{"gene":"TESK1","run_date":"2026-06-10T10:51:54","timeline":{"discoveries":[{"year":1995,"finding":"TESK1 is a serine/threonine kinase; protein expressed in COS cells exhibited serine/threonine kinase activity using myelin basic protein as a substrate. The protein contains an N-terminal kinase domain structurally similar to LIMK1/LIMK2 (49-50% identity) followed by a C-terminal proline-rich region.","method":"In vitro kinase assay with myelin basic protein as substrate; sequence analysis","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — direct in vitro kinase assay with defined substrate, replicated in multiple subsequent studies","pmids":["8537404"],"is_preprint":false},{"year":2001,"finding":"TESK1 has the potential to phosphorylate cofilin and induce actin cytoskeletal reorganization, with stage-specific expression in testicular germ cells driven by a 9.0-kb 5'-flanking region active after pachytene spermatocytes.","method":"Transgenic mouse lacZ reporter assay; promoter-driven cell-type-specific expression","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — transgenic reporter confirms promoter activity and stage-specificity; cofilin phosphorylation stated but not directly demonstrated in this paper","pmids":["11511097"],"is_preprint":false},{"year":2002,"finding":"TESK1 interacts with human Sprouty 4 (hSPRY4); the interaction was identified by yeast two-hybrid screen and confirmed by co-immunoprecipitation. The two proteins colocalize in cytoplasmic vesicles, and the interaction increases upon growth factor stimulation.","method":"Yeast two-hybrid, co-immunoprecipitation, colocalization imaging","journal":"European journal of biochemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — yeast two-hybrid plus reciprocal co-IP in a single lab; colocalization supporting","pmids":["12027893"],"is_preprint":false},{"year":2004,"finding":"Drosophila Cdi (TESK1 ortholog) functions as a downstream effector of Rac1 in spermatogenesis; eye-directed Cdi expression suppressed phenotypes of dominant-negative Rac1(N17), and Rac1 mutant infertility was enhanced by cdi loss-of-function, placing Cdi/TESK1 downstream of Rac1 in a Rac1-Cdi-Cofilin pathway.","method":"Drosophila genetic epistasis (gain-of-function screen, dominant-negative suppression, double-mutant fertility assay)","journal":"Journal of cell science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic epistasis in Drosophila with multiple alleles; ortholog relationship to human TESK1 established","pmids":["15169836"],"is_preprint":false},{"year":2005,"finding":"Actopaxin (parvin) directly binds TESK1 via the C-terminal regions of both proteins; this interaction inhibits TESK1 kinase activity in vitro, is negatively regulated by fibronectin adhesion, and a phosphomimetic actopaxin mutant shows impaired TESK1 binding. Retention of TESK1 by the actopaxin C-terminus prevents cofilin phosphorylation upon matrix adhesion and retards cell spreading, which is rescued by TESK1 overexpression.","method":"Co-immunoprecipitation, GST pulldown (direct binding), in vitro kinase assay, deletion/point mutant mapping, cell spreading assay, overexpression rescue","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — direct binding reconstituted, in vitro kinase inhibition assay, mutant mapping, and cellular functional rescue all in one study","pmids":["15817463"],"is_preprint":false},{"year":2006,"finding":"Drosophila Cdi (TESK1 ortholog) phosphorylates ADF/cofilin to regulate actin organization; loss- and gain-of-function of cdi alters actin organization, adherens junction proteins (DE-cadherin, β-catenin), and apical localization of Sevenless RTK in the eye epithelium. Cdi acts as a suppressor of activated Sevenless signaling.","method":"Drosophila EP gain-of-function screen, loss-of-function genetics, immunostaining of actin and junction proteins","journal":"Journal of cell science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic loss/gain-of-function with multiple cellular readouts in Drosophila ortholog","pmids":["17118962"],"is_preprint":false},{"year":2007,"finding":"TESK1 forms an endogenous complex with Spry2 in cell lines and mouse tissues; TESK1 co-expression relocalizes Spry2 to vesicles/endosomes, preventing its translocation to membrane ruffles upon growth factor stimulation. Independent of its kinase activity, TESK1 binding abrogates Spry2's inhibition of ERK phosphorylation by blocking Spry2–Grb2 interaction and impeding Spry2 dephosphorylation by PP2A catalytic subunit. TESK1 depletion in PC12 cells reduces bFGF-induced neurite outgrowth. TESK1 does not affect Spry2–c-Cbl interaction.","method":"Co-immunoprecipitation of endogenous proteins, subcellular localization imaging, kinase-dead mutant analysis, siRNA knockdown, neurite outgrowth assay, interaction