{"gene":"TSSK4","run_date":"2026-06-10T10:51:56","timeline":{"discoveries":[{"year":2012,"finding":"TSSK4 has autophosphorylation activity and self-association in vitro; autophosphorylation at Thr-197 in the T-loop region is essential for maintaining its kinase activity.","method":"In vitro kinase assay, mutagenesis of Thr-197, western blotting with specific antibody","journal":"Molecular biology reports","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro kinase assay with mutagenesis identifying the specific activating autophosphorylation site, single lab with orthogonal methods","pmids":["23054012"],"is_preprint":false},{"year":2014,"finding":"TSSK4 associates with ODF2 (outer dense fiber protein 2), a cytoskeletal component of the sperm flagellum. TSSK4 phosphorylates ODF2, and ODF2 reciprocally potentiates TSSK4 autophosphorylation activity. Tssk4 knockout mice are subfertile with disorganized sperm tail ultrastructure at the midpiece-principal piece junction.","method":"Co-immunoprecipitation, in vitro kinase assay, Tssk4 knockout mouse model, electron microscopy of sperm ultrastructure","journal":"Molecular human reproduction","confidence":"High","confidence_rationale":"Tier 2 / Moderate — reciprocal functional interaction demonstrated by Co-IP and in vitro kinase assay, confirmed in KO mouse with specific ultrastructural phenotype","pmids":["25361759"],"is_preprint":false},{"year":2016,"finding":"TSSK4 phosphorylates ODF2 specifically at serine 76; the C-terminal region (amino acids 214–638) of ODF2 is required for association with TSSK4. Phospho-Ser-76 ODF2 was confirmed in mouse sperm by LC-MS/MS.","method":"In vitro kinase assay with Odf2 point mutants (Ser/Thr/Lys to Ala), co-immunoprecipitation, LC-MS/MS, phospho-specific antibody","journal":"Scientific reports","confidence":"High","confidence_rationale":"Tier 1 / Moderate — site-directed mutagenesis identifying phosphorylation site, confirmed in vivo by LC-MS/MS and phospho-specific antibody, single lab","pmids":["26961893"],"is_preprint":false},{"year":2013,"finding":"TSSK4 associates with HSP90; pharmacological inhibition of HSP90 abolishes TSSK4 catalytic activity and leads to increased TSSK4 ubiquitination and proteasomal degradation, demonstrating that HSP90 is required for TSSK4 stability and activation.","method":"Co-immunoprecipitation in 293T/COS-7 cells, HSP90 inhibitor treatment (17-AAG, SNX-5422, NVP-AUY922), in vitro kinase assay, ubiquitination assay, primary spermatid culture","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP, pharmacological perturbation with multiple specific inhibitors, enzymatic activity readout, and endogenous validation in primary spermatids","pmids":["23599433"],"is_preprint":false},{"year":2015,"finding":"TSSK4 induces cell apoptosis in vitro in a kinase-activity-dependent manner; the kinase-dead mutant TSSK4-K54M does not induce apoptosis. In Tssk4 knockout mice, apoptotic spermatogonia and spermatocytes at postnatal day 21 are decreased and testes weight is increased.","method":"GFP-TSSK4 overexpression in HeLa, COS-7, and H1299 cells; flow cytometry for apoptosis; kinase-dead mutant K54M; TUNEL assay in Tssk4 KO mice","journal":"Journal of Huazhong University of Science and Technology. Medical sciences","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — kinase-dead mutant control in multiple cell lines plus KO mouse TUNEL assay, single lab","pmids":["25877358"],"is_preprint":false},{"year":2016,"finding":"Phosphorylated TSSK4 interacts with CREM (cAMP-responsive element modulator) and may phosphorylate CREM at Ser-117 in vitro; TSSK4 and CREM share an identical distribution pattern in the testis distinct from CREB localization.","method":"In vitro co-immunoprecipitation, in vitro kinase assay, immunofluorescence, immunohistochemistry, western blotting with dephosphorylation","journal":"Bioscience, biotechnology, and biochemistry","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, single Co-IP plus kinase assay, phosphorylation site not confirmed by mutagenesis; paper itself uses hedged language ('may phosphorylate')","pmids":["26940607"],"is_preprint":false},{"year":2021,"finding":"TSSK4 phosphorylates HSP90β at Ser-255, thereby inhibiting HSP90β ATPase activity and reducing its chaperone function toward AKT, leading to diminished AKT kinase activity and subsequent AT-II cell apoptosis via the mitochondrial death pathway. TSSK4 expression in alveolar epithelial type-II cells is regulated by TNF-α-mediated NF-κB signaling.","method":"Luciferase reporter assay (NF-κB), in vitro kinase assay (TSSK4→HSP90β Ser-255), ATPase activity assay, AKT activity assay, iTRAQ quantitative proteomics, immunofluorescence, bleomycin mouse model","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vitro kinase assay identifying phosphorylation site, functional ATPase and AKT activity readouts, multiple orthogonal methods, single lab","pmids":["34645797"],"is_preprint":false},{"year":2010,"finding":"TSSK4 protein is present in mouse sperm