{"gene":"STK4","run_date":"2026-06-10T10:51:54","timeline":{"discoveries":[{"year":1996,"finding":"STK4 (KRS2) is a serine/threonine kinase member of the Ste20p subfamily that is activated by a subset of stress conditions and apoptotic agents (but not mitogenic stimuli), purified and cloned as a stress-responsive kinase.","method":"Protein purification, cloning, and kinase activity assay under various stress conditions","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — purification and direct biochemical characterization in a single lab with clear activity assay, but limited mechanistic depth beyond activation specificity","pmids":["8816758"],"is_preprint":false},{"year":2012,"finding":"STK4-deficient lymphocytes and neutrophils exhibit enhanced loss of mitochondrial membrane potential and increased susceptibility to apoptosis, establishing STK4 as required for immune cell survival.","method":"Loss-of-function (homozygous truncation mutation in patients), mitochondrial membrane potential assay, apoptosis assay","journal":"Blood","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — patient-derived cells with defined genetic loss, two orthogonal cellular phenotype assays, single study","pmids":["22294732"],"is_preprint":false},{"year":2014,"finding":"STK4 (and STK3) directly phosphorylates LC3 at threonine 50 (Thr50), and this phosphorylation is essential for autophagosome-lysosome fusion; loss of STK3/STK4 blocks autophagy and impairs intracellular bacterial clearance, which is rescued by a phosphomimetic LC3-T50E mutant.","method":"In vitro kinase assay, phosphosite identification, genetic loss-of-function (STK3/STK4-deficient cells), autophagy flux assay, autophagosome-lysosome fusion assay, bacterial clearance assay, phosphomimetic rescue experiment across multiple species","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 1 / Strong — direct in vitro phosphorylation assay with site-specific mutagenesis, phosphomimetic rescue, multiple organisms, multiple orthogonal readouts","pmids":["25544559"],"is_preprint":false},{"year":2015,"finding":"STK4 dampens TLR4/9-induced proinflammatory cytokine secretion and enhances TLR3/4-triggered IFN-β production by binding to and phosphorylating IRAK1, leading to IRAK1 degradation in macrophages.","method":"Co-immunoprecipitation (binding), in vitro phosphorylation assay, macrophage-specific Stk4 knockout mice, cytokine ELISA, IRAK1 protein level measurement","journal":"The Journal of clinical investigation","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — direct binding demonstrated by Co-IP, direct phosphorylation assay, in vivo genetic knockout with defined phenotypic readouts, multiple orthogonal methods","pmids":["26457732"],"is_preprint":false},{"year":2015,"finding":"STK4 phosphorylates LC3 at Thr50 as a conserved regulatory mechanism for autophagy; this phosphorylation is critical for autophagosome-lysosome fusion and intracellular bacteria clearance.","method":"In vitro kinase assay, site-directed mutagenesis, autophagy assays","journal":"Autophagy","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — single commentary/follow-up paper confirming the same finding as PMID 25544559; no new independent methods","pmids":["25996575"],"is_preprint":false},{"year":2017,"finding":"STK4 localizes to the cytoplasm, lipid rafts, and nucleus in prostate cancer cells, and nuclear/lipid raft localization produces superior suppression of cell growth compared to cytoplasmic STK4, with each compartment activating distinct gene expression programs including oncogenic pathways (AR, PI3K/AKT, BMP/SMAD, WNT, RAS, JAK/STAT).","method":"Subcellular fractionation, ectopic expression in defined compartments, in vitro and in vivo growth assays, RNA sequencing","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct subcellular localization experiment with functional consequence (growth suppression), multiple compartments tested, RNA-seq for pathway mapping, single lab","pmids":["28880957"],"is_preprint":false},{"year":2019,"finding":"STK4/MST1 phosphorylates BECN1 at threonine 108 (T108) within its BH3 domain, increasing BECN1 affinity for anti-apoptotic proteins BCL2 and BCL2L1, thereby blocking autophagy; X-ray crystal structures of BCL2 and BCL2L1 complexed with T108-modified BECN1 BH3 peptides revealed the structural basis of this interaction, showing only minor (<2-fold) affinity increase.","method":"X-ray crystallography, surface plasmon resonance, microscale thermophoresis, biophysical binding assays, molecular dynamics simulation","journal":"Autophagy","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure determined, multiple biophysical methods (SPR, MST, MD), precise quantification of phosphorylation-dependent affinity change","pmids":["30626284"],"is_preprint":false},{"year":2019,"finding":"lncRNA TNRC6C-AS1 recruits methyltransferase to the STK4 promoter to increase STK4 promoter methylation and down-regulate STK4 expression, thereby activating the Hippo signaling pathway in thyroid carcinoma cells.","method":"ChIP/methyltransferase binding assay, STK4 promoter methylation analysis, gene silencing/overexpression, proliferation and apoptosis assays","journal":"Journal of cellular and molecular medicine","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — methyltransferase-STK4 promoter interaction demonstrated, promoter methylation quantified, functional rescue experiments; single lab","pmids":["31657132"],"is_preprint":false},{"year":2020,"finding":"TRAF6 mediates LPS-induced ubiquitination of STK4/MST1, activating STK4 which then inhibits TRAF6 autoubiquitination and downstream NF-κB signaling, constituting a negative feedback loop in macrophage TLR4 signaling.","method":"Myeloid-specific genetic ablation (KO mice), Co-immunoprecipitation, ubiquitination assay, NF-κB activation assay, cytokine measurement, LPS-induced septic shock model","journal":"Cellular and molecular life sciences : CMLS","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — in vivo knockout, direct ubiquitination assay, Co-IP for interaction, multiple orthogonal functional readouts, reciprocal regulation demonstrated","pmids":["32975614"],"is_preprint":false},{"year":2020,"finding":"STK4 deficiency impairs TBK1-IRF3 phosphorylation, leading to significantly reduced type I, II, and III interferon responses to TLR3, TLR9, and cytosolic RNA/DNA sensor ligands.","method":"Patient-derived cells (STK4 frameshift mutation), phospho-TBK1 and phospho-IRF3 immunoblot, cytokine ELISA, viral infection assay","journal":"Journal of clinical immunology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — patient-derived loss-of-function, direct phosphorylation readout of pathway components, single patient/lab","pmids":["33078349"],"is_preprint":false},{"year":2020,"finding":"STK4 directly phosphorylates β-catenin leading to its degradation via the ubiquitin-mediated pathway; STK4 colocalization with β-catenin was demonstrated, and STK4 knockdown causes β-catenin accumulation, promoting anchorage-independent growth and metastasis in colon cancer.","method":"Co-localization assay, in vitro