mapping","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — endogenous co-IP confirmed in multiple cell lines and mouse tissue, kinase-dead mutant separates kinase from localization function, siRNA knockdown with defined phenotype, multiple orthogonal methods","pmids":["17974561"],"is_preprint":false},{"year":2008,"finding":"TESK1 binds to and inhibits MARKK/TAO1 kinase; Spred1 binds TESK1 and inhibits its activity, thereby making F-actin stress fibers dynamic. The three-way Spred1–MARKK–TESK1 interaction links regulation of both microtubule (via MARK/Par1-MAPs) and F-actin (via TESK1-cofilin) cytoskeletons: elevated TESK1 increases stress fibers via cofilin phosphorylation, blocked by Spred1; elevated MARKK disrupts microtubules, blocked by TESK1.","method":"Yeast two-hybrid, co-immunoprecipitation, in vitro kinase assay, overexpression in CHO cells with cytoskeletal readouts","journal":"Molecular biology of the cell","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — yeast two-hybrid plus co-IP plus in vitro kinase inhibition, single lab with multiple orthogonal methods","pmids":["18216281"],"is_preprint":false},{"year":2018,"finding":"TESK1 phosphorylates cofilin-1 (CFL1) at the same serine residue as LIM kinases in glomerular podocytes; TESK1 knockout podocytes showed reduced phospho-CFL1 levels that were now sensitive to ROK inhibitor Y27632, and the motility-promoting effect of Y27632 was absent in TESK1 KO cells, establishing TESK1 as a ROK-independent regulator of podocyte cytoskeletal dynamics.","method":"TESK1 knockout cell line, Y27632 pharmacological inhibition, phospho-CFL1 western blot, podocyte motility assay","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — KO cells with defined biochemical and functional phenotype, single lab","pmids":["30115939"],"is_preprint":false}],"current_model":"TESK1 is a serine/threonine kinase of the LIM kinase subfamily that phosphorylates cofilin at Ser-3 to stabilize F-actin stress fibers and regulate cell spreading, adhesion, and cytoskeletal dynamics; its activity is negatively regulated by direct binding to actopaxin (parvin) and Spred1, while it positively modulates RTK-Ras-ERK signaling by binding Sprouty proteins (Spry2/Spry4) and re-localizing them to endosomal vesicles, thereby abrogating their inhibitory effect on ERK activation; additionally, TESK1 binds and inhibits MARKK/TAO1 to link actin and microtubule cytoskeletal regulation, and acts downstream of Rac1 (established by Drosophila ortholog genetics) in spermatogenesis."},"narrative":{"mechanistic_narrative":"TESK1 is a serine/threonine kinase with an N-terminal catalytic domain structurally related to the LIM kinases and a C-terminal proline-rich region, and it regulates actin cytoskeletal organization by phosphorylating cofilin [PMID:8537404, PMID:11511097]. Across podocytes and other cell systems it phosphorylates cofilin-1 at the same serine targeted by LIM kinases, acting as a Rho-kinase-independent route to cofilin inactivation that stabilizes F-actin and governs cell motility, spreading, and adhesion [PMID:30115939, PMID:17118962]. Its kinase activity is held in check by direct binding partners: actopaxin (parvin) binds the C-terminus of TESK1 to inhibit it in a manner relieved by fibronectin adhesion, coupling integrin engagement to cofilin phosphorylation and cell spreading [PMID:15817463], and Spred1 binds and inhibits TESK1 to keep stress fibers dynamic [PMID:18216281]. Through binding MARKK/TAO1 and inhibiting it, TESK1 links F-actin control to microtubule regulation within a Spred1–MARKK–TESK1 module [PMID:18216281]. Independently of its catalytic activity, TESK1 binds Sprouty proteins (Spry2, Spry4), relocalizing them to cytoplasmic vesicles/endosomes and blocking their Grb2 interaction and PP2A-mediated dephosphorylation, thereby abrogating Sprouty's inhibition of ERK and promoting growth-factor-induced neurite outgrowth [PMID:12027893, PMID:17974561]. Genetic epistasis in the Drosophila ortholog Cdi places TESK1 downstream of Rac1 in a Rac1–Cdi–cofilin pathway controlling spermatogenesis and epithelial actin/junction organization [PMID:15169836, PMID:17118962].","teleology":[{"year":1995,"claim":"Established that TESK1 is an active serine/threonine kinase and defined its domain architecture, placing it structurally near the LIM kinase family and framing the search for physiological substrates.","evidence":"In vitro kinase assay using myelin basic protein plus sequence analysis of the N-terminal kinase domain and C-terminal proline-rich region","pmids":["8537404"],"confidence":"High","gaps":["MBP is a generic substrate; physiological substrate not identified","Upstream regulators and activation mechanism unknown","No structural model of the kinase domain"]},{"year":2001,"claim":"Linked TESK1 to cofilin-driven