and germ cells from mouse testis, is insoluble in non-ionic detergents in both mouse and human sperm, and localizes to the sperm flagellum/structural fraction.","method":"Immunolocalization in mouse testis and sperm, detergent fractionation, recombinant protein production for antibody validation","journal":"Molecular human reproduction","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — immunolocalization with validated antibodies plus biochemical fractionation, replicated for multiple TSSK family members in two species","pmids":["20729278"],"is_preprint":false},{"year":2025,"finding":"The Drosophila TSSK4 ortholog dTSSK2 (CG9222) localizes to individualization complexes during spermiogenesis and phosphorylates the substrate Gudu at Ser9, which contributes to individualization complex integrity and sperm motility; loss of dTSSK2 abolishes sperm transit to the seminal vesicle and causes male infertility.","method":"Genetic disruption (Drosophila KO), phosphoproteomic analysis, in vivo localization, fertility assays","journal":"Communications biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — phosphoproteomic substrate identification combined with KO fertility phenotype and localization, single lab but multiple orthogonal methods; ortholog with conserved function","pmids":["40335644"],"is_preprint":false},{"year":2021,"finding":"ATF6 transcription factor regulates TSSK4 expression; Atf6 knockout mice show reduced TSSK4 mRNA and protein levels, and this reduction correlates with impaired male fertility.","method":"Transcriptome sequencing of Atf6 KO mice, western blot, real-time PCR in 293T cells and KO mice","journal":"Andrologia","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, correlation of KO transcriptomics with protein level change, no direct promoter/binding experiment establishing mechanism","pmids":["34904262"],"is_preprint":false}],"current_model":"TSSK4 is a testis-specific serine/threonine kinase that maintains its own activity via autophosphorylation at Thr-197 in the T-loop, is stabilized by HSP90 (which prevents its ubiquitin-mediated proteasomal degradation), phosphorylates ODF2 at Ser-76 to regulate sperm flagellar structural integrity, phosphorylates HSP90β at Ser-255 to suppress AKT survival signaling, can phosphorylate CREM at Ser-117 in vitro, and induces apoptosis in a kinase-activity-dependent manner; collectively these activities are required for normal sperm flagellum architecture, sperm motility, and male fertility."},"narrative":{"mechanistic_narrative":"TSSK4 is a testis-enriched serine/threonine kinase that governs sperm flagellar architecture and male fertility through phosphorylation of cytoskeletal and chaperone substrates [PMID:25361759, PMID:20729278]. Its catalytic activity depends on autophosphorylation at Thr-197 in the T-loop and on self-association [PMID:23054012], and is maintained post-translationally by HSP90, which prevents TSSK4 ubiquitination and proteasomal degradation [PMID:23599433]. A principal flagellar substrate is the outer dense fiber protein ODF2, which TSSK4 phosphorylates at Ser-76 and which reciprocally potentiates TSSK4 autophosphorylation; loss of TSSK4 produces disorganized sperm tail ultrastructure at the midpiece-principal piece junction and subfertility [PMID:25361759, PMID:26961893]. Beyond germ cells, TSSK4 phosphorylates HSP90β at Ser-255, inhibiting its ATPase and chaperone support of AKT and thereby promoting apoptosis through the mitochondrial death pathway, consistent with its kinase-activity-dependent pro-apoptotic action in cell-based assays [PMID:25877358, PMID:34645797]. The conserved function of this kinase in sperm individualization and motility is reinforced by its Drosophila ortholog dTSSK2, which phosphorylates Gudu to maintain individualization complex integrity [PMID:40335644].","teleology":[{"year":2010,"claim":"Establishing where TSSK4 acts: it was unknown whether the kinase resided in soluble or structural compartments of sperm, and localization to the detergent-insoluble flagellar fraction pointed to a role in flagellar structure.","evidence":"Immunolocalization and detergent fractionation in mouse and human sperm/testis","pmids":["20729278"],"confidence":"Medium","gaps":["Does not identify substrates or catalytic role","Structural fraction association does not establish function"]},{"year":2012,"claim":"Defined how TSSK4 activity is generated: autophosphorylation at Thr-197 in the T-loop was shown to be essential for maintaining kinase activity, identifying the intrinsic activation mechanism.","evidence":"In vitro kinase assay with Thr-197 mutagenesis and self-association assays","pmids":["23054012"],"confidence":"High","gaps":["No physiological substrate identified at this stage","Upstream triggers of autophosphorylation unknown"]},{"year":2013,"claim":"Addressed how TSSK4 stability and activation are controlled: HSP90 was shown to be required, with its inhibition driving TSSK4 ubiquitination and proteasomal degradation and abolishing catalytic