{"gene":"STK35","run_date":"2026-06-10T10:51:54","timeline":{"discoveries":[{"year":2002,"finding":"CLIK1 (STK35) is a nuclear kinase capable of autophosphorylation. Yeast two-hybrid analysis identified CLP-36 PDZ-LIM protein as a highly specific binding partner (36/37 clones). The association is mediated by the C-terminal LIM domain of CLP-36. Interaction with CLP-36 relocalizes the otherwise nuclear CLIK1 to actin stress fibers, where it disrupts the periodic staining pattern of CLP-36, establishing CLP-36 as an adapter that recruits CLIK1 to actin stress fibers.","method":"Yeast two-hybrid, autophosphorylation assay, colocalization/relocalization imaging","journal":"Journal of cell science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — yeast two-hybrid with high specificity (36/37 clones), autophosphorylation assay, relocalization imaging; single lab but multiple orthogonal methods","pmids":["11973348"],"is_preprint":false},{"year":2011,"finding":"STK35L1 (full-length STK35) localizes to the nucleus and nucleolus of endothelial cells via a bipartite nuclear localization signal and nucleolar localization sequences in its N-terminal region. Nuclear actin was identified as a novel binding partner mediated by a class III PDZ-binding domain motif in STK35L1. siRNA-mediated depletion of STK35L1 accelerated G1-to-S phase transition and prominently downregulated CDKN2A (p16INK4a) expression (~8.8-fold), indicating STK35L1 inhibits G1-to-S phase progression. STK35L1 depletion also impaired endothelial cell migration and sprouting.","method":"siRNA knockdown, cell cycle analysis, gene expression array, nuclear localization signal mapping, binding partner identification (PDZ-domain mediated actin interaction), wound healing assay, Matrigel sprouting assay","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — siRNA knockdown with multiple defined phenotypic readouts (cell cycle, migration, gene expression), nuclear localization experimentally mapped; single lab, multiple orthogonal methods","pmids":["21283756"],"is_preprint":false},{"year":2009,"finding":"The previously published STK35 coding sequence was found to be incomplete; the full-length protein (STK35L1) is 534 amino acids (58 kDa) with a 133 amino acid N-terminal extension, and is expressed in all human cell lines tested. EGFP-STK35L1 localizes to the nucleus and nucleolus.","method":"Molecular cloning, SDS-PAGE, siRNA silencing confirmation, EGFP fusion localization imaging","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct cloning and expression validation, subcellular localization by fluorescent imaging; single lab, multiple methods","pmids":["19756140"],"is_preprint":false},{"year":2022,"finding":"SCP4 phosphatase forms a complex with STK35 and its paralog PDIK1L; mass spectrometry of affinity-purified complexes identified STK35 and PDIK1L as binding partners and substrates of the SCP4 phosphatase domain. SCP4 regulates STK35 through two mechanisms: (1) catalytic removal of inhibitory phosphorylation on STK35, and (2) promoting STK35 protein stability. STK35 and PDIK1L function catalytically and redundantly in the same pathway as SCP4 to maintain AML proliferation and support amino acid biosynthesis and transport.","method":"CRISPR domain-focused screen, affinity purification–mass spectrometry, gene complementation assays, CRISPR exon scanning","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — affinity purification-MS identified complex and substrates, catalytic function confirmed by complementation, two distinct regulatory mechanisms demonstrated, domain-focused CRISPR screen with multiple orthogonal approaches","pmids":["35021089"],"is_preprint":false},{"year":2020,"finding":"NEDD4L E3 ubiquitin ligase associates with STK35 and ubiquitinates it (shown by co-immunoprecipitation). STK35 overexpression upregulates phospho-AKT, promotes glycolysis, and suppresses apoptosis in colorectal cancer cells; STK35 knockdown has the opposite effects, placing STK35 upstream of AKT signaling.","method":"Co-immunoprecipitation (ubiquitination assay), siRNA knockdown, STK35 overexpression, AKT phosphorylation assay, glycolysis measurement, apoptosis assay","journal":"Frontiers in cell and developmental biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal co-IP for ubiquitination, gain- and loss-of-function with multiple cellular readouts; single lab","pmids":["33117809"],"is_preprint":false},{"year":2021,"finding":"STK35L1 is upregulated during Plasmodium