Kb myogenic cells did not affect myoblast proliferation or migration, but significantly reduced myoblast differentiation indices (myosin expression, myosin-expressing cell size, and fusion index). Genetic analysis in CFW mice linked the Rps6ka6 locus to muscle fiber number in fast-twitch extensor digitorum longus muscle.","method":"CRISPR-Cas9 exon deletion in myogenic cells, myoblast proliferation/migration/differentiation assays, myosin immunostaining, fusion index quantification, CFW mouse genetic analysis with histology","journal":"G3 (Bethesda, Md.)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — CRISPR loss-of-function with specific differentiation phenotype; in vivo genetic association; single lab","pmids":["38577978"],"is_preprint":false},{"year":2018,"finding":"TRAF4 knockdown in breast cancer (MDA-MB-231) xenografts led to significant upregulation of RSK4 mRNA and protein, and coincident reduction of P-AKT, P-NF-κB, TGF-β1, TNF-α, MMP2, and MMP9. This places RSK4 as negatively regulated by TRAF4, upstream of the AKT/NF-κB pathway.","method":"Lentiviral TRAF4-shRNA in MDA-MB-231 cells, xenograft nude mouse model, IHC, Western blot, fluorescence quantitative RT-PCR","journal":"Biochemical and biophysical research communications","confidence":"Low","confidence_rationale":"Tier 3 / Weak — indirect relationship inferred from knockdown experiments without direct binding assay; single lab, in vivo only","pmids":["29684350"],"is_preprint":false},{"year":2020,"finding":"RSK4 overexpression in ccRCC cell lines enhances invasive and migratory abilities through upregulation of CD44 and MMP-9 expression; conversely, RSK4 knockdown reduces these abilities and CD44/MMP-9 expression.","method":"Ectopic RSK4 overexpression and knockdown in ccRCC cell lines, invasion/migration assays, IHC in patient samples, Western blot","journal":"Diagnostic pathology","confidence":"Low","confidence_rationale":"Tier 3 / Weak — functional assays link RSK4 to CD44/MMP-9, but mechanistic pathway is not delineated; single lab","pmids":["32209138"],"is_preprint":false},{"year":2020,"finding":"miR-548d-3p directly targets RSK4 and negatively regulates its expression. Dual-luciferase reporter assay confirmed binding of miR-548d-3p to the RSK4 3'UTR at a predicted interaction site (mutation of this site abolished the effect). miR-548d-3p overexpression phenocopies RSK4 knockdown in gastric cancer cells: increased proliferation, migration, invasion, and decreased apoptosis.","method":"Dual-luciferase reporter assay with wild-type and mutant RSK4 3'UTR, RT-PCR, Western blot, functional cell assays, nude mouse tumorigenesis","journal":"Cancer management and research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct miRNA-target interaction confirmed by mutagenesis in reporter assay; single lab","pmids":["33380838"],"is_preprint":false},{"year":2023,"finding":"RSK4 confers paclitaxel resistance in ovarian cancer cells. RSK4 elevation is associated with accelerated phosphorylation of the downstream translation factor eIF4B. RSK inhibitor (BI-D1870) co-treatment restored paclitaxel sensitivity. External RSK4 expression increased cell viability under paclitaxel treatment.","method":"DNA microarray, qRT-PCR, Western blotting, RSK inhibitor pharmacological treatment, RSK4 overexpression, cell viability assays","journal":"Biochemical and biophysical research communications","confidence":"Low","confidence_rationale":"Tier 3 / Weak — eIF4B phosphorylation identified as downstream event but direct RSK4→eIF4B relationship not fully established by mutagenesis; single lab","pmids":["37660640"],"is_preprint":false},{"year":2025,"finding":"RSK4 promotes metastasis and angiogenesis in clear cell renal cell carcinoma (ccRCC) by activating RUNX1-mediated transcription of EPHA2, leading to enhanced VEGF secretion and tube formation. Luciferase reporter and mutagenesis of the RUNX1 binding site in the EPHA2 promoter confirmed this regulatory axis.","method":"Transcriptomic and proteomic analysis, luciferase reporter assay with RUNX1/EPHA2 promoter binding site mutation, VEGF ELISA, tube formation assay, in vivo experiments","journal":"Cancer biology & therapy","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — luciferase reporter with mutagenesis confirms RUNX1→EPHA2 link downstream of RSK4; single lab","pmids":["39797421"],"is_preprint":false}],"current_model":"RSK4 (RPS6KA6) is a constitutively active, predominantly cytosolic serine/threonine kinase that—unlike RSK1-3—does not require growth factor stimulation, with its activation loop (Ser232) phosphorylated independently of PDK1; it inhibits the RAS-ERK signaling axis, promotes cellular senescence via p21 (but not p16 or p38), and its stability is regulated by antagonistic PTMs whereby GSK3β-mediated phosphorylation at Thr402/Ser406 targets it for FBXW7-dependent proteasomal degradation while OGT-mediated O-GlcNAcylation at Thr405 stabilizes it; floxacin antibiotics inhibit RSK4 through an allosteric site that mimics its hydrophobic-motif autoinhibitory mechanism; RSK4 expression is transcriptionally activated by FOXP2 and repressed by estrogen-driven DNMT3B-mediated promoter methylation, and it is negatively regulated post-transcriptionally by miR-548d-3p and by the TRAF4/USP7 ubiquitin axis, with context-dependent roles in drug resistance, metastasis, angiogenesis (via RUNX1/EPHA2/VEGF), immune modulation (via STAT3/sICAM-1/CCL22), and myoblast differentiation."