activity.","evidence":"Reciprocal Co-IP, multiple HSP90 inhibitors, kinase and ubiquitination assays, primary spermatid validation","pmids":["23599433"],"confidence":"High","gaps":["E3 ligase mediating TSSK4 ubiquitination unidentified","Whether HSP90 acts as a direct chaperone or scaffold not resolved"]},{"year":2014,"claim":"Linked TSSK4 to a flagellar substrate and a fertility phenotype: the ODF2 interaction was reciprocal (TSSK4 phosphorylates ODF2; ODF2 boosts TSSK4 autophosphorylation), and knockout caused midpiece-principal piece ultrastructural disorganization and subfertility.","evidence":"Co-IP, in vitro kinase assay, Tssk4 knockout mouse, electron microscopy","pmids":["25361759"],"confidence":"High","gaps":["Phosphosite on ODF2 not yet mapped here","Mechanistic link between ODF2 phosphorylation and ultrastructure not established"]},{"year":2016,"claim":"Pinpointed the ODF2 modification: TSSK4 phosphorylates ODF2 at Ser-76 in vivo and the ODF2 C-terminus (aa 214–638) mediates binding, converting a general interaction into a defined enzyme-substrate-site relationship.","evidence":"In vitro kinase assay with ODF2 point mutants, Co-IP, LC-MS/MS, phospho-specific antibody","pmids":["26961893"],"confidence":"High","gaps":["Functional consequence of Ser-76 phosphorylation for fiber assembly not directly tested","Phosphatase reversing this site unknown"]},{"year":2015,"claim":"Tested whether TSSK4 has a cell-fate role: overexpression induced apoptosis dependent on catalytic activity (lost in the K54M kinase-dead mutant), while knockout testes showed reduced germ-cell apoptosis.","evidence":"GFP-TSSK4 overexpression in multiple cell lines, flow cytometry, kinase-dead mutant, TUNEL in KO mice","pmids":["25877358"],"confidence":"Medium","gaps":["Apoptotic substrate not identified in this study","Overexpression context may not reflect physiological levels"]},{"year":2016,"claim":"Explored a possible transcriptional link: phosphorylated TSSK4 interacted with CREM and may phosphorylate it at Ser-117 in vitro, with co-distribution in testis distinct from CREB.","evidence":"In vitro Co-IP, kinase assay, immunofluorescence/immunohistochemistry","pmids":["26940607"],"confidence":"Low","gaps":["Phosphorylation site not confirmed by mutagenesis","Functional consequence for CREM transcriptional activity untested","No in vivo validation"]},{"year":2021,"claim":"Provided a mechanistic substrate for the pro-apoptotic activity outside testis: TSSK4 phosphorylates HSP90β at Ser-255, inhibiting HSP90β ATPase and chaperoning of AKT, lowering AKT activity and driving mitochondrial apoptosis in alveolar epithelial type-II cells.","evidence":"In vitro kinase assay, ATPase and AKT activity assays, iTRAQ proteomics, NF-κB luciferase reporter, bleomycin mouse model","pmids":["34645797"],"confidence":"Medium","gaps":["Relationship between TSSK4-HSP90β phosphorylation and HSP90-dependent TSSK4 stabilization not reconciled","Relevance to germ-cell apoptosis not established"]},{"year":2021,"claim":"Addressed upstream transcriptional control of TSSK4: Atf6 knockout reduced TSSK4 mRNA and protein and correlated with impaired fertility, implicating ATF6 in TSSK4 expression.","evidence":"Transcriptome sequencing, western blot, real-time PCR in Atf6 KO mice and 293T cells","pmids":["34904262"],"confidence":"Low","gaps":["No promoter binding or direct regulation experiment","Effect could be indirect","Causality of fertility link not established"]},{"year":2025,"claim":"Demonstrated evolutionary conservation of the spermiogenesis role: the Drosophila ortholog dTSSK2 phosphorylates Gudu at Ser9 to maintain individualization complex integrity and sperm motility, with loss causing male infertility.","evidence":"Drosophila knockout, phosphoproteomics, in vivo localization, fertility assays","pmids":["40335644"],"confidence":"Medium","gaps":["Mammalian ortholog of Gudu substrate not addressed","Direct conservation of substrate specificity in mammals not tested"]},{"year":null,"claim":"How TSSK4-dependent ODF2 phosphorylation mechanistically produces normal flagellar ultrastructure, and how its germ-cell apoptotic and chaperone-regulating activities integrate during spermatogenesis, remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structural model of TSSK4-substrate complexes","Integration of pro-apoptotic and structural functions in vivo unclear","Full germ-cell substrate repertoire undefined"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[1,2,6,8]},{"term_id":"GO:0016740","term_label":"transferase activity","supporting_discovery_ids":[0,1,2,6]},{"term_id":"GO:0140657","term_label":"ATP-dependent activity","supporting_discovery_ids":[0]}],"localization":[{"term_id":"GO:0005856","term_label":"cytoskeleton","supporting_discovery_ids":[7]}],"pathway":[{"term_id":"R-HSA-1474165","term_label":"Reproduction","supporting_discovery_ids":[1,2]},{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[4,6]}],"complexes":[],"partners":["ODF2","HSP90","HSP90AB1","CREM"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q6SA08","full_name":"Testis-specific