actin reorganization and to a defined developmental context by mapping a germ-cell, pachytene-stage-specific promoter, suggesting a spermatogenic role.","evidence":"Transgenic mouse lacZ reporter assay driven by a 9.0-kb 5'-flanking region","pmids":["11511097"],"confidence":"Medium","gaps":["Cofilin phosphorylation stated but not directly demonstrated in this work","Promoter activity does not establish endogenous protein function in germ cells"]},{"year":2002,"claim":"Identified the first non-cytoskeletal interactor, Sprouty4, connecting TESK1 to RTK signaling and showing the interaction is growth-factor responsive and vesicular.","evidence":"Yeast two-hybrid screen, reciprocal co-immunoprecipitation, and colocalization imaging in cytoplasmic vesicles","pmids":["12027893"],"confidence":"Medium","gaps":["Functional consequence of the interaction for ERK signaling not yet resolved","Single-lab interaction without orthogonal validation at the time"]},{"year":2004,"claim":"Used Drosophila genetics to position the TESK1 ortholog Cdi epistatically downstream of Rac1, defining a Rac1–Cdi–cofilin signaling axis in spermatogenesis.","evidence":"Genetic epistasis: dominant-negative Rac1(N17) suppression by Cdi and enhancement of Rac1 mutant infertility by cdi loss-of-function","pmids":["15169836"],"confidence":"Medium","gaps":["Direct biochemical link from Rac1 to TESK1 activation not shown","Mammalian conservation of the Rac1-TESK1 epistasis not tested"]},{"year":2005,"claim":"Resolved how TESK1 activity is gated by adhesion: actopaxin directly binds and inhibits TESK1, coupling matrix engagement to cofilin phosphorylation and cell spreading.","evidence":"GST pulldown for direct binding, in vitro kinase inhibition, deletion/point mutant mapping, fibronectin-regulated binding, and overexpression rescue of cell spreading","pmids":["15817463"],"confidence":"High","gaps":["Mechanism by which the actopaxin C-terminus structurally inhibits the kinase not defined","In vivo relevance of the adhesion switch not tested"]},{"year":2006,"claim":"Confirmed in the ortholog that Cdi/TESK1 phosphorylates ADF/cofilin to organize epithelial actin and junctions and to suppress activated Sevenless RTK signaling, reinforcing both the cytoskeletal and RTK-modulatory roles.","evidence":"Drosophila gain- and loss-of-function genetics with actin and adherens-junction (DE-cadherin, β-catenin) immunostaining and Sevenless localization readouts","pmids":["17118962"],"confidence":"Medium","gaps":["Direct phosphorylation of cofilin by Cdi not biochemically reconstituted here","Link between junction effects and RTK suppression mechanistically unresolved"]},{"year":2007,"claim":"Defined a kinase-independent function: TESK1 sequesters Spry2 on endosomes, blocking Spry2–Grb2 binding and PP2A dephosphorylation to relieve Sprouty inhibition of ERK and promote neurite outgrowth.","evidence":"Endogenous co-IP across cell lines and mouse tissue, kinase-dead mutant analysis, localization imaging, siRNA knockdown, and bFGF-induced neurite outgrowth assay in PC12 cells","pmids":["17974561"],"confidence":"High","gaps":["Structural basis of TESK1-Spry2 binding not defined","Whether cytoskeletal and Sprouty functions are coordinated in the same cell unknown"]},{"year":2008,"claim":"Integrated TESK1 into a cross-cytoskeletal regulatory module: TESK1 binds and inhibits MARKK/TAO1 while Spred1 binds and inhibits TESK1, coupling F-actin and microtubule dynamics.","evidence":"Yeast two-hybrid, co-immunoprecipitation, in vitro kinase inhibition, and overexpression in CHO cells with actin and microtubule readouts","pmids":["18216281"],"confidence":"Medium","gaps":["Stoichiometry and order of the three-way Spred1-MARKK-TESK1 interactions unresolved","Physiological context where this module operates not established"]},{"year":2018,"claim":"Established TESK1 as a ROK-independent cofilin-1 kinase in podocytes, showing it accounts for a Y27632-resistant pool of phospho-cofilin and controls cell motility.","evidence":"TESK1 knockout podocyte line, Y27632 inhibition, phospho-CFL1 western blot, and motility assay","pmids":["30115939"],"confidence":"Medium","gaps":["Upstream activator of TESK1 in podocytes unknown","Relevance to glomerular disease not directly demonstrated"]},{"year":null,"claim":"How TESK1 is activated and how its cytoskeletal kinase activity is coordinated with its kinase-independent Sprouty/ERK function within a single cell remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No defined activating signal or activation-loop phosphorylation mechanism","No structure of the kinase or its inhibitory complexes","No human disease association demonstrated by direct genetic evidence in the corpus"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,4,8]},{"term_id":"GO:0016740","term_label":"transferase