kinase assay (direct phosphorylation of β-catenin), ubiquitin degradation assay, STK4 knockdown with metastasis phenotype readout, in vivo tumor model","journal":"Molecular oncology","confidence":"Medium","confidence_rationale":"Tier 1-2 / Moderate — direct kinase assay showing phosphorylation of β-catenin, co-localization, ubiquitin pathway involvement, in vivo model; single lab","pmids":["32741119"],"is_preprint":false},{"year":2021,"finding":"STK3 and STK4 suppress mitochondrial capacity in adipocytes and regulate mitophagy by controlling phosphorylation and dimerization status of the mitophagy receptor BNIP3; genetic inactivation of Stk3/Stk4 increases mitochondrial mass and function, stabilizes UCP1 in beige adipose tissue, and confers resistance to metabolic dysfunction.","method":"Genetic inactivation (adipose-specific knockout mice), mitochondrial function assays, BNIP3 phosphorylation assay, dimerization assay, metabolic phenotyping, pharmacological inhibition","journal":"Nature metabolism","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — in vivo genetic knockout with multiple metabolic phenotype readouts, direct phosphorylation/dimerization of BNIP3 demonstrated, pharmacological validation, single lab with multiple orthogonal methods","pmids":["33758424"],"is_preprint":false},{"year":2021,"finding":"ROR2 inhibits STK4 phosphorylation (activity), which promotes nuclear translocation of FOXO1 that directly represses SMS1 transcription; this ROR2/STK4-FOXO1/SMS1 axis regulates sphingomyelin biosynthesis and dental pulp stem cell senescence.","method":"STK4 phosphorylation assay, FOXO1 nuclear translocation assay, FOXO1 ChIP on SMS1 promoter, gene knockdown/overexpression, proliferation assay","journal":"Aging cell","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — STK4 phosphorylation status measured, direct FOXO1 binding to SMS1 promoter shown by ChIP, functional rescue; single lab","pmids":["34278704"],"is_preprint":false},{"year":2021,"finding":"Crystallization of an STK4 inhibitor compound with STK4 revealed two-point hinge binding in the ATP-binding pocket, providing structural basis for STK4 inhibitor design.","method":"X-ray crystallography of STK4-inhibitor complex","journal":"Journal of medicinal chemistry","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — crystal structure determined but functional validation of binding mode limited to inhibitor potency data; single study","pmids":["34807584"],"is_preprint":false},{"year":2022,"finding":"TCR signaling induces nuclear translocation of STK4 in Treg cells, where STK4 forms a complex with NF-κB p65 and Foxp3; STK4 phosphorylates Foxp3 at serine-418 to stabilize this complex, which regulates Foxp3- and p65-dependent transcriptional programs required for Treg cell activation and immune tolerance.","method":"Nuclear translocation assay (live imaging/fractionation), Co-immunoprecipitation (STK4-p65-Foxp3 complex), in vitro phosphorylation assay (Foxp3 S418), phosphomimetic rescue (Foxp3-S418E), Treg-specific conditional knockout mice, adoptive immunotherapy model","journal":"Science immunology","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — direct phosphorylation of Foxp3-S418 demonstrated, trimeric complex identified by Co-IP, phosphomimetic rescue, in vivo Treg-specific KO, multiple orthogonal methods","pmids":["36149942"],"is_preprint":false},{"year":2025,"finding":"STK4 directly binds the RING2-LDD module of HOIP (the E3 ligase catalytic subunit of LUBAC) through its kinase domain, phosphorylates HOIP at T786 within its allosteric ubiquitin-binding site, thereby blocking ubiquitin binding to the allosteric site and attenuating HOIP's E3 ligase activity toward NF-κB signaling.","method":"Biochemical binding assay, mass spectrometry (phosphosite identification), X-ray crystallography (STK4-HOIP RING2-LDD complex structure), in vitro kinase assay, E3 ligase activity assay, ubiquitin-binding competition assay","journal":"Cell discovery","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure of STK4-substrate complex, direct phosphorylation site identified by MS, E3 activity assay with mechanistic explanation, multiple orthogonal methods in a single rigorous study","pmids":["40957900"],"is_preprint":false}],"current_model":"STK4 (MST1/KRS2) is a stress- and TCR-responsive serine/threonine kinase that directly phosphorylates multiple substrates—including LC3-Thr50 (driving autophagosome-lysosome fusion), BECN1-Thr108 (increasing BCL2 affinity to suppress autophagy), β-catenin (promoting ubiquitin-mediated degradation), IRAK1 (inducing its degradation to dampen TLR-NF-κB inflammation), BNIP3 (regulating mitophagy and mitochondrial capacity in adipocytes), Foxp3-Ser418 (stabilizing a nuclear STK4-p65-Foxp3 complex for Treg transcription), and HOIP-Thr786 (blocking the allosteric ubiquitin-binding site to inhibit LUBAC E3 activity)—and is itself activated by TRAF6-mediated ubiquitination; it also undergoes nuclear translocation upon TCR signaling, and its loss causes immune cell apoptosis, impaired interferon responses via reduced TBK1-IRF3 phosphorylation, and combined immunodeficiency in humans."},"narrative":{"mechanistic_narrative":"STK4 (MST1/KRS2) is a stress- and apoptosis-responsive serine/threonine kinase of the Ste20p subfamily that acts as a central node integrating immune signaling, autophagy, and cell-survival decisions [PMID:8816758]. It controls autophagy bidirectionally by directly phosphorylating LC3 at Thr50 to drive autophagosome-lysosome fusion and intracellular bacterial clearance [PMID:25544559], while phosphorylation of BECN1 at Thr108 within its BH3 domain enhances BECN1 affinity for BCL2/BCL2L1 to suppress autophagy [PMID:30626284]; in adipocytes it further regulates mitophagy and mitochondrial capacity through phosphorylation and dimerization control of BNIP3 [PMID:33758424]. In innate immunity STK4 enforces negative feedback on TLR-NF-κB signaling: it is activated by TRAF6-mediated ubiquitination and in turn suppresses TRAF6 autoubiquitination [PMID:32975614], binds and phosphorylates IRAK1 to trigger its degradation and dampen proinflammatory cytokines [PMID:26457732], and phosphorylates the LUBAC catalytic subunit HOIP at Thr786 to block its allosteric ubiquitin-binding site and attenuate E3 ligase activity [PMID:40957900]. Upon TCR signaling STK4 translocates to the nucleus, where it phosphorylates Foxp3 at Ser418 and stabilizes a nuclear STK4-p65-Foxp3 complex required for Treg activation and immune tolerance [PMID:36149942]. It also phosphorylates β-catenin to promote its ubiquitin-mediated degradation, restraining anchorage-independent growth and metastasis [PMID:32741119]. Loss-of-function in humans causes combined immunodeficiency, with patient cells showing heightened apoptosis and loss of mitochondrial membrane potential [PMID:22294732] and impaired type I/II/III interferon responses through reduced TBK1-IRF3 phosphorylation [PMID:33078349].","teleology":[{"year":1996,"claim":"Established STK4 as a stress-activated kinase distinct from mitogenic signaling, defining its activation specificity before