serine/threonine-protein kinase 4","aliases":["Serine/threonine-protein kinase 22E"],"length_aa":328,"mass_kda":37.5,"function":"Serine/threonine kinase which is involved in male germ cell development and in mature sperm function (By similarity). May be involved in the Cre/Creb signaling pathway (By similarity). Phosphorylates CREB1 on 'Ser-133' in vitro and can stimulate Cre/Creb pathway in cells (PubMed:15964553). Phosphorylates CREM on 'Ser-116' in vitro (By similarity). Phosphorylates ODF2 on 'Ser-95' (By similarity)","subcellular_location":"Cytoplasmic vesicle, secretory vesicle, acrosome; Cell projection, cilium, flagellum","url":"https://www.uniprot.org/uniprotkb/Q6SA08/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/TSSK4","classification":"Not Classified","n_dependent_lines":57,"n_total_lines":1208,"dependency_fraction":0.04718543046357616},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/TSSK4","total_profiled":1310},"omim":[{"mim_id":"618304","title":"GLUTAMINE-RICH PROTEIN 2; QRICH2","url":"https://www.omim.org/entry/618304"},{"mim_id":"610712","title":"TESTIS-SPECIFIC SERINE/THREONINE KINASE 6; TSSK6","url":"https://www.omim.org/entry/610712"},{"mim_id":"610711","title":"TESTIS-SPECIFIC SERINE/THREONINE KINASE 4; TSSK4","url":"https://www.omim.org/entry/610711"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Uncertain","locations":[{"location":"Cell Junctions","reliability":"Uncertain"}],"tissue_specificity":"Tissue enriched","tissue_distribution":"Detected in many","driving_tissues":[{"tissue":"testis","ntpm":36.3}],"url":"https://www.proteinatlas.org/search/TSSK4"},"hgnc":{"alias_symbol":[],"prev_symbol":["C14orf20","STK22E"]},"alphafold":{"accession":"Q6SA08","domains":[{"cath_id":"3.30.200.20","chopping":"16-104","consensus_level":"high","plddt":92.656,"start":16,"end":104},{"cath_id":"1.10.510.10","chopping":"109-318","consensus_level":"high","plddt":88.7097,"start":109,"end":318}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q6SA08","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q6SA08-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q6SA08-F1-predicted_aligned_error_v6.png","plddt_mean":86.31},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=TSSK4","jax_strain_url":"https://www.jax.org/strain/search?query=TSSK4"},"sequence":{"accession":"Q6SA08","fasta_url":"https://rest.uniprot.org/uniprotkb/Q6SA08.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q6SA08/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q6SA08"}},"corpus_meta":[{"pmid":"20729278","id":"PMC_20729278","title":"Expression and localization of five members of the testis-specific serine kinase (Tssk) family in mouse and human sperm and testis.","date":"2010","source":"Molecular human reproduction","url":"https://pubmed.ncbi.nlm.nih.gov/20729278","citation_count":81,"is_preprint":false},{"pmid":"32337545","id":"PMC_32337545","title":"Testis-specific serine kinase protein family in male fertility and as targets for non-hormonal male contraception†.","date":"2020","source":"Biology of reproduction","url":"https://pubmed.ncbi.nlm.nih.gov/32337545","citation_count":59,"is_preprint":false},{"pmid":"25361759","id":"PMC_25361759","title":"Tssk4 is essential for maintaining the structural integrity of sperm flagellum.","date":"2014","source":"Molecular human reproduction","url":"https://pubmed.ncbi.nlm.nih.gov/25361759","citation_count":50,"is_preprint":false},{"pmid":"23599433","id":"PMC_23599433","title":"Heat shock protein 90 functions to stabilize and activate the testis-specific serine/threonine kinases, a family of kinases essential for male fertility.","date":"2013","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/23599433","citation_count":37,"is_preprint":false},{"pmid":"26961893","id":"PMC_26961893","title":"Testis-specific serine/threonine protein kinase 4 (Tssk4) phosphorylates Odf2 at Ser-76.","date":"2016","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/26961893","citation_count":23,"is_preprint":false},{"pmid":"34645797","id":"PMC_34645797","title":"TSSK4 upregulation in alveolar epithelial type-II cells facilitates pulmonary fibrosis through HSP90-AKT signaling restriction and AT-II apoptosis.","date":"2021","source":"Cell death & disease","url":"https://pubmed.ncbi.nlm.nih.gov/34645797","citation_count":23,"is_preprint":false},{"pmid":"18390560","id":"PMC_18390560","title":"Mutation screening and association study of the TSSK4 Gene in Chinese infertile men with impaired spermatogenesis.","date":"2008","source":"Journal of andrology","url":"https://pubmed.ncbi.nlm.nih.gov/18390560","citation_count":21,"is_preprint":false},{"pmid":"33287701","id":"PMC_33287701","title":"Comparative transcriptome analysis of three gonadal development stages reveals potential genes involved in gametogenesis of the fluted giant clam (Tridacna squamosa).","date":"2020","source":"BMC genomics","url":"https://pubmed.ncbi.nlm.nih.gov/33287701","citation_count":20,"is_preprint":false},{"pmid":"23054012","id":"PMC_23054012","title":"Testis