},"narrative":{"mechanistic_narrative":"RSK4 (RPS6KA6) is a predominantly cytosolic serine/threonine kinase that, distinct from other RSK family members, is constitutively activated in serum-starved cells through constitutive phosphorylation of Ser232, Ser372, and Ser389 and does not require PDK1 for activation-loop phosphorylation [PMID:15632195]. Functionally, it acts as a negative regulator of growth signaling: it inhibits FGF-RAS-ERK (RTK) signaling through a region divergent from other RSK proteins [PMID:15121846] and drives cellular senescence in normal and malignant cells via a p21-dependent, p16/p38/ROS-independent program that requires intact Rb [PMID:19584160, PMID:21239520]. RSK4 abundance is tightly governed by antagonistic post-translational modifications, in which GSK3β phosphorylation at Thr402/Ser406 directs FBXW7-dependent proteasomal degradation while OGT-mediated O-GlcNAcylation at Thr405 stabilizes the protein and promotes cancer stem cell properties and radioresistance [PMID:41800247]. Its expression is further controlled transcriptionally by activation through FOXP2 and HNF4alpha [PMID:15615695, PMID:35607372], by estrogen-driven DNMT3B-mediated promoter methylation [PMID:31413588], and post-transcriptionally by miR-548d-3p and the USP7/TRAF4 axis [PMID:37006049, PMID:33380838]. RSK4 has context-dependent oncogenic and tumor-suppressive outputs across cancers, contributing to chemoresistance and metastasis, angiogenesis via a RUNX1/EPHA2/VEGF axis, and immune modulation through STAT3/sICAM-1/CCL22 signaling [PMID:34261798, PMID:38142759, PMID:39797421]; its kinase activity can be blocked by floxacin antibiotics binding an allosteric site that mimics the hydrophobic-motif autoinhibitory mechanism [PMID:34261798]. RSK4 also supports myoblast differentiation [PMID:38577978].","teleology":[{"year":2004,"claim":"Established that RSK4 is not merely another RSK paralog but a specific antagonist of RTK signaling, defining its first functional role.","evidence":"Overexpression and domain-deletion experiments with transcriptional reporters and ERK activation assays; expression vs pERK correlation in extraembryonic tissue","pmids":["15121846"],"confidence":"Medium","gaps":["No in vitro reconstitution of the inhibitory mechanism","Direct molecular target of the divergent inhibitory region not identified"]},{"year":2004,"claim":"Identified RSK4 as a direct transcriptional target, beginning to explain how its expression is set in specific tissues.","evidence":"ChIP cloning and EMSA mapping HNF4alpha binding sites in the RSK4 promoter; qRT-PCR in diabetic rat tissue","pmids":["15615695"],"confidence":"Medium","gaps":["Functional consequence of HNF4alpha-driven expression not tested","Restricted to rodent tissue context"]},{"year":2005,"claim":"Answered how RSK4 activation differs from RSK1-3 by showing it is constitutively phosphorylated and PDK1-independent, explaining its growth-factor-independent activity.","evidence":"In vitro kinase assays, phospho-specific antibodies, subcellular fractionation, PDK1-knockout mouse ES cells","pmids":["15632195"],"confidence":"High","gaps":["Kinase responsible for constitutive Ser232 phosphorylation not identified","Physiological substrates downstream of constitutive activity unresolved"]},{"year":2011,"claim":"Resolved the senescence mechanism by showing RSK4 enforces stress- and oncogene-induced senescence through p21, defining a tumor-suppressive role.","evidence":"shRNA knockdown in fibroblasts with oncogene overexpression, pathway inhibitors, and p21/Rb epistasis (across 2009 and 2011 studies)","pmids":["19584160","21239520"],"confidence":"Medium","gaps":["Direct link between RSK4 kinase activity and p21 induction not established","Single-lab findings"]},{"year":2016,"claim":"Began cataloging RSK4 protein partners and distinguishing the splice variant interactome to map its molecular context.","evidence":"SF-TAP tandem affinity purification with nano LC-MS/MS in MDA-MB-231 cells; bioinformatics","pmids":["27986634"],"confidence":"Low","gaps":["Interactome screen without functional validation of individual interactions","No reciprocal confirmation"]},{"year":2021,"claim":"Provided a structural mechanism of inhibition and a context-specific oncogenic role, showing floxacins block RSK4 via an allosteric site mimicking hydrophobic-motif autoinhibition.","evidence":"X-ray crystallography, HDX, Markov transient analysis, CRISPR/RNAi silencing, xenograft and bladder explant models","pmids":["34261798"],"confidence":"High","gaps":["Endogenous regulator engaging the allosteric site unknown","Reconciliation with tumor-suppressive roles in other contexts not addressed"]},{"year":2023,"claim":"Extended RSK4 into tumor-immune crosstalk, defining a STAT3-driven secretory program that reprograms macrophages.","evidence":"Co-culture, ELISA for sICAM-1/CCL22, phospho-STAT3 detection, RSK4 gain/loss in ESCC cells, xenografts, IHC","pmids":["38142759"],"confidence":"Medium","gaps":["Whether RSK4 phosphorylates STAT3 directly not fully resolved","Single-lab finding"]},{"year":2025,"claim":"Defined a transcriptional output of RSK4 driving angiogenesis through RUNX1-mediated EPHA2 and VEGF.","evidence":"Transcriptomic/proteomic analysis, luciferase reporter with RUNX1/EPHA2 binding-site mutagenesis, VEGF ELISA, tube formation, in vivo experiments","pmids":["39797421"],"confidence":"Medium","gaps":["Direct kinase substrate connecting RSK4 to RUNX1 activation not identified","Single-lab finding"]},{"year":2026,"claim":"Established how RSK4 protein levels are set by antagonistic PTMs, identifying GSK3β/FBXW7 degradation and OGT O-GlcNAcylation stabilization at adjacent residues.","evidence":"Site-directed mutagenesis at Thr402/Ser406/Thr405, co-IP with GSK3β and FBXW7, O-GlcNAc detection, proteasome inhibition, OSMI-4, PDX and organoid models","pmids":["41800247"],"confidence":"High","gaps":["Upstream signals controlling the GSK3β/OGT balance not defined","Single-lab