any substrate was known.","evidence":"Protein purification, cloning, and kinase assays under stress/apoptotic conditions","pmids":["8816758"],"confidence":"Medium","gaps":["No substrates identified","Upstream activators undefined","Physiological context unknown"]},{"year":2012,"claim":"Linked STK4 to immune cell survival by showing its loss causes mitochondrial depolarization and apoptosis, connecting the kinase to human immunodeficiency.","evidence":"Patient-derived cells with homozygous truncation, mitochondrial membrane potential and apoptosis assays","pmids":["22294732"],"confidence":"Medium","gaps":["Molecular mechanism linking STK4 loss to mitochondrial dysfunction not resolved","Substrates mediating survival unknown"]},{"year":2014,"claim":"Identified the first direct substrate-site relationship driving autophagy, showing STK4/STK3 phosphorylate LC3-Thr50 to enable autophagosome-lysosome fusion and bacterial clearance.","evidence":"In vitro kinase assay, phosphosite mapping, STK3/STK4-deficient cells, phosphomimetic LC3-T50E rescue across species","pmids":["25544559","25996575"],"confidence":"High","gaps":["How fusion machinery reads the LC3-T50 phosphomark not defined","Reconciliation with autophagy-suppressive roles unaddressed"]},{"year":2015,"claim":"Defined STK4 as a brake on TLR-driven inflammation by phosphorylating IRAK1 to trigger its degradation while enhancing IFN-β output.","evidence":"Co-IP, in vitro phosphorylation, macrophage-specific Stk4 knockout, cytokine ELISA","pmids":["26457732"],"confidence":"High","gaps":["IRAK1 phosphosite not specified","Ubiquitin ligase coupling degradation unknown"]},{"year":2017,"claim":"Showed STK4 function is compartment-dependent, with nuclear and lipid-raft pools driving distinct transcriptional programs and stronger growth suppression than cytoplasmic STK4.","evidence":"Subcellular fractionation, compartment-targeted expression, growth assays, RNA-seq in prostate cancer cells","pmids":["28880957"],"confidence":"Medium","gaps":["Mechanism of compartment targeting unknown","Nuclear substrates not identified here"]},{"year":2019,"claim":"Revealed an autophagy-suppressive arm: STK4 phosphorylates BECN1-Thr108 to raise BCL2/BCL2L1 affinity, with structural resolution of the modified interface.","evidence":"X-ray crystallography, SPR, MST, molecular dynamics","pmids":["30626284"],"confidence":"High","gaps":["Measured affinity increase was modest (<2-fold)","Cellular context dictating pro- vs anti-autophagy roles unresolved"]},{"year":2019,"claim":"Identified epigenetic silencing of STK4 via promoter methylation as a mechanism activating Hippo signaling in thyroid carcinoma.","evidence":"ChIP/methyltransferase binding, promoter methylation analysis, silencing/overexpression, proliferation and apoptosis assays","pmids":["31657132"],"confidence":"Medium","gaps":["Direct link to downstream Hippo effectors not mechanistically detailed","Single tumor context"]},{"year":2020,"claim":"Established a TRAF6-STK4 negative feedback loop in which TRAF6 ubiquitinates and activates STK4, which then suppresses TRAF6 autoubiquitination and NF-κB.","evidence":"Myeloid-specific KO, Co-IP, ubiquitination assays, NF-κB readouts, LPS septic shock model","pmids":["32975614"],"confidence":"High","gaps":["Ubiquitin chain types and STK4 ubiquitination sites not fully mapped","Mechanism of TRAF6 suppression by STK4 unclear"]},{"year":2020,"claim":"Connected STK4 to interferon immunity, showing its loss impairs TBK1-IRF3 phosphorylation and broad type I/II/III IFN responses.","evidence":"Patient-derived cells with frameshift mutation, phospho-immunoblot, cytokine ELISA, viral infection","pmids":["33078349"],"confidence":"Medium","gaps":["Whether STK4 acts directly on TBK1 not established","Single patient/lab"]},{"year":2020,"claim":"Defined a tumor-suppressive role through direct phosphorylation of β-catenin promoting its ubiquitin-mediated degradation.","evidence":"Co-localization, in vitro kinase assay, ubiquitin degradation assay, knockdown metastasis and in vivo tumor models","pmids":["32741119"],"confidence":"Medium","gaps":["β-catenin phosphosite not specified","Ligase coupling degradation unidentified"]},{"year":2021,"claim":"Extended STK4 into metabolic regulation by controlling BNIP3-dependent mitophagy and mitochondrial capacity in adipose tissue.","evidence":"Adipose-specific KO, mitochondrial and metabolic phenotyping, BNIP3 phosphorylation/dimerization assays, pharmacological inhibition","pmids":["33758424"],"confidence":"High","gaps":["BNIP3 phosphosite(s) not specified","Link to whole-body metabolism mechanistically partial"]},{"year":2021,"claim":"Placed STK4 within a ROR2-FOXO1 axis regulating sphingomyelin synthesis and stem cell senescence, showing inhibition of STK4 activity permits FOXO1 nuclear translocation.","evidence":"STK4 phosphorylation assay, FOXO1 nuclear translocation, FOXO1 ChIP on SMS1, knockdown/overexpression","pmids":["34278704"],"confidence":"Medium","gaps":["Direct STK4-FOXO1 phosphorylation not demonstrated here","Single lineage context"]},{"year":2021,"claim":"Provided a structural template for STK4 inhibitor design by resolving an inhibitor bound in the ATP pocket via two-point hinge binding.","evidence":"X-ray crystallography of STK4-inhibitor complex","pmids":["34807584"],"confidence":"Medium","gaps":["Cellular selectivity and on-target effects beyond potency not characterized"]},{"year":2022,"claim":"Revealed a nuclear, kinase-dependent transcriptional role in Tregs, where TCR-induced STK4 phosphorylates Foxp3-S418 and stabilizes a STK4-p65-Foxp3 complex for immune tolerance.","evidence":"Nuclear translocation imaging/fractionation, Co-IP of trimeric complex, in vitro kinase assay, Foxp3-S418E rescue, Treg-specific KO, adoptive immunotherapy model","pmids":["36149942"],"confidence":"High","gaps":["Signal driving nuclear translocation mechanistically incomplete","Genome-wide complex targets not mapped"]},{"year":2025,"claim":"Defined a structural mechanism by which STK4 inhibits LUBAC, phosphorylating HOIP-Thr786 in its allosteric ubiquitin-binding site to block ubiquitin binding and attenuate E3 activity.","evidence":"Biochemical binding, MS phosphosite mapping, X-ray crystallography of STK4-HOIP RING2-LDD complex, kinase and E3 activity assays, ubiquitin-binding competition","pmids":["40957900"],"confidence":"High","gaps":["In vivo consequence of HOIP-T786 phosphorylation not established","Integration with other STK4 NF-κB feedback arms unresolved"]},{"year":null,"claim":"How STK4 selects between its opposing roles (pro- vs anti-autophagy, kinase-dependent cytoplasmic feedback vs nuclear transcriptional complexes) in a given cell remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No unifying model of substrate selection by compartment or stimulus","Upstream determinants of nuclear translocation incompletely defined","Crosstalk among the multiple NF-κB-regulatory substrates not integrated"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[2,3,6,10,11,14,15]},{"term_id":"GO:0016740","term_label":"transferase