activity","supporting_discovery_ids":[0,8]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[6,7]}],"localization":[{"term_id":"GO:0031410","term_label":"cytoplasmic vesicle","supporting_discovery_ids":[2,6]},{"term_id":"GO:0005768","term_label":"endosome","supporting_discovery_ids":[6]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[4]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[6,7]}],"complexes":[],"partners":["SPRY2","SPRY4","PARVA","SPRED1","TAOK1","CFL1"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q15569","full_name":"Dual specificity testis-specific protein kinase 1","aliases":["Testicular protein kinase 1"],"length_aa":626,"mass_kda":67.7,"function":"Dual specificity protein kinase activity catalyzing autophosphorylation and phosphorylation of exogenous substrates on both serine/threonine and tyrosine residues (By similarity). Regulates the cellular cytoskeleton by enhancing actin stress fiber formation via phosphorylation of cofilin and by preventing microtubule breakdown via inhibition of TAOK1/MARKK kinase activity (By similarity). Inhibits podocyte motility via regulation of actin cytoskeletal dynamics and phosphorylation of CFL1 (By similarity). Positively regulates integrin-mediated cell spreading, via phosphorylation of cofilin (PubMed:15584898). Suppresses ciliogenesis via multiple pathways; phosphorylation of CFL1, suppression of ciliary vesicle directional trafficking to the ciliary base, and by facilitating YAP1 nuclear localization where it acts as a transcriptional corepressor of the TEAD4 target genes AURKA and PLK1 (PubMed:25849865). Probably plays a central role at and after the meiotic phase of spermatogenesis (By similarity)","subcellular_location":"Cytoplasm; Cytoplasm, perinuclear region; Cytoplasm, cytoskeleton, microtubule organizing center, centrosome; Cell projection, lamellipodium","url":"https://www.uniprot.org/uniprotkb/Q15569/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/TESK1","classification":"Not Classified","n_dependent_lines":11,"n_total_lines":1208,"dependency_fraction":0.009105960264900662},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/TESK1","total_profiled":1310},"omim":[{"mim_id":"607984","title":"SPROUTY RTK SIGNALING ANTAGONIST 4; SPRY4","url":"https://www.omim.org/entry/607984"},{"mim_id":"604746","title":"TESTIS-ASSOCIATED ACTIN-MODELING KINASE 2; TESK2","url":"https://www.omim.org/entry/604746"},{"mim_id":"601782","title":"TESTIS-ASSOCIATED ACTIN-MODELING KINASE 1; TESK1","url":"https://www.omim.org/entry/601782"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"","locations":[],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/TESK1"},"hgnc":{"alias_symbol":[],"prev_symbol":[]},"alphafold":{"accession":"Q15569","domains":[{"cath_id":"3.30.200.20","chopping":"49-131","consensus_level":"high","plddt":86.6542,"start":49,"end":131},{"cath_id":"1.10.510.10","chopping":"136-319_354-369_378-384","consensus_level":"high","plddt":90.7756,"start":136,"end":384}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q15569","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q15569-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q15569-F1-predicted_aligned_error_v6.png","plddt_mean":61.75},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=TESK1","jax_strain_url":"https://www.jax.org/strain/search?query=TESK1"},"sequence":{"accession":"Q15569","fasta_url":"https://rest.uniprot.org/uniprotkb/Q15569.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q15569/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q15569"}},"corpus_meta":[{"pmid":"12027893","id":"PMC_12027893","title":"Human sprouty 4, a new ras antagonist on 5q31, interacts with the dual specificity kinase TESK1.","date":"2002","source":"European journal of biochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/12027893","citation_count":89,"is_preprint":false},{"pmid":"8537404","id":"PMC_8537404","title":"Identification and characterization of a novel protein kinase, TESK1, specifically expressed in testicular germ cells.","date":"1995","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/8537404","citation_count":70,"is_preprint":false},{"pmid":"18216281","id":"PMC_18216281","title":"Spred1 and TESK1--two new interaction partners of the kinase MARKK/TAO1 that link the microtubule and actin cytoskeleton.","date":"2008","source":"Molecular biology of the cell","url":"https://pubmed.ncbi.nlm.nih.gov/18216281","citation_count":54,"is_preprint":false},{"pmid":"15817463","id":"PMC_15817463","title":"Actopaxin interacts with TESK1 to regulate cell spreading on fibronectin.","date":"2005","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/15817463","citation_count":42,"is_preprint":false},{"pmid":"9705840","id":"PMC_9705840","title":"Stage-specific expression of