finding"]},{"year":null,"claim":"How RSK4's constitutive kinase activity is mechanistically reconciled with its opposing tumor-suppressive (senescence, RTK inhibition) and oncogenic (metastasis, immune modulation, angiogenesis) outputs across tissues remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No unifying model linking direct substrates to context-dependent phenotypes","Bona fide physiological substrates of RSK4 kinase activity largely uncharacterized"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,5]},{"term_id":"GO:0016740","term_label":"transferase activity","supporting_discovery_ids":[0]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[0]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[1,7,9]},{"term_id":"R-HSA-8953897","term_label":"Cellular responses to stimuli","supporting_discovery_ids":[2,3]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[11]}],"complexes":[],"partners":["GSK3B","FBXW7","OGT","TRAF4"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q9UK32","full_name":"Ribosomal protein S6 kinase alpha-6","aliases":["90 kDa ribosomal protein S6 kinase 6","p90-RSK 6","p90RSK6","Ribosomal S6 kinase 4","RSK-4","pp90RSK4"],"length_aa":745,"mass_kda":83.9,"function":"Constitutively active serine/threonine-protein kinase that exhibits growth-factor-independent kinase activity and that may participate in p53/TP53-dependent cell growth arrest signaling and play an inhibitory role during embryogenesis","subcellular_location":"Cytoplasm, cytosol; Nucleus","url":"https://www.uniprot.org/uniprotkb/Q9UK32/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/RPS6KA6","classification":"Not Classified","n_dependent_lines":0,"n_total_lines":1208,"dependency_fraction":0.0},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/RPS6KA6","total_profiled":1310},"omim":[{"mim_id":"300303","title":"RIBOSOMAL PROTEIN S6 KINASE A6; RPS6KA6","url":"https://www.omim.org/entry/300303"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Nucleoplasm","reliability":"Supported"},{"location":"Mitochondria","reliability":"Supported"},{"location":"Nucleoli","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in many","driving_tissues":[],"url":"https://www.proteinatlas.org/search/RPS6KA6"},"hgnc":{"alias_symbol":["RSK4"],"prev_symbol":[]},"alphafold":{"accession":"Q9UK32","domains":[{"cath_id":"3.30.200.20","chopping":"60-156_357-399","consensus_level":"medium","plddt":76.6521,"start":60,"end":399},{"cath_id":"1.10.510.10","chopping":"158-350","consensus_level":"medium","plddt":86.9717,"start":158,"end":350},{"cath_id":"3.30.200.20","chopping":"413-497","consensus_level":"high","plddt":84.1719,"start":413,"end":497},{"cath_id":"1.10.510.10","chopping":"503-725","consensus_level":"high","plddt":77.3276,"start":503,"end":725}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9UK32","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9UK32-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9UK32-F1-predicted_aligned_error_v6.png","plddt_mean":73.94},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=RPS6KA6","jax_strain_url":"https://www.jax.org/strain/search?query=RPS6KA6"},"sequence":{"accession":"Q9UK32","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9UK32.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9UK32/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9UK32"}},"corpus_meta":[{"pmid":"10519135","id":"PMC_10519135","title":"Molecular and functional characterization of the small Ca(2+)-regulated K+ channel (rSK4) of colonic crypts.","date":"1999","source":"Pflugers Archiv : European journal of physiology","url":"https://pubmed.ncbi.nlm.nih.gov/10519135","citation_count":115,"is_preprint":false},{"pmid":"15632195","id":"PMC_15632195","title":"Functional characterization of human RSK4, a new 90-kDa ribosomal S6 kinase, reveals constitutive activation in most cell types.","date":"2005","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/15632195","citation_count":101,"is_preprint":false},{"pmid":"10644430","id":"PMC_10644430","title":"A novel ribosomal S6-kinase (RSK4; RPS6KA6) is commonly deleted in patients with complex X-linked mental retardation.","date":"1999","source":"Genomics","url":"https://pubmed.ncbi.nlm.nih.gov/10644430","citation_count":96,"is_preprint":false},{"pmid":"17314274","id":"PMC_17314274","title":"Aberrant expression of X-linked genes RbAp46, Rsk4, and Cldn2 in breast cancer.","date":"2007","source":"Molecular cancer research : MCR","url":"https://pubmed.ncbi.nlm.nih.gov/17314274","citation_count":66,"is_preprint":false},{"pmid":"15121846","id":"PMC_15121846","title":"Characterization of mouse Rsk4 as an inhibitor of fibroblast growth factor-RAS-extracellular signal-regulated kinase signaling.","date":"2004","source":"Molecular and cellular biology","url":"https://pubmed.ncbi.nlm.nih.gov/15121846","citation_count":54,"is_preprint":false},{"pmid":"22020623","id":"PMC_22020623","title":"PRKX, TTBK2 and RSK4 expression causes Sunitinib resistance in kidney carcinoma- and melanoma-cell lines.","date":"2012","source":"International journal of cancer","url":"https://pubmed.ncbi.nlm.nih.gov/22020623","citation_count":52,"is_preprint":false},{"pmid":"19584160","id":"PMC_19584160","title":"Regulation of replicative and stress-induced senescence by RSK4, which is down-regulated in human tumors.","date":"2009","source":"Clinical cancer research : an 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and metastasis of human breast adenocarcinoma cells.","date":"2015","source":"Oncology reports","url":"https://pubmed.ncbi.nlm.nih.gov/26397146","citation_count":20,"is_preprint":false},{"pmid":"26732474","id":"PMC_26732474","title":"Ribosomal S6 kinase 4 (RSK4) expression in ovarian