activity","supporting_discovery_ids":[2,3,6,14,15]},{"term_id":"GO:0140657","term_label":"ATP-dependent activity","supporting_discovery_ids":[13]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[5]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[5,14]},{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[5]}],"pathway":[{"term_id":"R-HSA-9612973","term_label":"Autophagy","supporting_discovery_ids":[2,6,11]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[3,8,9,14]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[8,10,15]},{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[1,6]}],"complexes":["STK4-p65-Foxp3 nuclear complex"],"partners":["IRAK1","TRAF6","BECN1","BNIP3","CTNNB1","FOXP3","RELA","RNF31"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q13043","full_name":"Serine/threonine-protein kinase 4","aliases":["Mammalian STE20-like protein kinase 1","MST-1","STE20-like kinase MST1","Serine/threonine-protein kinase Krs-2"],"length_aa":487,"mass_kda":55.6,"function":"Stress-activated, pro-apoptotic kinase which, following caspase-cleavage, enters the nucleus and induces chromatin condensation followed by internucleosomal DNA fragmentation. Key component of the Hippo signaling pathway which plays a pivotal role in organ size control and tumor suppression by restricting proliferation and promoting apoptosis. The core of this pathway is composed of a kinase cascade wherein STK3/MST2 and STK4/MST1, in complex with its regulatory protein SAV1, phosphorylates and activates LATS1/2 in complex with its regulatory protein MOB1, which in turn phosphorylates and inactivates YAP1 oncoprotein and WWTR1/TAZ. Phosphorylation of YAP1 by LATS2 inhibits its translocation into the nucleus to regulate cellular genes important for cell proliferation, cell death, and cell migration. STK3/MST2 and STK4/MST1 are required to repress proliferation of mature hepatocytes, to prevent activation of facultative adult liver stem cells (oval cells), and to inhibit tumor formation (By similarity). Phosphorylates 'Ser-14' of histone H2B (H2BS14ph) during apoptosis. Phosphorylates FOXO3 upon oxidative stress, which results in its nuclear translocation and cell death initiation. Phosphorylates MOBKL1A, MOBKL1B and RASSF2. Phosphorylates TNNI3 (cardiac Tn-I) and alters its binding affinity to TNNC1 (cardiac Tn-C) and TNNT2 (cardiac Tn-T). Phosphorylates FOXO1 on 'Ser-212' and regulates its activation and stimulates transcription of PMAIP1 in a FOXO1-dependent manner. Phosphorylates SIRT1 and inhibits SIRT1-mediated p53/TP53 deacetylation, thereby promoting p53/TP53 dependent transcription and apoptosis upon DNA damage. Acts as an inhibitor of PKB/AKT1. Phosphorylates AR on 'Ser-650' and suppresses its activity by intersecting with PKB/AKT1 signaling and antagonizing formation of AR-chromatin complexes","subcellular_location":"Cytoplasm; Nucleus","url":"https://www.uniprot.org/uniprotkb/Q13043/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/STK4","classification":"Not Classified","n_dependent_lines":2,"n_total_lines":1208,"dependency_fraction":0.0016556291390728477},"opencell":{"profiled":true,"resolved_as":"","ensg_id":"ENSG00000101109","cell_line_id":"CID001282","localizations":[{"compartment":"cytoplasmic","grade":3},{"compartment":"cytoskeleton","grade":1},{"compartment":"nucleoplasm","grade":1}],"interactors":[{"gene":"STK3","stoichiometry":4.0},{"gene":"MAP1LC3B","stoichiometry":0.2},{"gene":"MAP1S","stoichiometry":0.2},{"gene":"ITPRIP","stoichiometry":0.2},{"gene":"RASSF6","stoichiometry":0.2},{"gene":"SPCS3","stoichiometry":0.2},{"gene":"STRN3","stoichiometry":0.2},{"gene":"FGFR1OP2","stoichiometry":0.2},{"gene":"VAPA","stoichiometry":0.2},{"gene":"ACTR3B","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/target/CID001282","total_profiled":1310},"omim":[{"mim_id":"620929","title":"MOB KINASE ACTIVATOR 3A; MOB3A","url":"https://www.omim.org/entry/620929"},{"mim_id":"620110","title":"WW AND C2 DOMAINS-CONTAINING PROTEIN 2; WWC2","url":"https://www.omim.org/entry/620110"},{"mim_id":"617919","title":"STRIATIN-INTERACTING PROTEIN 2; STRIP2","url":"https://www.omim.org/entry/617919"},{"mim_id":"617918","title":"STRIATIN-INTERACTING PROTEIN 1; STRIP1","url":"https://www.omim.org/entry/617918"},{"mim_id":"616711","title":"TAO KINASE 3; TAOK3","url":"https://www.omim.org/entry/616711"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Nuclear bodies","reliability":"Supported"},{"location":"Nucleoplasm","reliability":"Additional"},{"location":"Cytosol","reliability":"Additional"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in all","driving_tissues":[{"tissue":"bone marrow","ntpm":46.4},{"tissue":"lymphoid tissue","ntpm":36.6}],"url":"https://www.proteinatlas.org/search/STK4"},"hgnc":{"alias_symbol":["MST1","KRS2","YSK3"],"prev_symbol":[]},"alphafold":{"accession":"Q13043","domains":[{"cath_id":"3.30.200.20","chopping":"20-104","consensus_level":"high","plddt":89.8005,"start":20,"end":104},{"cath_id":"1.10.510.10","chopping":"109-173_187-296","consensus_level":"high","plddt":93.8847,"start":109,"end":296}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q13043","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q13043-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q13043-F1-predicted_aligned_error_v6.png","plddt_mean":75.94},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=STK4","jax_strain_url":"https://www.jax.org/strain/search?query=STK4"},"sequence":{"accession":"Q13043","fasta_url":"https://rest.uniprot.org/uniprotkb/Q13043.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q13043/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q13043"}},"corpus_meta":[{"pmid":"22294732","id":"PMC_22294732","title":"The phenotype of human STK4 deficiency.","date":"2012","source":"Blood","url":"https://pubmed.ncbi.nlm.nih.gov/22294732","citation_count":256,"is_preprint":false},{"pmid":"25544559","id":"PMC_25544559","title":"Phosphorylation of LC3 by the Hippo kinases STK3/STK4 is essential for autophagy.","date":"2014","source":"Molecular cell","url":"https://pubmed.ncbi.nlm.nih.gov/25544559","citation_count":159,"is_preprint":false},{"pmid":"8816758","id":"PMC_8816758","title":"Newly identified stress-responsive protein kinases, Krs-1 and Krs-2.","date":"1996","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/8816758","citation_count":134,"is_preprint":false},{"pmid":"26457732","id":"PMC_26457732","title":"STK4 regulates TLR pathways and protects against chronic inflammation-related hepatocellular carcinoma.","date":"2015","source":"The Journal of clinical investigation","url":"https://pubmed.ncbi.nlm.nih.gov/26457732","citation_count":109,"is_preprint":false},{"pmid":"24752237","id":"PMC_24752237","title":"MicroRNA-18a is elevated in prostate cancer and promotes tumorigenesis through suppressing STK4 in vitro and in vivo.","date":"2014","source":"Oncogenesis","url":"https://pubmed.ncbi.nlm.nih.gov/24752237","citation_count":98,"is_preprint":false},{"pmid":"26117625","id":"PMC_26117625","title":"STK4 (MST1) deficiency in two siblings with autoimmune cytopenias: A novel mutation.","date":"2015","source":"Clinical immunology (Orlando, Fla.)","url":"https://pubmed.ncbi.nlm.nih.gov/26117625","citation_count":66,"is_preprint":false},{"pmid":"21228115","id":"PMC_21228115","title":"Identification of MST1/STK4 and SULF1 proteins as autoantibody targets for the diagnosis of colorectal cancer by using phage microarrays.","date":"2011","source":"Molecular & cellular proteomics : MCP","url":"https://pubmed.ncbi.nlm.nih.gov/21228115","citation_count":59,"is_preprint":false},{"pmid":"32555725","id":"PMC_32555725","title":"MicroRNA-18a targeting of the STK4/MST1 tumour suppressor is necessary for transformation in HPV positive cervical cancer.","date":"2020","source":"PLoS pathogens","url":"https://pubmed.ncbi.nlm.nih.gov/32555725","citation_count":58,"is_preprint":false},{"pmid":"33758424","id":"PMC_33758424","title":"STK3/STK4 signalling in adipocytes regulates mitophagy and energy expenditure.","date":"2021","source":"Nature metabolism","url":"https://pubmed.ncbi.nlm.nih.gov/33758424","citation_count":48,"is_preprint":false},{"pmid":"30626284","id":"PMC_30626284","title":"Structural insights into BCL2 pro-survival protein interactions with the key autophagy regulator BECN1 following phosphorylation by 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literature.","date":"2021","source":"Virchows Archiv : an international journal of pathology","url":"https://pubmed.ncbi.nlm.nih.gov/34604912","citation_count":10,"is_preprint":false},{"pmid":"34427831","id":"PMC_34427831","title":"A Novel STK4 Mutation Impairs T Cell Immunity Through Dysregulation of Cytokine-Induced Adhesion and Chemotaxis Genes.","date":"2021","source":"Journal of clinical immunology","url":"https://pubmed.ncbi.nlm.nih.gov/34427831","citation_count":10,"is_preprint":false},{"pmid":"35295003","id":"PMC_35295003","title":"LncRNA GAS5 enhances tumor stem cell-like medicated sensitivity of paclitaxel and inhibits epithelial-to-mesenchymal transition by targeting the miR-18a-5p/STK4 pathway in prostate cancer.","date":"2022","source":"Asian journal of andrology","url":"https://pubmed.ncbi.nlm.nih.gov/35295003","citation_count":10,"is_preprint":false},{"pmid":"36269304","id":"PMC_36269304","title":"Graphene oxide accelerates diabetic wound repair by inhibiting apoptosis of Ad-MSCs via Linc00324/miR-7977/STK4 pathway.","date":"2022","source":"FASEB journal : official publication of the Federation of American Societies for Experimental Biology","url":"https://pubmed.ncbi.nlm.nih.gov/36269304","citation_count":9,"is_preprint":false},{"pmid":"36394186","id":"PMC_36394186","title":"Hematopoietic stem cell transplantation in serine/threonine kinase 4 (STK4) deficiency: Report of two cases and literature review.","date":"2022","source":"Pediatric transplantation","url":"https://pubmed.ncbi.nlm.nih.gov/36394186","citation_count":9,"is_preprint":false},{"pmid":"30972994","id":"PMC_30972994","title":"MiR-1178-3p promotes the proliferation, migration and invasion of nasopharyngeal carcinoma Sune-1 cells by targeting STK4.","date":"2019","source":"Journal of biological regulators and homeostatic agents","url":"https://pubmed.ncbi.nlm.nih.gov/30972994","citation_count":9,"is_preprint":false},{"pmid":"32741119","id":"PMC_32741119","title":"Downregulation of STK4 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IJI","url":"https://pubmed.ncbi.nlm.nih.gov/33382390","citation_count":5,"is_preprint":false},{"pmid":"39110273","id":"PMC_39110273","title":"Epidermodysplasia Verruciformis and Vδ2 γδ T-cell Expansion in STK4 Deficiency.","date":"2024","source":"Journal of clinical immunology","url":"https://pubmed.ncbi.nlm.nih.gov/39110273","citation_count":3,"is_preprint":false},{"pmid":"38361950","id":"PMC_38361950","title":"A unique STK4 mutation truncating only the C-terminal SARAH domain results in a mild clinical phenotype despite severe T cell lymphopenia: Case report.","date":"2024","source":"Frontiers in immunology","url":"https://pubmed.ncbi.nlm.nih.gov/38361950","citation_count":3,"is_preprint":false},{"pmid":"40545108","id":"PMC_40545108","title":"circGAPVD1 inhibits the progression of gastric cancer through miR-4424/STK4 axis and encoding GAPVD1-137aa protein.","date":"2025","source":"International journal of biological 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letters","url":"https://pubmed.ncbi.nlm.nih.gov/42119789","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":24640,"output_tokens":4126,"usd":0.067905,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":12006,"output_tokens":4654,"usd":0.08819,"stage2_stop_reason":"end_turn"},"total_usd":0.156095,"stage1_batch_id":"msgbatch_017qqVz33Ga63SCDwzb35XPb","stage2_batch_id":"msgbatch_01Scr2Ei9x4HJMiRGxwrmvLK","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1996,\n      \"finding\": \"STK4 (KRS2) is a serine/threonine kinase member of the Ste20p subfamily that is activated by a subset of stress conditions and apoptotic agents (but not mitogenic stimuli), purified and cloned as a stress-responsive kinase.\",\n      \"method\": \"Protein purification, cloning, and kinase activity assay under various stress conditions\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — purification and direct biochemical characterization in a single lab with clear activity assay, but limited mechanistic depth beyond activation specificity\",\n      \"pmids\": [\"8816758\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"STK4-deficient lymphocytes and neutrophils exhibit enhanced loss of mitochondrial membrane potential and increased susceptibility to apoptosis, establishing STK4 as required for immune cell survival.\",\n      \"method\": \"Loss-of-function (homozygous truncation mutation in patients), mitochondrial membrane potential assay, apoptosis assay\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — patient-derived cells with defined genetic loss, two orthogonal cellular phenotype assays, single study\",\n      \"pmids\": [\"22294732\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"STK4 (and STK3) directly phosphorylates LC3 at threonine 50 (Thr50), and this phosphorylation is essential for autophagosome-lysosome fusion; loss of STK3/STK4 blocks autophagy and impairs intracellular bacterial clearance, which is rescued by a phosphomimetic LC3-T50E mutant.\",\n      \"method\": \"In vitro kinase assay, phosphosite identification, genetic loss-of-function (STK3/STK4-deficient cells), autophagy flux assay, autophagosome-lysosome fusion assay, bacterial clearance assay, phosphomimetic rescue experiment across multiple species\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — direct in vitro phosphorylation assay with site-specific mutagenesis, phosphomimetic rescue, multiple organisms, multiple orthogonal readouts\",\n      \"pmids\": [\"25544559\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"STK4 dampens TLR4/9-induced proinflammatory cytokine secretion and enhances TLR3/4-triggered IFN-β production by binding to and phosphorylating IRAK1, leading to IRAK1 degradation in macrophages.