testis-specific protein kinase 1 (TESK1) in rat spermatogenic cells.","date":"1998","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/9705840","citation_count":33,"is_preprint":false},{"pmid":"17974561","id":"PMC_17974561","title":"Tesk1 interacts with Spry2 to abrogate its inhibition of ERK phosphorylation downstream of receptor tyrosine kinase signaling.","date":"2007","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/17974561","citation_count":33,"is_preprint":false},{"pmid":"15169836","id":"PMC_15169836","title":"A screen for modifiers of RacGAP(84C) gain-of-function in the Drosophila eye revealed the LIM kinase Cdi/TESK1 as a downstream effector of Rac1 during spermatogenesis.","date":"2004","source":"Journal of cell science","url":"https://pubmed.ncbi.nlm.nih.gov/15169836","citation_count":21,"is_preprint":false},{"pmid":"25271995","id":"PMC_25271995","title":"Cloning and characterization of tesk1, a novel spermatogenesis-related gene, in the tongue sole (Cynoglossus semilaevis).","date":"2014","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/25271995","citation_count":20,"is_preprint":false},{"pmid":"11511097","id":"PMC_11511097","title":"Cell-type-specific expression of a TESK1 promoter-linked lacZ gene in transgenic mice.","date":"2001","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/11511097","citation_count":19,"is_preprint":false},{"pmid":"29396473","id":"PMC_29396473","title":"Decreased TESK1-mediated cofilin 1 phosphorylation in the jejunum of IBS-D patients may explain increased female predisposition to epithelial dysfunction.","date":"2018","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/29396473","citation_count":18,"is_preprint":false},{"pmid":"10390152","id":"PMC_10390152","title":"Drosophila center divider gene is expressed in CNS midline cells and encodes a developmentally regulated protein kinase orthologous to human TESK1.","date":"1999","source":"DNA and cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/10390152","citation_count":17,"is_preprint":false},{"pmid":"9469938","id":"PMC_9469938","title":"Structural organization and chromosomal localization of the mouse tesk1 (testis-specific protein kinase 1) gene.","date":"1998","source":"Gene","url":"https://pubmed.ncbi.nlm.nih.gov/9469938","citation_count":12,"is_preprint":false},{"pmid":"30115939","id":"PMC_30115939","title":"Regulation of cofilin phosphorylation in glomerular podocytes by testis specific kinase 1 (TESK1).","date":"2018","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/30115939","citation_count":10,"is_preprint":false},{"pmid":"17118962","id":"PMC_17118962","title":"The Cdi/TESK1 kinase is required for Sevenless signaling and epithelial organization in the Drosophila eye.","date":"2006","source":"Journal of cell science","url":"https://pubmed.ncbi.nlm.nih.gov/17118962","citation_count":7,"is_preprint":false},{"pmid":"26869317","id":"PMC_26869317","title":"Expression analysis and characterization of an autosome-localized tesk1 gene in half-smooth tongue sole (Cynoglossus semilaevis).","date":"2016","source":"Gene","url":"https://pubmed.ncbi.nlm.nih.gov/26869317","citation_count":5,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":9125,"output_tokens":2530,"usd":0.032662,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":9669,"output_tokens":3435,"usd":0.06711,"stage2_stop_reason":"end_turn"},"total_usd":0.099772,"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\": 1995,\n      \"finding\": \"TESK1 is a serine/threonine kinase; protein expressed in COS cells exhibited serine/threonine kinase activity using myelin basic protein as a substrate. The protein contains an N-terminal kinase domain structurally similar to LIMK1/LIMK2 (49-50% identity) followed by a C-terminal proline-rich region.\",\n      \"method\": \"In vitro kinase assay with myelin basic protein as substrate; sequence analysis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — direct in vitro kinase assay with defined substrate, replicated in multiple subsequent studies\",\n      \"pmids\": [\"8537404\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"TESK1 has the potential to phosphorylate cofilin and induce actin cytoskeletal reorganization, with stage-specific expression in testicular germ cells driven by a 9.0-kb 5'-flanking region active after pachytene spermatocytes.\",\n      \"method\": \"Transgenic mouse lacZ reporter assay; promoter-driven cell-type-specific expression\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — transgenic reporter confirms promoter activity and stage-specificity; cofilin phosphorylation stated but not directly demonstrated in this paper\",\n      \"pmids\": [\"11511097\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"TESK1 interacts with human Sprouty 4 (hSPRY4); the interaction was identified by yeast two-hybrid screen and confirmed by co-immunoprecipitation. The two proteins colocalize in cytoplasmic vesicles, and the interaction increases upon growth factor stimulation.