tumors and its regulation by antineoplastic drugs in ovarian cancer cell lines.","date":"2016","source":"Medical oncology (Northwood, London, England)","url":"https://pubmed.ncbi.nlm.nih.gov/26732474","citation_count":20,"is_preprint":false},{"pmid":"30579335","id":"PMC_30579335","title":"Effect of RSK4 on biological characteristics of colorectal cancer.","date":"2018","source":"World journal of surgical oncology","url":"https://pubmed.ncbi.nlm.nih.gov/30579335","citation_count":19,"is_preprint":false},{"pmid":"24338215","id":"PMC_24338215","title":"Frequent epigenetic inactivation of RSK4 by promoter methylation in cancerous and non-cancerous tissues of breast cancer.","date":"2013","source":"Medical 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Research","url":"https://pubmed.ncbi.nlm.nih.gov/21384559","citation_count":14,"is_preprint":false},{"pmid":"38142759","id":"PMC_38142759","title":"RSK4 promotes the macrophage recruitment and M2 polarization in esophageal squamous cell carcinoma.","date":"2023","source":"Biochimica et biophysica acta. 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with ribosomal S6 protein kinase 4 (RSK4) and its variant protein (RSK4m).","date":"2016","source":"International journal of biological macromolecules","url":"https://pubmed.ncbi.nlm.nih.gov/27986634","citation_count":8,"is_preprint":false},{"pmid":"34496560","id":"PMC_34496560","title":"Discovery, Optimization, and Structure-Activity Relationship Study of Novel and Potent RSK4 Inhibitors as Promising Agents for the Treatment of Esophageal Squamous Cell Carcinoma.","date":"2021","source":"Journal of medicinal chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/34496560","citation_count":7,"is_preprint":false},{"pmid":"32319586","id":"PMC_32319586","title":"Hypermethylation of the RSK4 promoter associated with BRAF V600E promotes papillary thyroid carcinoma.","date":"2020","source":"International journal of oncology","url":"https://pubmed.ncbi.nlm.nih.gov/32319586","citation_count":7,"is_preprint":false},{"pmid":"33380838","id":"PMC_33380838","title":"MiR-548d-3p Promotes Gastric Cancer by Targeting RSK4.","date":"2020","source":"Cancer management and research","url":"https://pubmed.ncbi.nlm.nih.gov/33380838","citation_count":6,"is_preprint":false},{"pmid":"39797421","id":"PMC_39797421","title":"RSK4 promotes the metastasis of clear cell renal cell carcinoma by activating RUNX1-mediated angiogenesis.","date":"2025","source":"Cancer biology & therapy","url":"https://pubmed.ncbi.nlm.nih.gov/39797421","citation_count":5,"is_preprint":false},{"pmid":"37660640","id":"PMC_37660640","title":"RSK4 confers paclitaxel resistance to ovarian cancer cells, which is resensitized by its inhibitor BI-D1870.","date":"2023","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/37660640","citation_count":4,"is_preprint":false},{"pmid":"30143490","id":"PMC_30143490","title":"A Pilot Study of RSK4 Expression in Patients with Human Non-Small Cell Lung Carcinoma.","date":"2018","source":"Annals of clinical and laboratory science","url":"https://pubmed.ncbi.nlm.nih.gov/30143490","citation_count":4,"is_preprint":false},{"pmid":"37402066","id":"PMC_37402066","title":"Ribosomal S6 kinase 4 (RSK4) tumor suppressor gene promoter methylation status in ovarian cancer.","date":"2023","source":"Molecular biology reports","url":"https://pubmed.ncbi.nlm.nih.gov/37402066","citation_count":2,"is_preprint":false},{"pmid":"36031631","id":"PMC_36031631","title":"Raltitrexed regulates proliferation and apoptosis of HGC-27 cells by upregulating RSK4.","date":"2022","source":"BMC pharmacology & toxicology","url":"https://pubmed.ncbi.nlm.nih.gov/36031631","citation_count":2,"is_preprint":false},{"pmid":"22944237","id":"PMC_22944237","title":"[Effects of breast cancer cells stably overexpressing RSK4 on growth of transplanted human breast cancer in severe combined immunodeficiency mice].","date":"2012","source":"Zhonghua yi xue za zhi","url":"https://pubmed.ncbi.nlm.nih.gov/22944237","citation_count":2,"is_preprint":false},{"pmid":"36498899","id":"PMC_36498899","title":"Differential Expression of RSK4 Transcript Isoforms in Cancer and Its Clinical Relevance.","date":"2022","source":"International journal of molecular sciences","url":"https://pubmed.ncbi.nlm.nih.gov/36498899","citation_count":1,"is_preprint":false},{"pmid":"39114716","id":"PMC_39114716","title":"Expression of RSK4 protein in non-small cell lung cancer tissues, adjacent tissues and its correlation with clinicopathological features.","date":"2024","source":"American journal of translational research","url":"https://pubmed.ncbi.nlm.nih.gov/39114716","citation_count":1,"is_preprint":false},{"pmid":"38577978","id":"PMC_38577978","title":"Validation of positional candidates Rps6ka6 and Pou3f4 for a locus associated with skeletal muscle mass variability.","date":"2024","source":"G3 (Bethesda, 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Constitutive activation results from constitutive phosphorylation of Ser232, Ser372, and Ser389. Low basal ERK activity in serum-starved cells is sufficient for ~50% of constitutive RSK4 activity. PDK1 gene deletion in mouse embryonic stem cells suggested PDK1 is not required for phosphorylation of the key activation-loop site Ser232, unlike other RSK family members.