\",\n      \"method\": \"Co-immunoprecipitation (binding), in vitro phosphorylation assay, macrophage-specific Stk4 knockout mice, cytokine ELISA, IRAK1 protein level measurement\",\n      \"journal\": \"The Journal of clinical investigation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — direct binding demonstrated by Co-IP, direct phosphorylation assay, in vivo genetic knockout with defined phenotypic readouts, multiple orthogonal methods\",\n      \"pmids\": [\"26457732\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"STK4 phosphorylates LC3 at Thr50 as a conserved regulatory mechanism for autophagy; this phosphorylation is critical for autophagosome-lysosome fusion and intracellular bacteria clearance.\",\n      \"method\": \"In vitro kinase assay, site-directed mutagenesis, autophagy assays\",\n      \"journal\": \"Autophagy\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — single commentary/follow-up paper confirming the same finding as PMID 25544559; no new independent methods\",\n      \"pmids\": [\"25996575\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"STK4 localizes to the cytoplasm, lipid rafts, and nucleus in prostate cancer cells, and nuclear/lipid raft localization produces superior suppression of cell growth compared to cytoplasmic STK4, with each compartment activating distinct gene expression programs including oncogenic pathways (AR, PI3K/AKT, BMP/SMAD, WNT, RAS, JAK/STAT).\",\n      \"method\": \"Subcellular fractionation, ectopic expression in defined compartments, in vitro and in vivo growth assays, RNA sequencing\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct subcellular localization experiment with functional consequence (growth suppression), multiple compartments tested, RNA-seq for pathway mapping, single lab\",\n      \"pmids\": [\"28880957\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"STK4/MST1 phosphorylates BECN1 at threonine 108 (T108) within its BH3 domain, increasing BECN1 affinity for anti-apoptotic proteins BCL2 and BCL2L1, thereby blocking autophagy; X-ray crystal structures of BCL2 and BCL2L1 complexed with T108-modified BECN1 BH3 peptides revealed the structural basis of this interaction, showing only minor (<2-fold) affinity increase.\",\n      \"method\": \"X-ray crystallography, surface plasmon resonance, microscale thermophoresis, biophysical binding assays, molecular dynamics simulation\",\n      \"journal\": \"Autophagy\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure determined, multiple biophysical methods (SPR, MST, MD), precise quantification of phosphorylation-dependent affinity change\",\n      \"pmids\": [\"30626284\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"lncRNA TNRC6C-AS1 recruits methyltransferase to the STK4 promoter to increase STK4 promoter methylation and down-regulate STK4 expression, thereby activating the Hippo signaling pathway in thyroid carcinoma cells.\",\n      \"method\": \"ChIP/methyltransferase binding assay, STK4 promoter methylation analysis, gene silencing/overexpression, proliferation and apoptosis assays\",\n      \"journal\": \"Journal of cellular and molecular medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — methyltransferase-STK4 promoter interaction demonstrated, promoter methylation quantified, functional rescue experiments; single lab\",\n      \"pmids\": [\"31657132\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"TRAF6 mediates LPS-induced ubiquitination of STK4/MST1, activating STK4 which then inhibits TRAF6 autoubiquitination and downstream NF-κB signaling, constituting a negative feedback loop in macrophage TLR4 signaling.\",\n      \"method\": \"Myeloid-specific genetic ablation (KO mice), Co-immunoprecipitation, ubiquitination assay, NF-κB activation assay, cytokine measurement, LPS-induced septic shock model\",\n      \"journal\": \"Cellular and molecular life sciences : CMLS\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — in vivo knockout, direct ubiquitination assay, Co-IP for interaction, multiple orthogonal functional readouts, reciprocal regulation demonstrated\",\n      \"pmids\": [\"32975614\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"STK4 deficiency impairs TBK1-IRF3 phosphorylation, leading to significantly reduced type I, II, and III interferon responses to TLR3, TLR9, and cytosolic RNA/DNA sensor ligands.\",\n      \"method\": \"Patient-derived cells (STK4 frameshift mutation), phospho-TBK1 and phospho-IRF3 immunoblot, cytokine ELISA, viral infection assay\",\n      \"journal\": \"Journal of clinical immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — patient-derived loss-of-function, direct phosphorylation readout of pathway components, single patient/lab\",\n      \"pmids\": [\"33078349\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"STK4 directly phosphorylates β-catenin leading to its degradation via the ubiquitin-mediated pathway; STK4 colocalization with β-catenin was demonstrated, and STK4 knockdown causes β-catenin accumulation, promoting anchorage-independent growth and metastasis in colon cancer.\",\n      \"method\": \"Co-localization assay, in vitro kinase assay (direct phosphorylation of β-catenin), ubiquitin degradation assay, STK4 knockdown with metastasis phenotype readout, in vivo tumor model\",\n      \"journal\": \"Molecular oncology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — direct kinase assay showing phosphorylation of β-catenin, co-localization, ubiquitin pathway involvement, in vivo model; single lab\",\n      \"pmids\": [\"32741119\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"STK3 and STK4 suppress mitochondrial capacity in adipocytes and regulate mitophagy by controlling phosphorylation and dimerization status of the mitophagy receptor BNIP3; genetic inactivation of Stk3/Stk4 increases mitochondrial mass and function, stabilizes UCP1 in beige adipose tissue, and confers resistance to metabolic dysfunction.\",\n      \"method\": \"Genetic inactivation (adipose-specific knockout mice), mitochondrial function assays, BNIP3 phosphorylation assay, dimerization assay, metabolic phenotyping, pharmacological inhibition\",\n      \"journal\": \"Nature metabolism\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — in vivo genetic knockout with multiple metabolic phenotype readouts, direct phosphorylation/dimerization of BNIP3 demonstrated, pharmacological validation, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"33758424\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"ROR2 inhibits STK4 phosphorylation (activity), which promotes nuclear translocation of FOXO1 that directly represses SMS1 transcription; this ROR2/STK4-FOXO1/SMS1 axis regulates sphingomyelin biosynthesis and dental pulp stem cell senescence.\",\n      \"method\": \"STK4 phosphorylation assay, FOXO1 nuclear translocation assay, FOXO1 ChIP on SMS1 promoter, gene knockdown/overexpression, proliferation assay\",\n      \"journal\": \"Aging cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — STK4 phosphorylation status measured, direct FOXO1 binding to SMS1 promoter shown by ChIP, functional rescue; single lab\",\n      \"pmids\": [\"34278704\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Crystallization of an STK4 inhibitor compound with STK4 revealed two-point hinge binding in the ATP-binding pocket, providing structural basis for STK4 inhibitor design.