\",\n      \"method\": \"Yeast two-hybrid, co-immunoprecipitation, colocalization imaging\",\n      \"journal\": \"European journal of biochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — yeast two-hybrid plus reciprocal co-IP in a single lab; colocalization supporting\",\n      \"pmids\": [\"12027893\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Drosophila Cdi (TESK1 ortholog) functions as a downstream effector of Rac1 in spermatogenesis; eye-directed Cdi expression suppressed phenotypes of dominant-negative Rac1(N17), and Rac1 mutant infertility was enhanced by cdi loss-of-function, placing Cdi/TESK1 downstream of Rac1 in a Rac1-Cdi-Cofilin pathway.\",\n      \"method\": \"Drosophila genetic epistasis (gain-of-function screen, dominant-negative suppression, double-mutant fertility assay)\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic epistasis in Drosophila with multiple alleles; ortholog relationship to human TESK1 established\",\n      \"pmids\": [\"15169836\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Actopaxin (parvin) directly binds TESK1 via the C-terminal regions of both proteins; this interaction inhibits TESK1 kinase activity in vitro, is negatively regulated by fibronectin adhesion, and a phosphomimetic actopaxin mutant shows impaired TESK1 binding. Retention of TESK1 by the actopaxin C-terminus prevents cofilin phosphorylation upon matrix adhesion and retards cell spreading, which is rescued by TESK1 overexpression.\",\n      \"method\": \"Co-immunoprecipitation, GST pulldown (direct binding), in vitro kinase assay, deletion/point mutant mapping, cell spreading assay, overexpression rescue\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — direct binding reconstituted, in vitro kinase inhibition assay, mutant mapping, and cellular functional rescue all in one study\",\n      \"pmids\": [\"15817463\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"Drosophila Cdi (TESK1 ortholog) phosphorylates ADF/cofilin to regulate actin organization; loss- and gain-of-function of cdi alters actin organization, adherens junction proteins (DE-cadherin, β-catenin), and apical localization of Sevenless RTK in the eye epithelium. Cdi acts as a suppressor of activated Sevenless signaling.\",\n      \"method\": \"Drosophila EP gain-of-function screen, loss-of-function genetics, immunostaining of actin and junction proteins\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic loss/gain-of-function with multiple cellular readouts in Drosophila ortholog\",\n      \"pmids\": [\"17118962\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"TESK1 forms an endogenous complex with Spry2 in cell lines and mouse tissues; TESK1 co-expression relocalizes Spry2 to vesicles/endosomes, preventing its translocation to membrane ruffles upon growth factor stimulation. Independent of its kinase activity, TESK1 binding abrogates Spry2's inhibition of ERK phosphorylation by blocking Spry2–Grb2 interaction and impeding Spry2 dephosphorylation by PP2A catalytic subunit. TESK1 depletion in PC12 cells reduces bFGF-induced neurite outgrowth. TESK1 does not affect Spry2–c-Cbl interaction.\",\n      \"method\": \"Co-immunoprecipitation of endogenous proteins, subcellular localization imaging, kinase-dead mutant analysis, siRNA knockdown, neurite outgrowth assay, interaction mapping\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — endogenous co-IP confirmed in multiple cell lines and mouse tissue, kinase-dead mutant separates kinase from localization function, siRNA knockdown with defined phenotype, multiple orthogonal methods\",\n      \"pmids\": [\"17974561\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"TESK1 binds to and inhibits MARKK/TAO1 kinase; Spred1 binds TESK1 and inhibits its activity, thereby making F-actin stress fibers dynamic. The three-way Spred1–MARKK–TESK1 interaction links regulation of both microtubule (via MARK/Par1-MAPs) and F-actin (via TESK1-cofilin) cytoskeletons: elevated TESK1 increases stress fibers via cofilin phosphorylation, blocked by Spred1; elevated MARKK disrupts microtubules, blocked by TESK1.\",\n      \"method\": \"Yeast two-hybrid, co-immunoprecipitation, in vitro kinase assay, overexpression in CHO cells with cytoskeletal readouts\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — yeast two-hybrid plus co-IP plus in vitro kinase inhibition, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"18216281\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"TESK1 phosphorylates cofilin-1 (CFL1) at the same serine residue as LIM kinases in glomerular podocytes; TESK1 knockout podocytes showed reduced phospho-CFL1 levels that were now sensitive to ROK inhibitor Y27632, and the motility-promoting effect of Y27632 was absent in TESK1 KO cells, establishing TESK1 as a ROK-independent regulator of podocyte cytoskeletal dynamics.