{"gene":"TG_254630","gene_symbol":"CRK6","run_date":"2026-09-11T11:01:00","timeline":{"discoveries":[{"year":2017,"finding":"TgCrk6 is essential for tachyzoite growth and is required for spindle function during mitosis; conditional tet-OFF knockdown of TgCrk6 caused mitotic defects consistent with a spindle function role. TgCrk6 did not interact with any tested cyclin, suggesting it may not require a cyclin timing mechanism.","method":"Conditional tet-OFF mutant generation, phenotypic analysis of mitotic structures, co-immunoprecipitation (negative result for cyclin interaction)","journal":"PLoS pathogens","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — conditional knockdown with defined mitotic phenotype and co-IP testing cyclin interactions; single lab, two orthogonal methods","pmids":["28671988"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2022,"finding":"TgCrk6 forms a stable complex with a parasite-specific atypical cyclin TgCyc1 (previously orphaned). Conditional knockdown of either TgCrk6 or TgCyc1 produces identical mitotic defects, demonstrating cooperative action. The TgCrk6/TgCyc1 complex regulates metaphase and controls the spindle assembly checkpoint (SAC), specifically controlling the centromere-associated network. Global phosphoproteomics showed that Toxoplasma metaphase is regulated by CDK, MAPK, and Aurora kinases, with TgCrk6/TgCyc1 specifically controlling centromere-associated processes.","method":"Co-immunoprecipitation/proteomics to identify cyclin partner TgCyc1; conditional expression models; quantitative fluorescence microscopy; global phosphoproteomics","journal":"mBio","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP identifying cyclin partner, conditional knockdown of both complex components with identical phenotypes, global phosphoproteomics; single lab but multiple orthogonal methods","pmids":["35130726"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]}],"current_model":"TgCrk6 is an essential Cdk-related kinase in Toxoplasma gondii tachyzoites that forms a stable complex with the parasite-specific atypical cyclin TgCyc1; this TgCrk6/TgCyc1 complex is required for mitotic spindle function and specifically controls the spindle assembly checkpoint (SAC) by regulating the centromere-associated network during metaphase, acting downstream of TgCrk4 in the cell cycle regulatory hierarchy."},"narrative":{"mechanistic_narrative":"TgCrk6 is an essential Cdk-related kinase in Toxoplasma gondii tachyzoites that governs mitotic spindle function and the spindle assembly checkpoint (SAC) [PMID:28671988, PMID:35130726]. Conditional knockdown of TgCrk6 arrests tachyzoite growth and produces mitotic defects consistent with a loss of spindle function [PMID:28671988]. TgCrk6 acts as the kinase partner of the parasite-specific atypical cyclin TgCyc1, and this stable TgCrk6/TgCyc1 complex controls metaphase by regulating the centromere-associated network, with knockdown of either component yielding identical mitotic defects [PMID:35130726].","teleology":[{"year":2017,"claim":"Establishing whether TgCrk6 has a cell-cycle role defined its function as an essential regulator of mitotic spindle integrity, and its failure to bind any tested cyclin raised the question of how it is temporally controlled.","evidence":"Conditional tet-OFF knockdown with mitotic phenotyping and co-IP testing cyclin interactions in T. gondii","pmids":["28671988"],"confidence":"Medium","gaps":["No cyclin partner identified at this stage (negative co-IP)","Direct kinase substrates not defined","Molecular basis of the spindle defect unresolved"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2022,"claim":"Identifying TgCyc1 as the binding partner resolved the earlier orphan-kinase puzzle and defined the TgCrk6/TgCyc1 complex as a metaphase regulator that specifically controls the SAC via the centromere-associated network.","evidence":"Reciprocal co-IP/proteomics, conditional knockdown of both components with identical phenotypes, and global phosphoproteomics in T. gondii","pmids":["35130726"],"confidence":"High","gaps":["Direct phosphorylation substrates within the centromere network not individually validated","Structural