berghei sporozoite infection in HepG2 cells and mouse liver, and this upregulation is dependent on STAT3 activation (which is phosphorylated upon infection). STK35L1 knockdown suppressed sporozoite infection and inhibited basal expression of ten cell cycle genes in infected hepatocytes (except CDKN3 and GTSE1), establishing STK35L1 as a host kinase required for the liver stage of malaria downstream of STAT3.","method":"siRNA knockdown, Plasmodium berghei infection assay, STAT3 phosphorylation analysis, cell cycle gene expression profiling","journal":"Experimental cell research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — siRNA loss-of-function with defined infection phenotype and pathway placement (STAT3→STK35L1→cell cycle genes); single lab, multiple readouts","pmids":["34358525"],"is_preprint":false},{"year":2026,"finding":"STK35 functions as a broad regulator of the DNA damage response (DDR); its depletion sensitizes diverse human cell lines to DNA-damaging agents (especially DNA adduct-forming agents such as cisplatin and MMS). Saturated mutagenesis base-editor screening identified specific residues within the conserved kinase domain—notably A423 and W445—whose mutation markedly sensitizes cells to multiple DNA-damaging agents, indicating these residues are functionally critical. SCP4 phenocopies STK35 loss under cisplatin/MMS treatment.","method":"siRNA/CRISPR depletion, saturated adenine base editor (ABE) and cytosine base editor (CBE) screens, genotoxic drug sensitivity assays","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 1-2 / Moderate — systematic saturated mutagenesis at single-amino-acid resolution with functional readout; single lab, but comprehensive mutagenesis screen with multiple orthogonal base editors","pmids":["42161060"],"is_preprint":false}],"current_model":"STK35 (CLIK1/STK35L1) is a nuclear serine/threonine kinase capable of autophosphorylation that localizes to the nucleus and nucleolus via N-terminal NLS sequences; it is recruited to actin stress fibers by the CLP-36 PDZ-LIM adapter protein via CLP-36's LIM domain, interacts with nuclear actin through a PDZ-binding motif, inhibits G1-to-S phase cell cycle progression partly by maintaining CDKN2A (p16INK4a) expression, regulates endothelial cell migration and angiogenesis, forms a phospho-catalytic complex with the SCP4 phosphatase (which removes inhibitory phosphorylation from STK35 and stabilizes it) and its paralog PDIK1L to support amino acid biosynthesis and AML proliferation, is ubiquitinated by NEDD4L and acts upstream of AKT signaling to promote glycolysis and suppress apoptosis, serves as a broad regulator of the DNA damage response with functionally critical kinase domain residues A423 and W445, and plays an obligatory host role in Plasmodium liver-stage infection downstream of STAT3 activation."},"narrative":{"mechanistic_narrative":"STK35 (CLIK1/STK35L1) is a nuclear and nucleolar serine/threonine kinase that couples cell-cycle control to actin-associated signaling and, more recently, to nutrient metabolism and genome maintenance [PMID:11973348, PMID:19756140, PMID:35021089]. The full-length 534-residue protein localizes to the nucleus and nucleolus through N-terminal bipartite nuclear and nucleolar localization signals, undergoes autophosphorylation, and engages the actin cytoskeleton via two routes: recruitment to actin stress fibers by the CLP-36 PDZ-LIM adapter through CLP-36's LIM domain, and direct binding to nuclear actin through its class III PDZ-binding motif [PMID:11973348, PMID:21283756, PMID:19756140]. STK35 restrains G1-to-S progression and sustains CDKN2A (p16INK4a) expression, and its depletion impairs endothelial cell migration and sprouting [PMID:21283756]. Its activity is set by post-translational control: the SCP4 phosphatase forms a complex with STK35 and its paralog PDIK1L, removing inhibitory phosphorylation and stabilizing STK35, with all three acting catalytically and redundantly to support amino acid biosynthesis/transport and AML proliferation, while the NEDD4L E3 ligase ubiquitinates STK35 in a pathway placing STK35 upstream of AKT to promote glycolysis and suppress apoptosis [PMID:35021089, PMID:33117809]. STK35 additionally acts as a broad regulator of the DNA damage response—depletion sensitizes cells to DNA adduct-forming agents and the kinase-domain residues A423 and W445 are functionally critical—and serves as a host kinase required for Plasmodium liver-stage infection downstream of