\",\n      \"method\": \"In vitro kinase assays, phospho-specific antibodies, subcellular fractionation, PDK1-knockout mouse embryonic stem cells, immunoblotting\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — direct biochemical characterization with mutagenesis-equivalent site mapping, knockout cells, and multiple orthogonal methods in a single rigorous study\",\n      \"pmids\": [\"15632195\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Rsk4 functions as an inhibitor of fibroblast growth factor-RAS-ERK (RTK) signaling. RTK inhibition is specific to RSK4 among RSK family members and depends on a region of RSK4 that is divergent from other RSK members. Rsk4 inhibits transcriptional activation of specific RTK signaling targets as well as ERK activation. Rsk4 expression in extraembryonic tissue is inversely correlated with activated ERK1/2 levels.\",\n      \"method\": \"Overexpression and domain-deletion experiments in cell lines, transcriptional reporter assays, ERK activation assays, in situ hybridization/immunostaining for expression vs. pERK correlation\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — multiple functional assays in one lab, domain specificity established, but no in vitro reconstitution\",\n      \"pmids\": [\"15121846\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"RSK4 overexpression induces cell cycle arrest and senescence features in normal fibroblasts (IMR90) and malignant colon carcinoma cells (HCT116). RSK4 mRNA levels increase during both replicative and stress-induced senescence. RSK4-induced senescence is mediated through p21, but is independent of p16, p38 MAPKs, and reactive oxygen species. Cells with RSK4 shRNA knockdown partially escape senescence and show increased resistance to cisplatin. E1A expression or Rb siRNA confers resistance to RSK4-mediated senescence.\",\n      \"method\": \"RSK4 cDNA overexpression and shRNA knockdown in IMR90 and HCT116 cells; senescence assays; pharmacological inhibitors of p16, p38, ROS; E1A overexpression; Rb siRNA epistasis\",\n      \"journal\": \"Clinical cancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — epistasis experiments with multiple pathway components, loss- and gain-of-function in multiple cell lines, single lab\",\n      \"pmids\": [\"19584160\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"RSK4 inhibition via shRNA allows normal human fibroblasts to bypass stress-induced senescence (oxidative stress, DNA damage) and oncogene-induced senescence (KRAS-V12, BRAF-E600 overexpression). RSK4-induced senescence is mediated through p21 but is independent of p16, p38 MAPKs, and reactive oxygen species induction.\",\n      \"method\": \"shRNA knockdown of RSK4 in normal human fibroblasts; oncogene overexpression; pharmacological inhibitors; senescence assays; p21 inhibition epistasis\",\n      \"journal\": \"Carcinogenesis\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — epistasis with multiple pathway inhibitors, replicated in multiple stress contexts, single lab\",\n      \"pmids\": [\"21239520\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"RSK4 (and PAK5) are direct transcriptional targets of HNF4alpha. HNF4alpha binds to novel sites in the RSK4 promoter as confirmed by chromatin immunoprecipitation (ChIP) cloning and electrophoretic mobility shift assays (EMSA). RSK4 expression is repressed in streptozotocin-induced diabetic rat kidney and brain coincident with HNF4alpha repression.\",\n      \"method\": \"ChIP cloning, EMSA with oligonucleotides harboring HNF4alpha binding sites, quantitative RT-PCR in diabetic rat tissue\",\n      \"journal\": \"Molecular pharmacology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — two orthogonal methods (ChIP + EMSA) establish direct binding, single lab\",\n      \"pmids\": [\"15615695\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"RSK4 promotes drug resistance and metastasis in lung and bladder cancer. CRISPR and RNAi silencing of RSK4 sensitized tumor cells to chemotherapy and hindered metastasis in vitro and in vivo. X-ray crystallography identified an allosteric binding site on RSK4 through which floxacin antibiotics (e.g., trovafloxacin) block RSK4 kinase activation by mimicking a kinase autoinhibitory mechanism involving RSK4's hydrophobic motif. Deuterium exchange and Markov transient analyses confirmed the allosteric mechanism.\",\n      \"method\": \"CRISPR knockdown, RNAi, X-ray crystallography, hydrogen-deuterium exchange, Markov transient analysis, xenograft models, genetically engineered mouse models, bladder tumor explants\",\n      \"journal\": \"Science translational medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure with allosteric mechanism validated by HDX and functional assays; multiple model systems\",\n      \"pmids\": [\"34261798\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Tandem affinity purification coupled with nano LC-MS/MS identified 82 RSK4-associated proteins and 137 RSK4m (alternative splice variant)-associated proteins in MDA-MB-231 cells. Bioinformatics analysis indicated enrichment in cell senescence pathways. RSK4 and RSK4m have distinct interactomes, with RSK4m associated with more molecular functions.\",\n      \"method\": \"SF-TAP (tandem affinity purification with STREP II + Flag tags) coupled to nano LC-MS/MS, bioinformatics (GO, IPA)\",\n      \"journal\": \"International journal of biological macromolecules\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — interactome screen without functional validation of individual interactions; single lab, no reciprocal confirmation\",\n      \"pmids\": [\"27986634\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"FOXP2 transcription factor directly binds to the RSK4 (RPS6KA6) promoter and transcriptionally activates its expression. Knockdown of RSK4 reverses the anti-proliferative and pro-apoptotic effects of FOXP2 overexpression in thyroid cancer cells, placing RSK4 downstream of FOXP2. FOXP2/RSK4 regulation is associated with the PI3K/AKT pathway.