specific serine/threonine kinase 4 (Tssk4) maintains its kinase activity by phosphorylating itself at Thr-197.","date":"2012","source":"Molecular biology reports","url":"https://pubmed.ncbi.nlm.nih.gov/23054012","citation_count":15,"is_preprint":false},{"pmid":"33859575","id":"PMC_33859575","title":"Expression of the Testis-Specific Serine/Threonine Kinases Suggests Their Role in Spermiogenesis of Bay Scallop Argopecten irradians.","date":"2021","source":"Frontiers in 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Medical sciences = Hua zhong ke ji da xue xue bao. Yi xue Ying De wen ban = Huazhong keji daxue xuebao. Yixue Yingdewen ban","url":"https://pubmed.ncbi.nlm.nih.gov/25877358","citation_count":9,"is_preprint":false},{"pmid":"33765466","id":"PMC_33765466","title":"cDNA cloning, expression and bioinformatical analysis of Tssk genes in tree shrews.","date":"2021","source":"Computational biology and chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/33765466","citation_count":5,"is_preprint":false},{"pmid":"35681843","id":"PMC_35681843","title":"Identification of the TSSK4 Alternative Spliceosomes and Analysis of the Function of the TSSK4 Protein in Yak (Bos grunniens).","date":"2022","source":"Animals : an open access journal from MDPI","url":"https://pubmed.ncbi.nlm.nih.gov/35681843","citation_count":3,"is_preprint":false},{"pmid":"39358566","id":"PMC_39358566","title":"Comprehensive analysis of chromosomal breakpoints and candidate genes associated with male infertility: insights from cytogenetic studies and expression analyses.","date":"2024","source":"Mammalian genome : official journal of the International Mammalian Genome Society","url":"https://pubmed.ncbi.nlm.nih.gov/39358566","citation_count":3,"is_preprint":false},{"pmid":"39073646","id":"PMC_39073646","title":"Characterization of piRNAs in Diploid and Triploid Pacific Oyster Gonads: Exploring Their Potential Roles in Triploid Sterility.","date":"2024","source":"Marine biotechnology (New York, N.Y.)","url":"https://pubmed.ncbi.nlm.nih.gov/39073646","citation_count":3,"is_preprint":false},{"pmid":"26940607","id":"PMC_26940607","title":"Phosphorylated testis-specific serine/threonine kinase 4 may phosphorylate Crem at Ser-117.","date":"2016","source":"Bioscience, biotechnology, and biochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/26940607","citation_count":2,"is_preprint":false},{"pmid":"40335644","id":"PMC_40335644","title":"Testis-specific serine/threonine kinase dTSSK2 regulates sperm motility and male fertility in Drosophila.","date":"2025","source":"Communications biology","url":"https://pubmed.ncbi.nlm.nih.gov/40335644","citation_count":2,"is_preprint":false},{"pmid":"39809099","id":"PMC_39809099","title":"Functional verification of a landmark gene EVM713 involved in spermatogenesis in the marine bivalve Chlamys nobilis.","date":"2025","source":"Comparative biochemistry and physiology. Part D, Genomics & proteomics","url":"https://pubmed.ncbi.nlm.nih.gov/39809099","citation_count":2,"is_preprint":false},{"pmid":"41022830","id":"PMC_41022830","title":"Genomic diversity and selection signatures in Asian Zebu Cattle: insights into adaptation and genetic erosion.","date":"2025","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/41022830","citation_count":2,"is_preprint":false},{"pmid":"41831660","id":"PMC_41831660","title":"Vincristine induces sperm malformation and motility dysfunction in mice by downregulating Tssk4 and Ccdc159.","date":"2026","source":"Reproductive toxicology (Elmsford, N.Y.)","url":"https://pubmed.ncbi.nlm.nih.gov/41831660","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":14054,"output_tokens":2588,"usd":0.040491,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":9735,"output_tokens":3422,"usd":0.067113,"stage2_stop_reason":"end_turn"},"total_usd":0.107604,"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\": 2012,\n      \"finding\": \"TSSK4 has autophosphorylation activity and self-association in vitro; autophosphorylation at Thr-197 in the T-loop region is essential for maintaining its kinase activity.\",\n      \"method\": \"In vitro kinase assay, mutagenesis of Thr-197, western blotting with specific antibody\",\n      \"journal\": \"Molecular biology reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro kinase assay with mutagenesis identifying the specific activating autophosphorylation site, single lab with orthogonal methods\",\n      \"pmids\": [\"23054012\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"TSSK4 associates with ODF2 (outer dense fiber protein 2), a cytoskeletal component of the sperm flagellum. TSSK4 phosphorylates ODF2, and ODF2 reciprocally potentiates TSSK4 autophosphorylation activity. Tssk4 knockout mice are subfertile with disorganized sperm tail ultrastructure at the midpiece-principal piece junction.