STAT3 [PMID:34358525, PMID:42161060].","teleology":[{"year":2002,"claim":"Established STK35/CLIK1 as a nuclear autophosphorylating kinase and identified a physical and spatial link to the actin cytoskeleton, answering whether this kinase has defined binding partners and a regulated localization.","evidence":"Yeast two-hybrid (36/37 clones), autophosphorylation assay, and relocalization imaging in cells","pmids":["11973348"],"confidence":"Medium","gaps":["No substrate of the kinase identified","Functional consequence of stress-fiber recruitment not determined","Single-lab interaction not reciprocally validated in vivo"]},{"year":2009,"claim":"Corrected the gene model, defining the full-length 534-aa STK35L1 protein and confirming broad expression with nuclear/nucleolar localization, providing the correct molecular entity for subsequent study.","evidence":"Molecular cloning, SDS-PAGE, siRNA validation, and EGFP fusion imaging","pmids":["19756140"],"confidence":"Medium","gaps":["Functional role of the N-terminal extension not tested","No catalytic characterization of full-length protein"]},{"year":2011,"claim":"Mapped the localization signals and a nuclear actin interaction and assigned a cell-cycle and angiogenic function, showing STK35L1 inhibits G1-to-S transition and supports endothelial migration.","evidence":"siRNA knockdown with cell-cycle analysis, expression arrays, NLS/NoLS mapping, wound-healing and Matrigel sprouting assays","pmids":["21283756"],"confidence":"Medium","gaps":["Mechanism linking STK35L1 to CDKN2A expression not resolved","Direct kinase substrates in the cell cycle not identified","Whether nuclear actin binding mediates the cell-cycle effect untested"]},{"year":2020,"claim":"Identified NEDD4L-mediated ubiquitination of STK35 and placed STK35 upstream of AKT-driven glycolysis and apoptosis suppression, connecting the kinase to metabolic survival signaling in cancer.","evidence":"Reciprocal co-immunoprecipitation/ubiquitination assay, siRNA knockdown and overexpression with AKT phosphorylation, glycolysis, and apoptosis readouts","pmids":["33117809"],"confidence":"Medium","gaps":["Whether STK35 phosphorylates an AKT-pathway component directly is unknown","Functional consequence of NEDD4L ubiquitination (degradation vs. signaling) not defined","Single-lab findings in colorectal cells only"]},{"year":2021,"claim":"Demonstrated an obligatory host role for STK35L1 in malaria liver-stage infection downstream of STAT3, linking the kinase to a host-pathogen cell-cycle gene program.","evidence":"siRNA knockdown with Plasmodium berghei infection assays, STAT3 phosphorylation analysis, and cell-cycle gene profiling in hepatocytes","pmids":["34358525"],"confidence":"Medium","gaps":["Direct kinase target underlying infection support not identified","Mechanism by which STAT3 induces STK35L1 unresolved","Single-lab loss-of-function evidence"]},{"year":2022,"claim":"Resolved the post-translational regulation of STK35 by showing SCP4 phosphatase forms a complex with STK35 and PDIK1L, removing inhibitory phosphorylation and stabilizing it, with the kinases acting redundantly to support amino acid biosynthesis and AML proliferation.","evidence":"Domain-focused CRISPR screen, affinity purification–mass spectrometry, and gene complementation/exon-scanning assays","pmids":["35021089"],"confidence":"High","gaps":["Phosphosites removed by SCP4 not individually mapped to function","Direct kinase substrates in amino acid metabolism not identified","Whether SCP4 regulation operates outside AML untested"]},{"year":2026,"claim":"Established STK35 as a broad DNA damage response regulator and pinpointed kinase-domain residues A423 and W445 as functionally critical, defining a genome-maintenance role and structure-function landmarks.","evidence":"siRNA/CRISPR depletion, saturated adenine and cytosine base-editor mutagenesis screens, and genotoxic drug sensitivity assays","pmids":["42161060"],"confidence":"Medium","gaps":["DDR substrate(s) of STK35 not identified","Step in the DDR at which STK35 acts not defined","Mechanistic role of A423/W445 (catalysis vs. structure) not resolved"]},{"year":null,"claim":"The direct physiological substrate(s) of STK35 kinase activity remain unidentified across all its assigned roles (cell cycle, AKT signaling, amino acid metabolism, DDR, malaria infection).","evidence":"","pmids":[],"confidence":"Medium","gaps":["No phosphorylation substrate experimentally established","Unclear whether the diverse