\",\n      \"method\": \"Dual-luciferase reporter assay, chromatin immunoprecipitation (ChIP), JASPAR binding site prediction, rescue/knockdown experiments in TPC-1 cells\",\n      \"journal\": \"Experimental and therapeutic medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct promoter binding confirmed by ChIP and luciferase reporter; epistasis by rescue experiment; single lab\",\n      \"pmids\": [\"35607372\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"17β-estradiol (E2) reduces RSK4 expression in ER+ breast cancer cells by promoting DNMT3B-mediated methylation of the RSK4 promoter, thereby decreasing RSK4 transcriptional activity. E2-induced RSK4 silencing is associated with increased cell proliferation, clone formation, and invasion.\",\n      \"method\": \"Bisulfite genomic sequencing for methylation, luciferase reporter assay for transcriptional activity, Western blotting for DNMT1/3A/3B, MTT/Transwell/flow cytometry functional assays, RT-PCR\",\n      \"journal\": \"OncoTargets and therapy\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — methylation mechanism identified with specific DNMT isoform, luciferase confirms transcriptional regulation, single lab\",\n      \"pmids\": [\"31413588\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"TRAF4 negatively regulates RSK4; TRAF4 knockdown leads to increased RSK4 expression in ovarian cancer cells. USP7 deubiquitinates and stabilizes TRAF4 via co-immunoprecipitation-confirmed interaction, and TRAF4 destabilization leads to RSK4 upregulation and suppression of the PI3K/AKT pathway. This USP7/TRAF4/RSK4/PI3K/AKT axis was confirmed in a xenograft mouse model.\",\n      \"method\": \"Co-immunoprecipitation for TRAF4 ubiquitination, siRNA knockdown of USP7 and TRAF4, RSK4 overexpression, Western blotting, xenograft mouse model\",\n      \"journal\": \"Journal of cancer research and therapeutics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — Co-IP for ubiquitination, multiple knockdowns, in vivo confirmation; single lab\",\n      \"pmids\": [\"37006049\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"RSK4 promotes macrophage recruitment and M2 polarization in esophageal squamous cell carcinoma (ESCC) by enhancing secretion of soluble ICAM-1 (sICAM-1) via direct and indirect STAT3 phosphorylation. RSK4-conditioned macrophages then secrete CCL22 to enhance tumor proliferation, migration, and invasion.\",\n      \"method\": \"In vitro co-culture experiments, ELISA for sICAM-1 and CCL22, phospho-STAT3 detection, RSK4 overexpression/knockdown in ESCC cells, xenograft mouse model, IHC in human ESCC tissues\",\n      \"journal\": \"Biochimica et biophysica acta. Molecular basis of disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — mechanistic pathway (RSK4→STAT3→sICAM-1→macrophage M2→CCL22) established with multiple assays; single lab\",\n      \"pmids\": [\"38142759\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"RSK4 stability is regulated by antagonistic post-translational modifications: GSK3β phosphorylates RSK4 at Thr402/Ser406, promoting its proteasomal degradation via the FBXW7-dependent ubiquitin-proteasome pathway. OGT-mediated O-GlcNAcylation of RSK4 at Thr405 antagonizes GSK3β phosphorylation, thereby stabilizing RSK4 and enhancing cancer stem cell properties and radioresistance in ESCC. Inhibition of O-GlcNAcylation with OSMI-4 destabilizes RSK4 and sensitizes ESCC to radiotherapy.\",\n      \"method\": \"Site-directed mutagenesis at Thr402/Ser406/Thr405, co-immunoprecipitation with GSK3β and FBXW7, O-GlcNAc detection, proteasome inhibitor experiments, OSMI-4 treatment, patient-derived xenograft and organoid models\",\n      \"journal\": \"International journal of biological sciences\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — site-specific mutagenesis identifies exact PTM residues, writer (OGT, GSK3β) and reader/eraser (FBXW7) identified, multiple orthogonal methods, in vivo validation; single lab\",\n      \"pmids\": [\"41800247\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Deletion of exon 7 of Rps6ka6 (creating a severely truncated RSK4 protein) in H2Kb myogenic cells did not affect myoblast proliferation or migration, but significantly reduced myoblast differentiation indices (myosin expression, myosin-expressing cell size, and fusion index). Genetic analysis in CFW mice linked the Rps6ka6 locus to muscle fiber number in fast-twitch extensor digitorum longus muscle.\",\n      \"method\": \"CRISPR-Cas9 exon deletion in myogenic cells, myoblast proliferation/migration/differentiation assays, myosin immunostaining, fusion index quantification, CFW mouse genetic analysis with histology\",\n      \"journal\": \"G3 (Bethesda, Md.)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — CRISPR loss-of-function with specific differentiation phenotype; in vivo genetic association; single lab\",\n      \"pmids\": [\"38577978\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"TRAF4 knockdown in breast cancer (MDA-MB-231) xenografts led to significant upregulation of RSK4 mRNA and protein, and coincident reduction of P-AKT, P-NF-κB, TGF-β1, TNF-α, MMP2, and MMP9. This places RSK4 as negatively regulated by TRAF4, upstream of the AKT/NF-κB pathway.\",\n      \"method\": \"Lentiviral TRAF4-shRNA in MDA-MB-231 cells, xenograft nude mouse model, IHC, Western blot, fluorescence quantitative RT-PCR\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — indirect relationship inferred from knockdown experiments without direct binding assay; single lab, in vivo only\",\n      \"pmids\": [\"29684350\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"RSK4 overexpression in ccRCC cell lines enhances invasive and migratory abilities through upregulation of CD44 and MMP-9 expression; conversely, RSK4 knockdown reduces these abilities and CD44/MMP-9 expression.