\",\n      \"method\": \"X-ray crystallography of STK4-inhibitor complex\",\n      \"journal\": \"Journal of medicinal chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — crystal structure determined but functional validation of binding mode limited to inhibitor potency data; single study\",\n      \"pmids\": [\"34807584\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"TCR signaling induces nuclear translocation of STK4 in Treg cells, where STK4 forms a complex with NF-κB p65 and Foxp3; STK4 phosphorylates Foxp3 at serine-418 to stabilize this complex, which regulates Foxp3- and p65-dependent transcriptional programs required for Treg cell activation and immune tolerance.\",\n      \"method\": \"Nuclear translocation assay (live imaging/fractionation), Co-immunoprecipitation (STK4-p65-Foxp3 complex), in vitro phosphorylation assay (Foxp3 S418), phosphomimetic rescue (Foxp3-S418E), Treg-specific conditional knockout mice, adoptive immunotherapy model\",\n      \"journal\": \"Science immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — direct phosphorylation of Foxp3-S418 demonstrated, trimeric complex identified by Co-IP, phosphomimetic rescue, in vivo Treg-specific KO, multiple orthogonal methods\",\n      \"pmids\": [\"36149942\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"STK4 directly binds the RING2-LDD module of HOIP (the E3 ligase catalytic subunit of LUBAC) through its kinase domain, phosphorylates HOIP at T786 within its allosteric ubiquitin-binding site, thereby blocking ubiquitin binding to the allosteric site and attenuating HOIP's E3 ligase activity toward NF-κB signaling.\",\n      \"method\": \"Biochemical binding assay, mass spectrometry (phosphosite identification), X-ray crystallography (STK4-HOIP RING2-LDD complex structure), in vitro kinase assay, E3 ligase activity assay, ubiquitin-binding competition assay\",\n      \"journal\": \"Cell discovery\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure of STK4-substrate complex, direct phosphorylation site identified by MS, E3 activity assay with mechanistic explanation, multiple orthogonal methods in a single rigorous study\",\n      \"pmids\": [\"40957900\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"STK4 (MST1/KRS2) is a stress- and TCR-responsive serine/threonine kinase that directly phosphorylates multiple substrates—including LC3-Thr50 (driving autophagosome-lysosome fusion), BECN1-Thr108 (increasing BCL2 affinity to suppress autophagy), β-catenin (promoting ubiquitin-mediated degradation), IRAK1 (inducing its degradation to dampen TLR-NF-κB inflammation), BNIP3 (regulating mitophagy and mitochondrial capacity in adipocytes), Foxp3-Ser418 (stabilizing a nuclear STK4-p65-Foxp3 complex for Treg transcription), and HOIP-Thr786 (blocking the allosteric ubiquitin-binding site to inhibit LUBAC E3 activity)—and is itself activated by TRAF6-mediated ubiquitination; it also undergoes nuclear translocation upon TCR signaling, and its loss causes immune cell apoptosis, impaired interferon responses via reduced TBK1-IRF3 phosphorylation, and combined immunodeficiency in humans.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"STK4 (MST1/KRS2) is a stress- and apoptosis-responsive serine/threonine kinase of the Ste20p subfamily that acts as a central node integrating immune signaling, autophagy, and cell-survival decisions [#0]. It controls autophagy bidirectionally by directly phosphorylating LC3 at Thr50 to drive autophagosome-lysosome fusion and intracellular bacterial clearance [#2], while phosphorylation of BECN1 at Thr108 within its BH3 domain enhances BECN1 affinity for BCL2/BCL2L1 to suppress autophagy [#6]; in adipocytes it further regulates mitophagy and mitochondrial capacity through phosphorylation and dimerization control of BNIP3 [#11]. In innate immunity STK4 enforces negative feedback on TLR-NF-\\u03baB signaling: it is activated by TRAF6-mediated ubiquitination and in turn suppresses TRAF6 autoubiquitination [#8], binds and phosphorylates IRAK1 to trigger its degradation and dampen proinflammatory cytokines [#3], and phosphorylates the LUBAC catalytic subunit HOIP at Thr786 to block its allosteric ubiquitin-binding site and attenuate E3 ligase activity [#15]. Upon TCR signaling STK4 translocates to the nucleus, where it phosphorylates Foxp3 at Ser418 and stabilizes a nuclear STK4-p65-Foxp3 complex required for Treg activation and immune tolerance [#14]. It also phosphorylates \\u03b2-catenin to promote its ubiquitin-mediated degradation, restraining anchorage-independent growth and metastasis [#10]. Loss-of-function in humans causes combined immunodeficiency, with patient cells showing heightened apoptosis and loss of mitochondrial membrane potential [#1] and impaired type I/II/III interferon responses through reduced TBK1-IRF3 phosphorylation [#9].\",\n  \"teleology\": [\n    {\n      \"year\": 1996,\n      \"claim\": \"Established STK4 as a stress-activated kinase distinct from mitogenic signaling, defining its activation specificity before any substrate was known.\",\n      \"evidence\": \"Protein purification, cloning, and kinase assays under stress/apoptotic conditions\",\n      \"pmids\": [\"8816758\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No substrates identified\", \"Upstream activators undefined\", \"Physiological context unknown\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Linked STK4 to immune cell survival by showing its loss causes mitochondrial depolarization and apoptosis, connecting the kinase to human immunodeficiency.\",\n      \"evidence\": \"Patient-derived cells with homozygous truncation, mitochondrial membrane potential and apoptosis assays\",\n      \"pmids\": [\"22294732\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular mechanism linking STK4 loss to mitochondrial dysfunction not resolved\", \"Substrates mediating survival unknown\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Identified the first direct substrate-site relationship driving autophagy, showing STK4/STK3 phosphorylate LC3-Thr50 to enable autophagosome-lysosome fusion and bacterial clearance.\",\n      \"evidence\": \"In vitro kinase assay, phosphosite mapping, STK3/STK4-deficient cells, phosphomimetic LC3-T50E rescue across species\",\n      \"pmids\": [\"25544559\", \"25996575\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How fusion machinery reads the LC3-T50 phosphomark not defined\", \"Reconciliation with autophagy-suppressive roles unaddressed\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Defined STK4 as a brake on TLR-driven inflammation by phosphorylating IRAK1 to trigger its degradation while enhancing IFN-\\u03b2 output.\",\n      \"evidence\": \"Co-IP, in vitro phosphorylation, macrophage-specific Stk4 knockout, cytokine ELISA\",\n      \"pmids\": [\"26457732\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"IRAK1 phosphosite not specified\", \"Ubiquitin ligase coupling degradation unknown\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Showed STK4 function is compartment-dependent, with nuclear and lipid-raft pools driving distinct transcriptional programs and stronger growth suppression than cytoplasmic STK4.