phenotypes converge on a common molecular target","No structural model linking A423/W445 to substrate engagement"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,3,6]},{"term_id":"GO:0016740","term_label":"transferase activity","supporting_discovery_ids":[0,3]},{"term_id":"GO:0008092","term_label":"cytoskeletal protein binding","supporting_discovery_ids":[0,1]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[0,1,2]},{"term_id":"GO:0005730","term_label":"nucleolus","supporting_discovery_ids":[1,2]},{"term_id":"GO:0005856","term_label":"cytoskeleton","supporting_discovery_ids":[0]}],"pathway":[{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[1,5]},{"term_id":"R-HSA-73894","term_label":"DNA Repair","supporting_discovery_ids":[6]},{"term_id":"R-HSA-1430728","term_label":"Metabolism","supporting_discovery_ids":[3,4]}],"complexes":["SCP4–STK35–PDIK1L phospho-catalytic complex"],"partners":["CLP-36","SCP4","PDIK1L","NEDD4L"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q8TDR2","full_name":"Serine/threonine-protein kinase 35","aliases":["CLP-36-interacting kinase 1","CLIK-1","PDLIM1-interacting kinase 1","Serine/threonine-protein kinase 35 L1"],"length_aa":534,"mass_kda":58.1,"function":"","subcellular_location":"Nucleus; Nucleus, nucleolus; Cytoplasm","url":"https://www.uniprot.org/uniprotkb/Q8TDR2/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/STK35","classification":"Not Classified","n_dependent_lines":33,"n_total_lines":1208,"dependency_fraction":0.027317880794701987},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/STK35","total_profiled":1310},"omim":[{"mim_id":"610785","title":"PDLIM1-INTERACTING KINASE 1-LIKE; PDIK1L","url":"https://www.omim.org/entry/610785"},{"mim_id":"609370","title":"SERINE/THREONINE KINASE 35; STK35","url":"https://www.omim.org/entry/609370"},{"mim_id":"605900","title":"PDZ AND LIM DOMAIN PROTEIN 1; PDLIM1","url":"https://www.omim.org/entry/605900"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Nucleoplasm","reliability":"Supported"},{"location":"Nuclear bodies","reliability":"Supported"}],"tissue_specificity":"Tissue enriched","tissue_distribution":"Detected in all","driving_tissues":[{"tissue":"retina","ntpm":110.2}],"url":"https://www.proteinatlas.org/search/STK35"},"hgnc":{"alias_symbol":["bA550O8.2","CLIK1","STK35L1"],"prev_symbol":[]},"alphafold":{"accession":"Q8TDR2","domains":[{"cath_id":"3.30.200.20","chopping":"203-317","consensus_level":"high","plddt":85.5257,"start":203,"end":317},{"cath_id":"1.10.510.10","chopping":"322-390_408-534","consensus_level":"high","plddt":89.2417,"start":322,"end":534}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q8TDR2","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q8TDR2-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q8TDR2-F1-predicted_aligned_error_v6.png","plddt_mean":66.19},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=STK35","jax_strain_url":"https://www.jax.org/strain/search?query=STK35"},"sequence":{"accession":"Q8TDR2","fasta_url":"https://rest.uniprot.org/uniprotkb/Q8TDR2.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q8TDR2/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q8TDR2"}},"corpus_meta":[{"pmid":"11973348","id":"PMC_11973348","title":"Clik1: a novel kinase targeted to actin stress fibers by the CLP-36 PDZ-LIM protein.","date":"2002","source":"Journal of cell science","url":"https://pubmed.ncbi.nlm.nih.gov/11973348","citation_count":61,"is_preprint":false},{"pmid":"33117809","id":"PMC_33117809","title":"STK35 Is Ubiquitinated by NEDD4L and Promotes Glycolysis and Inhibits Apoptosis Through Regulating the AKT Signaling Pathway, Influencing Chemoresistance of Colorectal Cancer.","date":"2020","source":"Frontiers in cell and developmental biology","url":"https://pubmed.ncbi.nlm.nih.gov/33117809","citation_count":29,"is_preprint":false},{"pmid":"21283756","id":"PMC_21283756","title":"STK35L1 associates with nuclear actin and regulates cell cycle and migration of endothelial cells.","date":"2011","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/21283756","citation_count":26,"is_preprint":false},{"pmid":"19756140","id":"PMC_19756140","title":"Identifying and characterizing a novel protein kinase STK35L1 and deciphering its orthologs and close-homologs in vertebrates.","date":"2009","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/19756140","citation_count":10,"is_preprint":false},{"pmid":"35780559","id":"PMC_35780559","title":"Promoter polymorphisms in STK35 