\",\n      \"method\": \"Ectopic RSK4 overexpression and knockdown in ccRCC cell lines, invasion/migration assays, IHC in patient samples, Western blot\",\n      \"journal\": \"Diagnostic pathology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — functional assays link RSK4 to CD44/MMP-9, but mechanistic pathway is not delineated; single lab\",\n      \"pmids\": [\"32209138\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"miR-548d-3p directly targets RSK4 and negatively regulates its expression. Dual-luciferase reporter assay confirmed binding of miR-548d-3p to the RSK4 3'UTR at a predicted interaction site (mutation of this site abolished the effect). miR-548d-3p overexpression phenocopies RSK4 knockdown in gastric cancer cells: increased proliferation, migration, invasion, and decreased apoptosis.\",\n      \"method\": \"Dual-luciferase reporter assay with wild-type and mutant RSK4 3'UTR, RT-PCR, Western blot, functional cell assays, nude mouse tumorigenesis\",\n      \"journal\": \"Cancer management and research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct miRNA-target interaction confirmed by mutagenesis in reporter assay; single lab\",\n      \"pmids\": [\"33380838\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"RSK4 confers paclitaxel resistance in ovarian cancer cells. RSK4 elevation is associated with accelerated phosphorylation of the downstream translation factor eIF4B. RSK inhibitor (BI-D1870) co-treatment restored paclitaxel sensitivity. External RSK4 expression increased cell viability under paclitaxel treatment.\",\n      \"method\": \"DNA microarray, qRT-PCR, Western blotting, RSK inhibitor pharmacological treatment, RSK4 overexpression, cell viability assays\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — eIF4B phosphorylation identified as downstream event but direct RSK4→eIF4B relationship not fully established by mutagenesis; single lab\",\n      \"pmids\": [\"37660640\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"RSK4 promotes metastasis and angiogenesis in clear cell renal cell carcinoma (ccRCC) by activating RUNX1-mediated transcription of EPHA2, leading to enhanced VEGF secretion and tube formation. Luciferase reporter and mutagenesis of the RUNX1 binding site in the EPHA2 promoter confirmed this regulatory axis.\",\n      \"method\": \"Transcriptomic and proteomic analysis, luciferase reporter assay with RUNX1/EPHA2 promoter binding site mutation, VEGF ELISA, tube formation assay, in vivo experiments\",\n      \"journal\": \"Cancer biology & therapy\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — luciferase reporter with mutagenesis confirms RUNX1→EPHA2 link downstream of RSK4; single lab\",\n      \"pmids\": [\"39797421\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"RSK4 (RPS6KA6) is a constitutively active, predominantly cytosolic serine/threonine kinase that—unlike RSK1-3—does not require growth factor stimulation, with its activation loop (Ser232) phosphorylated independently of PDK1; it inhibits the RAS-ERK signaling axis, promotes cellular senescence via p21 (but not p16 or p38), and its stability is regulated by antagonistic PTMs whereby GSK3β-mediated phosphorylation at Thr402/Ser406 targets it for FBXW7-dependent proteasomal degradation while OGT-mediated O-GlcNAcylation at Thr405 stabilizes it; floxacin antibiotics inhibit RSK4 through an allosteric site that mimics its hydrophobic-motif autoinhibitory mechanism; RSK4 expression is transcriptionally activated by FOXP2 and repressed by estrogen-driven DNMT3B-mediated promoter methylation, and it is negatively regulated post-transcriptionally by miR-548d-3p and by the TRAF4/USP7 ubiquitin axis, with context-dependent roles in drug resistance, metastasis, angiogenesis (via RUNX1/EPHA2/VEGF), immune modulation (via STAT3/sICAM-1/CCL22), and myoblast differentiation.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"RSK4 (RPS6KA6) is a predominantly cytosolic serine/threonine kinase that, distinct from other RSK family members, is constitutively activated in serum-starved cells through constitutive phosphorylation of Ser232, Ser372, and Ser389 and does not require PDK1 for activation-loop phosphorylation [#0]. Functionally, it acts as a negative regulator of growth signaling: it inhibits FGF-RAS-ERK (RTK) signaling through a region divergent from other RSK proteins [#1] and drives cellular senescence in normal and malignant cells via a p21-dependent, p16/p38/ROS-independent program that requires intact Rb [#2, #3]. RSK4 abundance is tightly governed by antagonistic post-translational modifications, in which GSK3\\u03b2 phosphorylation at Thr402/Ser406 directs FBXW7-dependent proteasomal degradation while OGT-mediated O-GlcNAcylation at Thr405 stabilizes the protein and promotes cancer stem cell properties and radioresistance [#11]. Its expression is further controlled transcriptionally by activation through FOXP2 and HNF4alpha [#4, #7], by estrogen-driven DNMT3B-mediated promoter methylation [#8], and post-transcriptionally by miR-548d-3p and the USP7/TRAF4 axis [#9, #15]. RSK4 has context-dependent oncogenic and tumor-suppressive outputs across cancers, contributing to chemoresistance and metastasis, angiogenesis via a RUNX1/EPHA2/VEGF axis, and immune modulation through STAT3/sICAM-1/CCL22 signaling [#5, #10, #17]; its kinase activity can be blocked by floxacin antibiotics binding an allosteric site that mimics the hydrophobic-motif autoinhibitory mechanism [#5]. RSK4 also supports myoblast differentiation [#12].\",\n  \"teleology\": [\n    {\n      \"year\": 2004,\n      \"claim\": \"Established that RSK4 is not merely another RSK paralog but a specific antagonist of RTK signaling, defining its first functional role.