basis of the atypical cyclin-kinase interaction unknown","Mechanism by which the complex enforces checkpoint arrest not resolved"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]}],"mechanism_profile":{"molecular_activity":[],"localization":[],"pathway":[],"complexes":["TgCrk6/TgCyc1 kinase-cyclin complex"],"partners":["TGCYC1 (PARASITE-SPECIFIC ATYPICAL CYCLIN)"],"other_free_text":["TgCrk6/TgCyc1 controls the centromere-associated network during metaphase","Toxoplasma metaphase phosphoregulation involves CDK, MAPK, and Aurora kinases, with TgCrk6/TgCyc1 acting on centromere-associated processes","SAC role resolved within the composite S/M/C phase via the ToxoFUCCIS indicator"]},"strain_conflicts":[]},"prefetch_data":{},"corpus_meta":[{"pmid":"10873834","id":"PMC_10873834","title":"Intracellular targets of cyclin-dependent kinase inhibitors: identification by affinity chromatography using immobilised inhibitors.","date":"2000","source":"Chemistry & biology","url":"https://pubmed.ncbi.nlm.nih.gov/10873834","citation_count":202,"is_preprint":false},{"pmid":"15023358","id":"PMC_15023358","title":"Protein kinases as targets for anti-parasitic chemotherapy.","date":"2004","source":"Biochimica et biophysica acta","url":"https://pubmed.ncbi.nlm.nih.gov/15023358","citation_count":113,"is_preprint":false},{"pmid":"18703066","id":"PMC_18703066","title":"The cell cycle and Toxoplasma gondii cell division: tightly knit or loosely stitched?","date":"2008","source":"International journal for parasitology","url":"https://pubmed.ncbi.nlm.nih.gov/18703066","citation_count":82,"is_preprint":false},{"pmid":"28671988","id":"PMC_28671988","title":"Checkpoints of apicomplexan cell division identified in Toxoplasma gondii.","date":"2017","source":"PLoS pathogens","url":"https://pubmed.ncbi.nlm.nih.gov/28671988","citation_count":59,"is_preprint":false},{"pmid":"15811523","id":"PMC_15811523","title":"Characterization of two T. gondii CK1 isoforms.","date":"2005","source":"Molecular and biochemical parasitology","url":"https://pubmed.ncbi.nlm.nih.gov/15811523","citation_count":26,"is_preprint":false},{"pmid":"35130726","id":"PMC_35130726","title":"Novel CRK-Cyclin Complex Controls Spindle Assembly Checkpoint in Toxoplasma Endodyogeny.","date":"2022","source":"mBio","url":"https://pubmed.ncbi.nlm.nih.gov/35130726","citation_count":17,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":9774,"output_tokens":1213,"usd":0.023758,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":6237,"output_tokens":2327,"usd":0.04468,"stage2_stop_reason":"end_turn"},"total_usd":0.068438,"stage1_batch_id":"msgbatch_01TEPNZ4MCzJ7xtsNwosiDRk","stage2_batch_id":"msgbatch_01FQShM7n1sfCr4pWTDShmki","note":"batch pricing = 50% of standard","corrective_rerun":"2026-09-10 wrong-gene rerun (round-4 rules)","corrective_reason":"summary amended","hand_review_patch":"2026-09-11 AK hand-review verdicts (handoff/tools_2026-09-11/patch)","hand_review_verdict":"edit"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2017,\n      \"finding\": \"TgCrk6 is essential for tachyzoite growth and is required for spindle function during mitosis; conditional tet-OFF knockdown of TgCrk6 caused mitotic defects consistent with a spindle function role. TgCrk6 did not interact with any tested cyclin, suggesting it may not require a cyclin timing mechanism.\",\n      \"method\": \"Conditional tet-OFF mutant generation, phenotypic analysis of mitotic structures, co-immunoprecipitation (negative result for cyclin interaction)\",\n      \"journal\": \"PLoS pathogens\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — conditional knockdown with defined mitotic phenotype and co-IP testing cyclin interactions; single lab, two orthogonal methods\",\n      \"pmids\": [\"28671988\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"TgCrk6 forms a stable complex with a parasite-specific atypical cyclin TgCyc1 (previously orphaned). Conditional knockdown of either TgCrk6 or TgCyc1 produces identical mitotic defects, demonstrating cooperative action. The TgCrk6/TgCyc1 complex regulates metaphase and controls the spindle assembly checkpoint (SAC), specifically controlling the centromere-associated network. Global phosphoproteomics showed that Toxoplasma metaphase is regulated by CDK, MAPK, and Aurora kinases, with TgCrk6/TgCyc1 specifically controlling centromere-associated processes.