and IFT27 genes and their associations with boar sperm freezability.","date":"2022","source":"Theriogenology","url":"https://pubmed.ncbi.nlm.nih.gov/35780559","citation_count":7,"is_preprint":false},{"pmid":"35021089","id":"PMC_35021089","title":"SCP4-STK35/PDIK1L complex is a dual phospho-catalytic signaling dependency in acute myeloid leukemia.","date":"2022","source":"Cell reports","url":"https://pubmed.ncbi.nlm.nih.gov/35021089","citation_count":4,"is_preprint":false},{"pmid":"34358525","id":"PMC_34358525","title":"STK35L1 regulates host cell cycle-related genes and is essential for Plasmodium infection during the liver stage of malaria.","date":"2021","source":"Experimental cell research","url":"https://pubmed.ncbi.nlm.nih.gov/34358525","citation_count":4,"is_preprint":false},{"pmid":"29970477","id":"PMC_29970477","title":"The STK35 locus contributes to normal gametogenesis and encodes a lncRNA responsive to oxidative stress.","date":"2018","source":"Biology open","url":"https://pubmed.ncbi.nlm.nih.gov/29970477","citation_count":4,"is_preprint":false},{"pmid":"35097018","id":"PMC_35097018","title":"STK35 Gene Therapy Attenuates Endothelial Dysfunction and Improves Cardiac Function in Diabetes.","date":"2022","source":"Frontiers in cardiovascular medicine","url":"https://pubmed.ncbi.nlm.nih.gov/35097018","citation_count":2,"is_preprint":false},{"pmid":"37229227","id":"PMC_37229227","title":"Retraction: STK35 gene therapy attenuates endothelial dysfunction and improves cardiac function in diabetes.","date":"2023","source":"Frontiers in cardiovascular medicine","url":"https://pubmed.ncbi.nlm.nih.gov/37229227","citation_count":0,"is_preprint":false},{"pmid":"42161060","id":"PMC_42161060","title":"The functional landscape of STK35 residues at single-amino-acid resolution in the DNA damage response and genotoxic drug vulnerability.","date":"2026","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/42161060","citation_count":0,"is_preprint":false},{"pmid":"35218030","id":"PMC_35218030","title":"A novel bactericidal small molecule, STK-35, and its derivative, STK-66, as antibacterial agents against Gram-negative pathogenic bacteria in vitro and in vivo.","date":"2022","source":"Letters in applied microbiology","url":"https://pubmed.ncbi.nlm.nih.gov/35218030","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":7712,"output_tokens":2309,"usd":0.028886,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":9411,"output_tokens":3153,"usd":0.06294,"stage2_stop_reason":"end_turn"},"total_usd":0.091826,"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\": 2002,\n      \"finding\": \"CLIK1 (STK35) is a nuclear kinase capable of autophosphorylation. Yeast two-hybrid analysis identified CLP-36 PDZ-LIM protein as a highly specific binding partner (36/37 clones). The association is mediated by the C-terminal LIM domain of CLP-36. Interaction with CLP-36 relocalizes the otherwise nuclear CLIK1 to actin stress fibers, where it disrupts the periodic staining pattern of CLP-36, establishing CLP-36 as an adapter that recruits CLIK1 to actin stress fibers.\",\n      \"method\": \"Yeast two-hybrid, autophosphorylation assay, colocalization/relocalization imaging\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — yeast two-hybrid with high specificity (36/37 clones), autophosphorylation assay, relocalization imaging; single lab but multiple orthogonal methods\",\n      \"pmids\": [\"11973348\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"STK35L1 (full-length STK35) localizes to the nucleus and nucleolus of endothelial cells via a bipartite nuclear localization signal and nucleolar localization sequences in its N-terminal region. Nuclear actin was identified as a novel binding partner mediated by a class III PDZ-binding domain motif in STK35L1. siRNA-mediated depletion of STK35L1 accelerated G1-to-S phase transition and prominently downregulated CDKN2A (p16INK4a) expression (~8.8-fold), indicating STK35L1 inhibits G1-to-S phase progression. STK35L1 depletion also impaired endothelial cell migration and sprouting.