\",\n      \"evidence\": \"Overexpression and domain-deletion experiments with transcriptional reporters and ERK activation assays; expression vs pERK correlation in extraembryonic tissue\",\n      \"pmids\": [\"15121846\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No in vitro reconstitution of the inhibitory mechanism\", \"Direct molecular target of the divergent inhibitory region not identified\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Identified RSK4 as a direct transcriptional target, beginning to explain how its expression is set in specific tissues.\",\n      \"evidence\": \"ChIP cloning and EMSA mapping HNF4alpha binding sites in the RSK4 promoter; qRT-PCR in diabetic rat tissue\",\n      \"pmids\": [\"15615695\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Functional consequence of HNF4alpha-driven expression not tested\", \"Restricted to rodent tissue context\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Answered how RSK4 activation differs from RSK1-3 by showing it is constitutively phosphorylated and PDK1-independent, explaining its growth-factor-independent activity.\",\n      \"evidence\": \"In vitro kinase assays, phospho-specific antibodies, subcellular fractionation, PDK1-knockout mouse ES cells\",\n      \"pmids\": [\"15632195\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Kinase responsible for constitutive Ser232 phosphorylation not identified\", \"Physiological substrates downstream of constitutive activity unresolved\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Resolved the senescence mechanism by showing RSK4 enforces stress- and oncogene-induced senescence through p21, defining a tumor-suppressive role.\",\n      \"evidence\": \"shRNA knockdown in fibroblasts with oncogene overexpression, pathway inhibitors, and p21/Rb epistasis (across 2009 and 2011 studies)\",\n      \"pmids\": [\"19584160\", \"21239520\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct link between RSK4 kinase activity and p21 induction not established\", \"Single-lab findings\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Began cataloging RSK4 protein partners and distinguishing the splice variant interactome to map its molecular context.\",\n      \"evidence\": \"SF-TAP tandem affinity purification with nano LC-MS/MS in MDA-MB-231 cells; bioinformatics\",\n      \"pmids\": [\"27986634\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Interactome screen without functional validation of individual interactions\", \"No reciprocal confirmation\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Provided a structural mechanism of inhibition and a context-specific oncogenic role, showing floxacins block RSK4 via an allosteric site mimicking hydrophobic-motif autoinhibition.\",\n      \"evidence\": \"X-ray crystallography, HDX, Markov transient analysis, CRISPR/RNAi silencing, xenograft and bladder explant models\",\n      \"pmids\": [\"34261798\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Endogenous regulator engaging the allosteric site unknown\", \"Reconciliation with tumor-suppressive roles in other contexts not addressed\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Extended RSK4 into tumor-immune crosstalk, defining a STAT3-driven secretory program that reprograms macrophages.\",\n      \"evidence\": \"Co-culture, ELISA for sICAM-1/CCL22, phospho-STAT3 detection, RSK4 gain/loss in ESCC cells, xenografts, IHC\",\n      \"pmids\": [\"38142759\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether RSK4 phosphorylates STAT3 directly not fully resolved\", \"Single-lab finding\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Defined a transcriptional output of RSK4 driving angiogenesis through RUNX1-mediated EPHA2 and VEGF.\",\n      \"evidence\": \"Transcriptomic/proteomic analysis, luciferase reporter with RUNX1/EPHA2 binding-site mutagenesis, VEGF ELISA, tube formation, in vivo experiments\",\n      \"pmids\": [\"39797421\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct kinase substrate connecting RSK4 to RUNX1 activation not identified\", \"Single-lab finding\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Established how RSK4 protein levels are set by antagonistic PTMs, identifying GSK3\\u03b2/FBXW7 degradation and OGT O-GlcNAcylation stabilization at adjacent residues.\",\n      \"evidence\": \"Site-directed mutagenesis at Thr402/Ser406/Thr405, co-IP with GSK3\\u03b2 and FBXW7, O-GlcNAc detection, proteasome inhibition, OSMI-4, PDX and organoid models\",\n      \"pmids\": [\"41800247\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Upstream signals controlling the GSK3\\u03b2/OGT balance not defined\", \"Single-lab finding\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How RSK4's constitutive kinase activity is mechanistically reconciled with its opposing tumor-suppressive (senescence, RTK inhibition) and oncogenic (metastasis, immune modulation, angiogenesis) outputs across tissues remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No unifying model linking direct substrates to context-dependent phenotypes\", \"Bona fide physiological substrates of RSK4 kinase activity largely uncharacterized\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 5]},\n      {\"term_id\": \"GO:0016740\", \"supporting_discovery_ids\": [0]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [0]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [1, 7, 9]},\n      {\"term_id\": \"R-HSA-8953897\", \"supporting_discovery_ids\": [2, 3]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [11]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"GSK3B\", \"FBXW7\", \"OGT\", \"TRAF4\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}