\",\n      \"evidence\": \"Subcellular fractionation, compartment-targeted expression, growth assays, RNA-seq in prostate cancer cells\",\n      \"pmids\": [\"28880957\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism of compartment targeting unknown\", \"Nuclear substrates not identified here\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Revealed an autophagy-suppressive arm: STK4 phosphorylates BECN1-Thr108 to raise BCL2/BCL2L1 affinity, with structural resolution of the modified interface.\",\n      \"evidence\": \"X-ray crystallography, SPR, MST, molecular dynamics\",\n      \"pmids\": [\"30626284\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Measured affinity increase was modest (<2-fold)\", \"Cellular context dictating pro- vs anti-autophagy roles unresolved\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Identified epigenetic silencing of STK4 via promoter methylation as a mechanism activating Hippo signaling in thyroid carcinoma.\",\n      \"evidence\": \"ChIP/methyltransferase binding, promoter methylation analysis, silencing/overexpression, proliferation and apoptosis assays\",\n      \"pmids\": [\"31657132\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct link to downstream Hippo effectors not mechanistically detailed\", \"Single tumor context\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Established a TRAF6-STK4 negative feedback loop in which TRAF6 ubiquitinates and activates STK4, which then suppresses TRAF6 autoubiquitination and NF-\\u03baB.\",\n      \"evidence\": \"Myeloid-specific KO, Co-IP, ubiquitination assays, NF-\\u03baB readouts, LPS septic shock model\",\n      \"pmids\": [\"32975614\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Ubiquitin chain types and STK4 ubiquitination sites not fully mapped\", \"Mechanism of TRAF6 suppression by STK4 unclear\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Connected STK4 to interferon immunity, showing its loss impairs TBK1-IRF3 phosphorylation and broad type I/II/III IFN responses.\",\n      \"evidence\": \"Patient-derived cells with frameshift mutation, phospho-immunoblot, cytokine ELISA, viral infection\",\n      \"pmids\": [\"33078349\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether STK4 acts directly on TBK1 not established\", \"Single patient/lab\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Defined a tumor-suppressive role through direct phosphorylation of \\u03b2-catenin promoting its ubiquitin-mediated degradation.\",\n      \"evidence\": \"Co-localization, in vitro kinase assay, ubiquitin degradation assay, knockdown metastasis and in vivo tumor models\",\n      \"pmids\": [\"32741119\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"\\u03b2-catenin phosphosite not specified\", \"Ligase coupling degradation unidentified\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Extended STK4 into metabolic regulation by controlling BNIP3-dependent mitophagy and mitochondrial capacity in adipose tissue.\",\n      \"evidence\": \"Adipose-specific KO, mitochondrial and metabolic phenotyping, BNIP3 phosphorylation/dimerization assays, pharmacological inhibition\",\n      \"pmids\": [\"33758424\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"BNIP3 phosphosite(s) not specified\", \"Link to whole-body metabolism mechanistically partial\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Placed STK4 within a ROR2-FOXO1 axis regulating sphingomyelin synthesis and stem cell senescence, showing inhibition of STK4 activity permits FOXO1 nuclear translocation.\",\n      \"evidence\": \"STK4 phosphorylation assay, FOXO1 nuclear translocation, FOXO1 ChIP on SMS1, knockdown/overexpression\",\n      \"pmids\": [\"34278704\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct STK4-FOXO1 phosphorylation not demonstrated here\", \"Single lineage context\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Provided a structural template for STK4 inhibitor design by resolving an inhibitor bound in the ATP pocket via two-point hinge binding.\",\n      \"evidence\": \"X-ray crystallography of STK4-inhibitor complex\",\n      \"pmids\": [\"34807584\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Cellular selectivity and on-target effects beyond potency not characterized\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Revealed a nuclear, kinase-dependent transcriptional role in Tregs, where TCR-induced STK4 phosphorylates Foxp3-S418 and stabilizes a STK4-p65-Foxp3 complex for immune tolerance.\",\n      \"evidence\": \"Nuclear translocation imaging/fractionation, Co-IP of trimeric complex, in vitro kinase assay, Foxp3-S418E rescue, Treg-specific KO, adoptive immunotherapy model\",\n      \"pmids\": [\"36149942\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Signal driving nuclear translocation mechanistically incomplete\", \"Genome-wide complex targets not mapped\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Defined a structural mechanism by which STK4 inhibits LUBAC, phosphorylating HOIP-Thr786 in its allosteric ubiquitin-binding site to block ubiquitin binding and attenuate E3 activity.\",\n      \"evidence\": \"Biochemical binding, MS phosphosite mapping, X-ray crystallography of STK4-HOIP RING2-LDD complex, kinase and E3 activity assays, ubiquitin-binding competition\",\n      \"pmids\": [\"40957900\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo consequence of HOIP-T786 phosphorylation not established\", \"Integration with other STK4 NF-\\u03baB feedback arms unresolved\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How STK4 selects between its opposing roles (pro- vs anti-autophagy, kinase-dependent cytoplasmic feedback vs nuclear transcriptional complexes) in a given cell remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No unifying model of substrate selection by compartment or stimulus\", \"Upstream determinants of nuclear translocation incompletely defined\", \"Crosstalk among the multiple NF-\\u03baB-regulatory substrates not integrated\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [2, 3, 6, 10, 11, 14, 15]},\n      {\"term_id\": \"GO:0016740\", \"supporting_discovery_ids\": [2, 3, 6, 14, 15]},\n      {\"term_id\": \"GO:0140657\", \"supporting_discovery_ids\": [13]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [5]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [5, 14]},\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [5]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-9612973\", \"supporting_discovery_ids\": [2, 6, 11]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [3, 8, 9, 14]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [8, 10, 15]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [1, 6]}\n    ],\n    \"complexes\": [\"STK4-p65-Foxp3 nuclear complex\"],\n    \"partners\": [\"IRAK1\", \"TRAF6\", \"BECN1\", \"BNIP3\", \"CTNNB1\", \"FOXP3\", \"RELA\", \"RNF31\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}