\",\n      \"method\": \"siRNA knockdown, cell cycle analysis, gene expression array, nuclear localization signal mapping, binding partner identification (PDZ-domain mediated actin interaction), wound healing assay, Matrigel sprouting assay\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — siRNA knockdown with multiple defined phenotypic readouts (cell cycle, migration, gene expression), nuclear localization experimentally mapped; single lab, multiple orthogonal methods\",\n      \"pmids\": [\"21283756\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"The previously published STK35 coding sequence was found to be incomplete; the full-length protein (STK35L1) is 534 amino acids (58 kDa) with a 133 amino acid N-terminal extension, and is expressed in all human cell lines tested. EGFP-STK35L1 localizes to the nucleus and nucleolus.\",\n      \"method\": \"Molecular cloning, SDS-PAGE, siRNA silencing confirmation, EGFP fusion localization imaging\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct cloning and expression validation, subcellular localization by fluorescent imaging; single lab, multiple methods\",\n      \"pmids\": [\"19756140\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"SCP4 phosphatase forms a complex with STK35 and its paralog PDIK1L; mass spectrometry of affinity-purified complexes identified STK35 and PDIK1L as binding partners and substrates of the SCP4 phosphatase domain. SCP4 regulates STK35 through two mechanisms: (1) catalytic removal of inhibitory phosphorylation on STK35, and (2) promoting STK35 protein stability. STK35 and PDIK1L function catalytically and redundantly in the same pathway as SCP4 to maintain AML proliferation and support amino acid biosynthesis and transport.\",\n      \"method\": \"CRISPR domain-focused screen, affinity purification–mass spectrometry, gene complementation assays, CRISPR exon scanning\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — affinity purification-MS identified complex and substrates, catalytic function confirmed by complementation, two distinct regulatory mechanisms demonstrated, domain-focused CRISPR screen with multiple orthogonal approaches\",\n      \"pmids\": [\"35021089\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"NEDD4L E3 ubiquitin ligase associates with STK35 and ubiquitinates it (shown by co-immunoprecipitation). STK35 overexpression upregulates phospho-AKT, promotes glycolysis, and suppresses apoptosis in colorectal cancer cells; STK35 knockdown has the opposite effects, placing STK35 upstream of AKT signaling.\",\n      \"method\": \"Co-immunoprecipitation (ubiquitination assay), siRNA knockdown, STK35 overexpression, AKT phosphorylation assay, glycolysis measurement, apoptosis assay\",\n      \"journal\": \"Frontiers in cell and developmental biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal co-IP for ubiquitination, gain- and loss-of-function with multiple cellular readouts; single lab\",\n      \"pmids\": [\"33117809\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"STK35L1 is upregulated during Plasmodium berghei sporozoite infection in HepG2 cells and mouse liver, and this upregulation is dependent on STAT3 activation (which is phosphorylated upon infection). STK35L1 knockdown suppressed sporozoite infection and inhibited basal expression of ten cell cycle genes in infected hepatocytes (except CDKN3 and GTSE1), establishing STK35L1 as a host kinase required for the liver stage of malaria downstream of STAT3.\",\n      \"method\": \"siRNA knockdown, Plasmodium berghei infection assay, STAT3 phosphorylation analysis, cell cycle gene expression profiling\",\n      \"journal\": \"Experimental cell research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — siRNA loss-of-function with defined infection phenotype and pathway placement (STAT3→STK35L1→cell cycle genes); single lab, multiple readouts\",\n      \"pmids\": [\"34358525\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"STK35 functions as a broad regulator of the DNA damage response (DDR); its depletion sensitizes diverse human cell lines to DNA-damaging agents (especially DNA adduct-forming agents such as cisplatin and MMS). Saturated mutagenesis base-editor screening identified specific residues within the conserved kinase domain—notably A423 and W445—whose mutation markedly sensitizes cells to multiple DNA-damaging agents, indicating these residues are functionally critical. SCP4 phenocopies STK35 loss under cisplatin/MMS treatment.