\",\n      \"method\": \"Co-immunoprecipitation/proteomics to identify cyclin partner TgCyc1; conditional expression models; quantitative fluorescence microscopy; global phosphoproteomics\",\n      \"journal\": \"mBio\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP identifying cyclin partner, conditional knockdown of both complex components with identical phenotypes, global phosphoproteomics; single lab but multiple orthogonal methods\",\n      \"pmids\": [\"35130726\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"TgCrk6 acts downstream of TgCrk4 in the cell cycle: TgCrk4 regulates G2/M transition and acts upstream of the spindle assembly checkpoint, placing TgCrk6 (which controls the SAC) downstream in the pathway.\",\n      \"method\": \"Conditional knockdown epistasis analysis; quantitative microscopy; global phosphoproteomics\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic epistasis via conditional knockdowns with defined phenotypes; single lab, pathway placement inferred from sequential phenotypes\",\n      \"pmids\": [\"38600241\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"The TgCrk6/TgCyc1-dependent spindle assembly checkpoint was functionally detected using the ToxoFUCCIS cell cycle indicator system, confirming TgCrk6's role in SAC within the composite S/M/C phase of the Toxoplasma cell cycle.\",\n      \"method\": \"Fluorescent Ubiquitination-based Cell Cycle Indicator (ToxoFUCCIS), live and immunofluorescence microscopy\",\n      \"journal\": \"mBio\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — novel cell cycle indicator with live imaging confirming SAC checkpoint; single lab, corroborates prior findings with orthogonal tool\",\n      \"pmids\": [\"40590555\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    }\n  ],\n  \"current_model\": \"TgCrk6 is an essential Cdk-related kinase in Toxoplasma gondii tachyzoites that forms a stable complex with the parasite-specific atypical cyclin TgCyc1; this TgCrk6/TgCyc1 complex is required for mitotic spindle function and specifically controls the spindle assembly checkpoint (SAC) by regulating the centromere-associated network during metaphase, acting downstream of TgCrk4 in the cell cycle regulatory hierarchy.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"TgCrk6 is an essential Cdk-related kinase in Toxoplasma gondii tachyzoites that governs mitotic spindle function and the spindle assembly checkpoint (SAC) [#0, #1]. Conditional knockdown of TgCrk6 arrests tachyzoite growth and produces mitotic defects consistent with a loss of spindle function [#0]. TgCrk6 acts as the kinase partner of the parasite-specific atypical cyclin TgCyc1, and this stable TgCrk6/TgCyc1 complex controls metaphase by regulating the centromere-associated network, with knockdown of either component yielding identical mitotic defects [#1]. Within the cell cycle regulatory hierarchy, TgCrk6 acts downstream of TgCrk4, which controls the G2/M transition upstream of the checkpoint [#2], and its SAC-controlling activity has been directly resolved within the composite S/M/C phase using the ToxoFUCCIS cell cycle indicator [#3].\",\n  \"teleology\": [\n    {\n      \"year\": 2017,\n      \"claim\": \"Establishing whether TgCrk6 has a cell-cycle role defined its function as an essential regulator of mitotic spindle integrity, and its failure to bind any tested cyclin raised the question of how it is temporally controlled.\",\n      \"evidence\": \"Conditional tet-OFF knockdown with mitotic phenotyping and co-IP testing cyclin interactions in T. gondii\",\n      \"pmids\": [\"28671988\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"No cyclin partner identified at this stage (negative co-IP)\",\n        \"Direct kinase substrates not defined\",\n        \"Molecular basis of the spindle defect unresolved\"\n      ],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Identifying TgCyc1 as the binding partner resolved the earlier orphan-kinase puzzle and defined the TgCrk6/TgCyc1 complex as a metaphase regulator that specifically controls the SAC via the centromere-associated network.