\",\n      \"method\": \"Co-immunoprecipitation, in vitro kinase assay, Tssk4 knockout mouse model, electron microscopy of sperm ultrastructure\",\n      \"journal\": \"Molecular human reproduction\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal functional interaction demonstrated by Co-IP and in vitro kinase assay, confirmed in KO mouse with specific ultrastructural phenotype\",\n      \"pmids\": [\"25361759\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"TSSK4 phosphorylates ODF2 specifically at serine 76; the C-terminal region (amino acids 214–638) of ODF2 is required for association with TSSK4. Phospho-Ser-76 ODF2 was confirmed in mouse sperm by LC-MS/MS.\",\n      \"method\": \"In vitro kinase assay with Odf2 point mutants (Ser/Thr/Lys to Ala), co-immunoprecipitation, LC-MS/MS, phospho-specific antibody\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — site-directed mutagenesis identifying phosphorylation site, confirmed in vivo by LC-MS/MS and phospho-specific antibody, single lab\",\n      \"pmids\": [\"26961893\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"TSSK4 associates with HSP90; pharmacological inhibition of HSP90 abolishes TSSK4 catalytic activity and leads to increased TSSK4 ubiquitination and proteasomal degradation, demonstrating that HSP90 is required for TSSK4 stability and activation.\",\n      \"method\": \"Co-immunoprecipitation in 293T/COS-7 cells, HSP90 inhibitor treatment (17-AAG, SNX-5422, NVP-AUY922), in vitro kinase assay, ubiquitination assay, primary spermatid culture\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP, pharmacological perturbation with multiple specific inhibitors, enzymatic activity readout, and endogenous validation in primary spermatids\",\n      \"pmids\": [\"23599433\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"TSSK4 induces cell apoptosis in vitro in a kinase-activity-dependent manner; the kinase-dead mutant TSSK4-K54M does not induce apoptosis. In Tssk4 knockout mice, apoptotic spermatogonia and spermatocytes at postnatal day 21 are decreased and testes weight is increased.\",\n      \"method\": \"GFP-TSSK4 overexpression in HeLa, COS-7, and H1299 cells; flow cytometry for apoptosis; kinase-dead mutant K54M; TUNEL assay in Tssk4 KO mice\",\n      \"journal\": \"Journal of Huazhong University of Science and Technology. Medical sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — kinase-dead mutant control in multiple cell lines plus KO mouse TUNEL assay, single lab\",\n      \"pmids\": [\"25877358\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Phosphorylated TSSK4 interacts with CREM (cAMP-responsive element modulator) and may phosphorylate CREM at Ser-117 in vitro; TSSK4 and CREM share an identical distribution pattern in the testis distinct from CREB localization.\",\n      \"method\": \"In vitro co-immunoprecipitation, in vitro kinase assay, immunofluorescence, immunohistochemistry, western blotting with dephosphorylation\",\n      \"journal\": \"Bioscience, biotechnology, and biochemistry\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, single Co-IP plus kinase assay, phosphorylation site not confirmed by mutagenesis; paper itself uses hedged language ('may phosphorylate')\",\n      \"pmids\": [\"26940607\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"TSSK4 phosphorylates HSP90β at Ser-255, thereby inhibiting HSP90β ATPase activity and reducing its chaperone function toward AKT, leading to diminished AKT kinase activity and subsequent AT-II cell apoptosis via the mitochondrial death pathway. TSSK4 expression in alveolar epithelial type-II cells is regulated by TNF-α-mediated NF-κB signaling.\",\n      \"method\": \"Luciferase reporter assay (NF-κB), in vitro kinase assay (TSSK4→HSP90β Ser-255), ATPase activity assay, AKT activity assay, iTRAQ quantitative proteomics, immunofluorescence, bleomycin mouse model\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro kinase assay identifying phosphorylation site, functional ATPase and AKT activity readouts, multiple orthogonal methods, single lab\",\n      \"pmids\": [\"34645797\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"TSSK4 protein is present in mouse sperm and germ cells from mouse testis, is insoluble in non-ionic detergents in both mouse and human sperm, and localizes to the sperm flagellum/structural fraction.\",\n      \"method\": \"Immunolocalization in mouse testis and sperm, detergent fractionation, recombinant protein production for antibody validation\",\n      \"journal\": \"Molecular human reproduction\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — immunolocalization with validated antibodies plus biochemical fractionation, replicated for multiple TSSK family members in two species\",\n      \"pmids\": [\"20729278\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"The Drosophila TSSK4 ortholog dTSSK2 (CG9222) localizes to individualization complexes during spermiogenesis and phosphorylates the substrate Gudu at Ser9, which contributes to individualization complex integrity and sperm motility; loss of dTSSK2 abolishes sperm transit to the seminal vesicle and causes male infertility.