\",\n      \"method\": \"siRNA/CRISPR depletion, saturated adenine base editor (ABE) and cytosine base editor (CBE) screens, genotoxic drug sensitivity assays\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — systematic saturated mutagenesis at single-amino-acid resolution with functional readout; single lab, but comprehensive mutagenesis screen with multiple orthogonal base editors\",\n      \"pmids\": [\"42161060\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"STK35 (CLIK1/STK35L1) is a nuclear serine/threonine kinase capable of autophosphorylation that localizes to the nucleus and nucleolus via N-terminal NLS sequences; it is recruited to actin stress fibers by the CLP-36 PDZ-LIM adapter protein via CLP-36's LIM domain, interacts with nuclear actin through a PDZ-binding motif, inhibits G1-to-S phase cell cycle progression partly by maintaining CDKN2A (p16INK4a) expression, regulates endothelial cell migration and angiogenesis, forms a phospho-catalytic complex with the SCP4 phosphatase (which removes inhibitory phosphorylation from STK35 and stabilizes it) and its paralog PDIK1L to support amino acid biosynthesis and AML proliferation, is ubiquitinated by NEDD4L and acts upstream of AKT signaling to promote glycolysis and suppress apoptosis, serves as a broad regulator of the DNA damage response with functionally critical kinase domain residues A423 and W445, and plays an obligatory host role in Plasmodium liver-stage infection downstream of STAT3 activation.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"STK35 (CLIK1/STK35L1) is a nuclear and nucleolar serine/threonine kinase that couples cell-cycle control to actin-associated signaling and, more recently, to nutrient metabolism and genome maintenance [#0, #2, #3]. The full-length 534-residue protein localizes to the nucleus and nucleolus through N-terminal bipartite nuclear and nucleolar localization signals, undergoes autophosphorylation, and engages the actin cytoskeleton via two routes: recruitment to actin stress fibers by the CLP-36 PDZ-LIM adapter through CLP-36's LIM domain, and direct binding to nuclear actin through its class III PDZ-binding motif [#0, #1, #2]. STK35 restrains G1-to-S progression and sustains CDKN2A (p16INK4a) expression, and its depletion impairs endothelial cell migration and sprouting [#1]. Its activity is set by post-translational control: the SCP4 phosphatase forms a complex with STK35 and its paralog PDIK1L, removing inhibitory phosphorylation and stabilizing STK35, with all three acting catalytically and redundantly to support amino acid biosynthesis/transport and AML proliferation, while the NEDD4L E3 ligase ubiquitinates STK35 in a pathway placing STK35 upstream of AKT to promote glycolysis and suppress apoptosis [#3, #4]. STK35 additionally acts as a broad regulator of the DNA damage response—depletion sensitizes cells to DNA adduct-forming agents and the kinase-domain residues A423 and W445 are functionally critical—and serves as a host kinase required for Plasmodium liver-stage infection downstream of STAT3 [#5, #6].\",\n  \"teleology\": [\n    {\n      \"year\": 2002,\n      \"claim\": \"Established STK35/CLIK1 as a nuclear autophosphorylating kinase and identified a physical and spatial link to the actin cytoskeleton, answering whether this kinase has defined binding partners and a regulated localization.\",\n      \"evidence\": \"Yeast two-hybrid (36/37 clones), autophosphorylation assay, and relocalization imaging in cells\",\n      \"pmids\": [\"11973348\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"No substrate of the kinase identified\",\n        \"Functional consequence of stress-fiber recruitment not determined\",\n        \"Single-lab interaction not reciprocally validated in vivo\"\n      ]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Corrected the gene model, defining the full-length 534-aa STK35L1 protein and confirming broad expression with nuclear/nucleolar localization, providing the correct molecular entity for subsequent study.\",\n      \"evidence\": \"Molecular cloning, SDS-PAGE, siRNA validation, and EGFP fusion imaging\",\n      \"pmids\": [\"19756140\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Functional role of the N-terminal extension not tested\",\n        \"No catalytic characterization of full-length protein\"\n      ]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Mapped the localization signals and a nuclear actin interaction and assigned a cell-cycle and angiogenic function, showing STK35L1 inhibits G1-to-S transition and supports endothelial migration.