\",\n      \"method\": \"TESK1 knockout cell line, Y27632 pharmacological inhibition, phospho-CFL1 western blot, podocyte motility assay\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — KO cells with defined biochemical and functional phenotype, single lab\",\n      \"pmids\": [\"30115939\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"TESK1 is a serine/threonine kinase of the LIM kinase subfamily that phosphorylates cofilin at Ser-3 to stabilize F-actin stress fibers and regulate cell spreading, adhesion, and cytoskeletal dynamics; its activity is negatively regulated by direct binding to actopaxin (parvin) and Spred1, while it positively modulates RTK-Ras-ERK signaling by binding Sprouty proteins (Spry2/Spry4) and re-localizing them to endosomal vesicles, thereby abrogating their inhibitory effect on ERK activation; additionally, TESK1 binds and inhibits MARKK/TAO1 to link actin and microtubule cytoskeletal regulation, and acts downstream of Rac1 (established by Drosophila ortholog genetics) in spermatogenesis.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"TESK1 is a serine/threonine kinase with an N-terminal catalytic domain structurally related to the LIM kinases and a C-terminal proline-rich region, and it regulates actin cytoskeletal organization by phosphorylating cofilin [#0, #1]. Across podocytes and other cell systems it phosphorylates cofilin-1 at the same serine targeted by LIM kinases, acting as a Rho-kinase-independent route to cofilin inactivation that stabilizes F-actin and governs cell motility, spreading, and adhesion [#8, #5]. Its kinase activity is held in check by direct binding partners: actopaxin (parvin) binds the C-terminus of TESK1 to inhibit it in a manner relieved by fibronectin adhesion, coupling integrin engagement to cofilin phosphorylation and cell spreading [#4], and Spred1 binds and inhibits TESK1 to keep stress fibers dynamic [#7]. Through binding MARKK/TAO1 and inhibiting it, TESK1 links F-actin control to microtubule regulation within a Spred1\\u2013MARKK\\u2013TESK1 module [#7]. Independently of its catalytic activity, TESK1 binds Sprouty proteins (Spry2, Spry4), relocalizing them to cytoplasmic vesicles/endosomes and blocking their Grb2 interaction and PP2A-mediated dephosphorylation, thereby abrogating Sprouty's inhibition of ERK and promoting growth-factor-induced neurite outgrowth [#2, #6]. Genetic epistasis in the Drosophila ortholog Cdi places TESK1 downstream of Rac1 in a Rac1\\u2013Cdi\\u2013cofilin pathway controlling spermatogenesis and epithelial actin/junction organization [#3, #5].\",\n  \"teleology\": [\n    {\n      \"year\": 1995,\n      \"claim\": \"Established that TESK1 is an active serine/threonine kinase and defined its domain architecture, placing it structurally near the LIM kinase family and framing the search for physiological substrates.\",\n      \"evidence\": \"In vitro kinase assay using myelin basic protein plus sequence analysis of the N-terminal kinase domain and C-terminal proline-rich region\",\n      \"pmids\": [\"8537404\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"MBP is a generic substrate; physiological substrate not identified\", \"Upstream regulators and activation mechanism unknown\", \"No structural model of the kinase domain\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Linked TESK1 to cofilin-driven actin reorganization and to a defined developmental context by mapping a germ-cell, pachytene-stage-specific promoter, suggesting a spermatogenic role.\",\n      \"evidence\": \"Transgenic mouse lacZ reporter assay driven by a 9.0-kb 5'-flanking region\",\n      \"pmids\": [\"11511097\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Cofilin phosphorylation stated but not directly demonstrated in this work\", \"Promoter activity does not establish endogenous protein function in germ cells\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Identified the first non-cytoskeletal interactor, Sprouty4, connecting TESK1 to RTK signaling and showing the interaction is growth-factor responsive and vesicular.\",\n      \"evidence\": \"Yeast two-hybrid screen, reciprocal co-immunoprecipitation, and colocalization imaging in cytoplasmic vesicles\",\n      \"pmids\": [\"12027893\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Functional consequence of the interaction for ERK signaling not yet resolved\", \"Single-lab interaction without orthogonal validation at the time\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Used Drosophila genetics to position the TESK1 ortholog Cdi epistatically downstream of Rac1, defining a Rac1\\u2013Cdi\\u2013cofilin signaling axis in spermatogenesis.