\",\n      \"evidence\": \"Reciprocal co-IP/proteomics, conditional knockdown of both components with identical phenotypes, and global phosphoproteomics in T. gondii\",\n      \"pmids\": [\"35130726\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Direct phosphorylation substrates within the centromere network not individually validated\",\n        \"Structural basis of the atypical cyclin-kinase interaction unknown\",\n        \"Mechanism by which the complex enforces checkpoint arrest not resolved\"\n      ],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Epistasis analysis placed TgCrk6 downstream of TgCrk4, ordering the checkpoint machinery relative to the G2/M transition and defining the sequence of kinase control across mitosis.\",\n      \"evidence\": \"Conditional knockdown epistasis, quantitative microscopy, and global phosphoproteomics in T. gondii\",\n      \"pmids\": [\"38600241\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Pathway order inferred from sequential phenotypes rather than direct biochemical linkage\",\n        \"Whether TgCrk4 directly regulates TgCrk6/TgCyc1 not established\"\n      ],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"A dedicated cell-cycle reporter directly localized TgCrk6-dependent SAC activity within the composite S/M/C phase, confirming the checkpoint role with an orthogonal live-imaging tool.\",\n      \"evidence\": \"ToxoFUCCIS fluorescent cell-cycle indicator with live and immunofluorescence microscopy in T. gondii\",\n      \"pmids\": [\"40590555\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Does not add new molecular substrates or interactors\",\n        \"Kinetics of checkpoint engagement not quantified\"\n      ],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The direct phosphorylation substrates of the TgCrk6/TgCyc1 complex at the centromere-associated network, and the structural basis of its atypical cyclin partnership, remain unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"No validated direct substrate identified\",\n        \"No structural model of the TgCrk6/TgCyc1 complex\",\n        \"Mechanism connecting TgCrk4 upstream control to TgCrk6 activation unknown\"\n      ],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0004693\", \"supporting_discovery_ids\": [0, 1], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]}\n    ],\n    \"localization\": [],\n    \"pathway\": [\n      {\"term_id\": \"GO:0007094\", \"supporting_discovery_ids\": [1, 3], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]},\n      {\"term_id\": \"GO:0007052\", \"supporting_discovery_ids\": [0, 1], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]},\n      {\"term_id\": \"GO:0000278\", \"supporting_discovery_ids\": [0, 2], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]}\n    ],\n    \"complexes\": [\n      \"TgCrk6/TgCyc1 kinase-cyclin complex\"\n    ],\n    \"partners\": [\n      \"TgCyc1 (parasite-specific atypical cyclin)\",\n      \"TgCrk4 (acts upstream in the cell cycle hierarchy)\"\n    ],\n    \"other_free_text\": [\n      \"TgCrk6/TgCyc1 controls the centromere-associated network during metaphase\",\n      \"Toxoplasma metaphase phosphoregulation involves CDK, MAPK, and Aurora kinases, with TgCrk6/TgCyc1 acting on centromere-associated processes\",\n      \"SAC role resolved within the composite S/M/C phase via the ToxoFUCCIS indicator\"\n    ]\n  },\n  \"strain_conflicts\": []\n}","audit_flag":null,"strains":[{"strain":"ME49","gene_id":"TGME49_254630","name":"CRK6","product":"CDK-related protein kinase CRK6","uniprot_acc":"S8GII9","chromosome":"TGME49_chrIII","start_pos":1867147},{"strain":"GT1","gene_id":"TGGT1_254630","name":null,"product":"CMGC kinase","uniprot_acc":null,"chromosome":"TGGT1_chrIII","start_pos":1848333},{"strain":"RH88","gene_id":"TGRH88_003700","name":null,"product":"CMGC kinase","uniprot_acc":null,"chromosome":"CM023084","start_pos":735444},{"strain":"VEG","gene_id":"TGVEG_254630","name":null,"product":"CMGC kinase","uniprot_acc":"A0A125YWH7","chromosome":"TGVEG_chrIII","start_pos":1877064}]}