\",\n      \"evidence\": \"siRNA knockdown with cell-cycle analysis, expression arrays, NLS/NoLS mapping, wound-healing and Matrigel sprouting assays\",\n      \"pmids\": [\"21283756\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Mechanism linking STK35L1 to CDKN2A expression not resolved\",\n        \"Direct kinase substrates in the cell cycle not identified\",\n        \"Whether nuclear actin binding mediates the cell-cycle effect untested\"\n      ]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Identified NEDD4L-mediated ubiquitination of STK35 and placed STK35 upstream of AKT-driven glycolysis and apoptosis suppression, connecting the kinase to metabolic survival signaling in cancer.\",\n      \"evidence\": \"Reciprocal co-immunoprecipitation/ubiquitination assay, siRNA knockdown and overexpression with AKT phosphorylation, glycolysis, and apoptosis readouts\",\n      \"pmids\": [\"33117809\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Whether STK35 phosphorylates an AKT-pathway component directly is unknown\",\n        \"Functional consequence of NEDD4L ubiquitination (degradation vs. signaling) not defined\",\n        \"Single-lab findings in colorectal cells only\"\n      ]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Demonstrated an obligatory host role for STK35L1 in malaria liver-stage infection downstream of STAT3, linking the kinase to a host-pathogen cell-cycle gene program.\",\n      \"evidence\": \"siRNA knockdown with Plasmodium berghei infection assays, STAT3 phosphorylation analysis, and cell-cycle gene profiling in hepatocytes\",\n      \"pmids\": [\"34358525\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Direct kinase target underlying infection support not identified\",\n        \"Mechanism by which STAT3 induces STK35L1 unresolved\",\n        \"Single-lab loss-of-function evidence\"\n      ]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Resolved the post-translational regulation of STK35 by showing SCP4 phosphatase forms a complex with STK35 and PDIK1L, removing inhibitory phosphorylation and stabilizing it, with the kinases acting redundantly to support amino acid biosynthesis and AML proliferation.\",\n      \"evidence\": \"Domain-focused CRISPR screen, affinity purification–mass spectrometry, and gene complementation/exon-scanning assays\",\n      \"pmids\": [\"35021089\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Phosphosites removed by SCP4 not individually mapped to function\",\n        \"Direct kinase substrates in amino acid metabolism not identified\",\n        \"Whether SCP4 regulation operates outside AML untested\"\n      ]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Established STK35 as a broad DNA damage response regulator and pinpointed kinase-domain residues A423 and W445 as functionally critical, defining a genome-maintenance role and structure-function landmarks.\",\n      \"evidence\": \"siRNA/CRISPR depletion, saturated adenine and cytosine base-editor mutagenesis screens, and genotoxic drug sensitivity assays\",\n      \"pmids\": [\"42161060\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"DDR substrate(s) of STK35 not identified\",\n        \"Step in the DDR at which STK35 acts not defined\",\n        \"Mechanistic role of A423/W445 (catalysis vs. structure) not resolved\"\n      ]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The direct physiological substrate(s) of STK35 kinase activity remain unidentified across all its assigned roles (cell cycle, AKT signaling, amino acid metabolism, DDR, malaria infection).\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"No phosphorylation substrate experimentally established\",\n        \"Unclear whether the diverse phenotypes converge on a common molecular target\",\n        \"No structural model linking A423/W445 to substrate engagement\"\n      ]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 3, 6]},\n      {\"term_id\": \"GO:0016740\", \"supporting_discovery_ids\": [0, 3]},\n      {\"term_id\": \"GO:0008092\", \"supporting_discovery_ids\": [0, 1]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [0, 1, 2]},\n      {\"term_id\": \"GO:0005730\", \"supporting_discovery_ids\": [1, 2]},\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [0]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [1, 5]},\n      {\"term_id\": \"R-HSA-73894\", \"supporting_discovery_ids\": [6]},\n      {\"term_id\": \"R-HSA-1430728\", \"supporting_discovery_ids\": [3, 4]}\n    ],\n    \"complexes\": [\n      \"SCP4–STK35–PDIK1L phospho-catalytic complex\"\n    ],\n    \"partners\": [\n      \"CLP-36\",\n      \"SCP4\",\n      \"PDIK1L\",\n      \"NEDD4L\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```","audit_flag":null,"evaluation":{"faith_supported":5,"faith_total":5,"faith_pct":100.0}}