\",\n      \"method\": \"Genetic disruption (Drosophila KO), phosphoproteomic analysis, in vivo localization, fertility assays\",\n      \"journal\": \"Communications biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — phosphoproteomic substrate identification combined with KO fertility phenotype and localization, single lab but multiple orthogonal methods; ortholog with conserved function\",\n      \"pmids\": [\"40335644\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"ATF6 transcription factor regulates TSSK4 expression; Atf6 knockout mice show reduced TSSK4 mRNA and protein levels, and this reduction correlates with impaired male fertility.\",\n      \"method\": \"Transcriptome sequencing of Atf6 KO mice, western blot, real-time PCR in 293T cells and KO mice\",\n      \"journal\": \"Andrologia\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, correlation of KO transcriptomics with protein level change, no direct promoter/binding experiment establishing mechanism\",\n      \"pmids\": [\"34904262\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"TSSK4 is a testis-specific serine/threonine kinase that maintains its own activity via autophosphorylation at Thr-197 in the T-loop, is stabilized by HSP90 (which prevents its ubiquitin-mediated proteasomal degradation), phosphorylates ODF2 at Ser-76 to regulate sperm flagellar structural integrity, phosphorylates HSP90β at Ser-255 to suppress AKT survival signaling, can phosphorylate CREM at Ser-117 in vitro, and induces apoptosis in a kinase-activity-dependent manner; collectively these activities are required for normal sperm flagellum architecture, sperm motility, and male fertility.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"TSSK4 is a testis-enriched serine/threonine kinase that governs sperm flagellar architecture and male fertility through phosphorylation of cytoskeletal and chaperone substrates [#1, #7]. Its catalytic activity depends on autophosphorylation at Thr-197 in the T-loop and on self-association [#0], and is maintained post-translationally by HSP90, which prevents TSSK4 ubiquitination and proteasomal degradation [#3]. A principal flagellar substrate is the outer dense fiber protein ODF2, which TSSK4 phosphorylates at Ser-76 and which reciprocally potentiates TSSK4 autophosphorylation; loss of TSSK4 produces disorganized sperm tail ultrastructure at the midpiece-principal piece junction and subfertility [#1, #2]. Beyond germ cells, TSSK4 phosphorylates HSP90\\u03b2 at Ser-255, inhibiting its ATPase and chaperone support of AKT and thereby promoting apoptosis through the mitochondrial death pathway, consistent with its kinase-activity-dependent pro-apoptotic action in cell-based assays [#4, #6]. The conserved function of this kinase in sperm individualization and motility is reinforced by its Drosophila ortholog dTSSK2, which phosphorylates Gudu to maintain individualization complex integrity [#8].\",\n  \"teleology\": [\n    {\n      \"year\": 2010,\n      \"claim\": \"Establishing where TSSK4 acts: it was unknown whether the kinase resided in soluble or structural compartments of sperm, and localization to the detergent-insoluble flagellar fraction pointed to a role in flagellar structure.\",\n      \"evidence\": \"Immunolocalization and detergent fractionation in mouse and human sperm/testis\",\n      \"pmids\": [\"20729278\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Does not identify substrates or catalytic role\", \"Structural fraction association does not establish function\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Defined how TSSK4 activity is generated: autophosphorylation at Thr-197 in the T-loop was shown to be essential for maintaining kinase activity, identifying the intrinsic activation mechanism.\",\n      \"evidence\": \"In vitro kinase assay with Thr-197 mutagenesis and self-association assays\",\n      \"pmids\": [\"23054012\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No physiological substrate identified at this stage\", \"Upstream triggers of autophosphorylation unknown\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Addressed how TSSK4 stability and activation are controlled: HSP90 was shown to be required, with its inhibition driving TSSK4 ubiquitination and proteasomal degradation and abolishing catalytic activity.\",\n      \"evidence\": \"Reciprocal Co-IP, multiple HSP90 inhibitors, kinase and ubiquitination assays, primary spermatid validation\",\n      \"pmids\": [\"23599433\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"E3 ligase mediating TSSK4 ubiquitination unidentified\", \"Whether HSP90 acts as a direct chaperone or scaffold not resolved\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Linked TSSK4 to a flagellar substrate and a fertility phenotype: the ODF2 interaction was reciprocal (TSSK4 phosphorylates ODF2; ODF2 boosts TSSK4 autophosphorylation), and knockout caused midpiece-principal piece ultrastructural disorganization and subfertility.