\",\n      \"evidence\": \"Genetic epistasis: dominant-negative Rac1(N17) suppression by Cdi and enhancement of Rac1 mutant infertility by cdi loss-of-function\",\n      \"pmids\": [\"15169836\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct biochemical link from Rac1 to TESK1 activation not shown\", \"Mammalian conservation of the Rac1-TESK1 epistasis not tested\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Resolved how TESK1 activity is gated by adhesion: actopaxin directly binds and inhibits TESK1, coupling matrix engagement to cofilin phosphorylation and cell spreading.\",\n      \"evidence\": \"GST pulldown for direct binding, in vitro kinase inhibition, deletion/point mutant mapping, fibronectin-regulated binding, and overexpression rescue of cell spreading\",\n      \"pmids\": [\"15817463\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism by which the actopaxin C-terminus structurally inhibits the kinase not defined\", \"In vivo relevance of the adhesion switch not tested\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Confirmed in the ortholog that Cdi/TESK1 phosphorylates ADF/cofilin to organize epithelial actin and junctions and to suppress activated Sevenless RTK signaling, reinforcing both the cytoskeletal and RTK-modulatory roles.\",\n      \"evidence\": \"Drosophila gain- and loss-of-function genetics with actin and adherens-junction (DE-cadherin, \\u03b2-catenin) immunostaining and Sevenless localization readouts\",\n      \"pmids\": [\"17118962\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct phosphorylation of cofilin by Cdi not biochemically reconstituted here\", \"Link between junction effects and RTK suppression mechanistically unresolved\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Defined a kinase-independent function: TESK1 sequesters Spry2 on endosomes, blocking Spry2\\u2013Grb2 binding and PP2A dephosphorylation to relieve Sprouty inhibition of ERK and promote neurite outgrowth.\",\n      \"evidence\": \"Endogenous co-IP across cell lines and mouse tissue, kinase-dead mutant analysis, localization imaging, siRNA knockdown, and bFGF-induced neurite outgrowth assay in PC12 cells\",\n      \"pmids\": [\"17974561\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of TESK1-Spry2 binding not defined\", \"Whether cytoskeletal and Sprouty functions are coordinated in the same cell unknown\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Integrated TESK1 into a cross-cytoskeletal regulatory module: TESK1 binds and inhibits MARKK/TAO1 while Spred1 binds and inhibits TESK1, coupling F-actin and microtubule dynamics.\",\n      \"evidence\": \"Yeast two-hybrid, co-immunoprecipitation, in vitro kinase inhibition, and overexpression in CHO cells with actin and microtubule readouts\",\n      \"pmids\": [\"18216281\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Stoichiometry and order of the three-way Spred1-MARKK-TESK1 interactions unresolved\", \"Physiological context where this module operates not established\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Established TESK1 as a ROK-independent cofilin-1 kinase in podocytes, showing it accounts for a Y27632-resistant pool of phospho-cofilin and controls cell motility.\",\n      \"evidence\": \"TESK1 knockout podocyte line, Y27632 inhibition, phospho-CFL1 western blot, and motility assay\",\n      \"pmids\": [\"30115939\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Upstream activator of TESK1 in podocytes unknown\", \"Relevance to glomerular disease not directly demonstrated\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How TESK1 is activated and how its cytoskeletal kinase activity is coordinated with its kinase-independent Sprouty/ERK function within a single cell remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No defined activating signal or activation-loop phosphorylation mechanism\", \"No structure of the kinase or its inhibitory complexes\", \"No human disease association demonstrated by direct genetic evidence in the corpus\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 4, 8]},\n      {\"term_id\": \"GO:0016740\", \"supporting_discovery_ids\": [0, 8]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [6, 7]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0031410\", \"supporting_discovery_ids\": [2, 6]},\n      {\"term_id\": \"GO:0005768\", \"supporting_discovery_ids\": [6]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [4]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [6, 7]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"SPRY2\", \"SPRY4\", \"PARVA\", \"SPRED1\", \"TAOK1\", \"CFL1\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":5,"faith_total":6,"faith_pct":83.33333333333333}}