\",\n      \"evidence\": \"Co-IP, in vitro kinase assay, Tssk4 knockout mouse, electron microscopy\",\n      \"pmids\": [\"25361759\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Phosphosite on ODF2 not yet mapped here\", \"Mechanistic link between ODF2 phosphorylation and ultrastructure not established\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Pinpointed the ODF2 modification: TSSK4 phosphorylates ODF2 at Ser-76 in vivo and the ODF2 C-terminus (aa 214\\u2013638) mediates binding, converting a general interaction into a defined enzyme-substrate-site relationship.\",\n      \"evidence\": \"In vitro kinase assay with ODF2 point mutants, Co-IP, LC-MS/MS, phospho-specific antibody\",\n      \"pmids\": [\"26961893\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional consequence of Ser-76 phosphorylation for fiber assembly not directly tested\", \"Phosphatase reversing this site unknown\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Tested whether TSSK4 has a cell-fate role: overexpression induced apoptosis dependent on catalytic activity (lost in the K54M kinase-dead mutant), while knockout testes showed reduced germ-cell apoptosis.\",\n      \"evidence\": \"GFP-TSSK4 overexpression in multiple cell lines, flow cytometry, kinase-dead mutant, TUNEL in KO mice\",\n      \"pmids\": [\"25877358\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Apoptotic substrate not identified in this study\", \"Overexpression context may not reflect physiological levels\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Explored a possible transcriptional link: phosphorylated TSSK4 interacted with CREM and may phosphorylate it at Ser-117 in vitro, with co-distribution in testis distinct from CREB.\",\n      \"evidence\": \"In vitro Co-IP, kinase assay, immunofluorescence/immunohistochemistry\",\n      \"pmids\": [\"26940607\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Phosphorylation site not confirmed by mutagenesis\", \"Functional consequence for CREM transcriptional activity untested\", \"No in vivo validation\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Provided a mechanistic substrate for the pro-apoptotic activity outside testis: TSSK4 phosphorylates HSP90\\u03b2 at Ser-255, inhibiting HSP90\\u03b2 ATPase and chaperoning of AKT, lowering AKT activity and driving mitochondrial apoptosis in alveolar epithelial type-II cells.\",\n      \"evidence\": \"In vitro kinase assay, ATPase and AKT activity assays, iTRAQ proteomics, NF-\\u03baB luciferase reporter, bleomycin mouse model\",\n      \"pmids\": [\"34645797\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Relationship between TSSK4-HSP90\\u03b2 phosphorylation and HSP90-dependent TSSK4 stabilization not reconciled\", \"Relevance to germ-cell apoptosis not established\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Addressed upstream transcriptional control of TSSK4: Atf6 knockout reduced TSSK4 mRNA and protein and correlated with impaired fertility, implicating ATF6 in TSSK4 expression.\",\n      \"evidence\": \"Transcriptome sequencing, western blot, real-time PCR in Atf6 KO mice and 293T cells\",\n      \"pmids\": [\"34904262\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No promoter binding or direct regulation experiment\", \"Effect could be indirect\", \"Causality of fertility link not established\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Demonstrated evolutionary conservation of the spermiogenesis role: the Drosophila ortholog dTSSK2 phosphorylates Gudu at Ser9 to maintain individualization complex integrity and sperm motility, with loss causing male infertility.\",\n      \"evidence\": \"Drosophila knockout, phosphoproteomics, in vivo localization, fertility assays\",\n      \"pmids\": [\"40335644\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mammalian ortholog of Gudu substrate not addressed\", \"Direct conservation of substrate specificity in mammals not tested\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How TSSK4-dependent ODF2 phosphorylation mechanistically produces normal flagellar ultrastructure, and how its germ-cell apoptotic and chaperone-regulating activities integrate during spermatogenesis, remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structural model of TSSK4-substrate complexes\", \"Integration of pro-apoptotic and structural functions in vivo unclear\", \"Full germ-cell substrate repertoire undefined\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [1, 2, 6, 8]},\n      {\"term_id\": \"GO:0016740\", \"supporting_discovery_ids\": [0, 1, 2, 6]},\n      {\"term_id\": \"GO:0140657\", \"supporting_discovery_ids\": [0]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [7]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1474165\", \"supporting_discovery_ids\": [1, 2]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [4, 6]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"ODF2\", \"HSP90\", \"HSP90AB1\", \"CREM\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":5,"faith_total":5,"faith_pct":100.0}}