{"gene":"TG_233010","gene_symbol":"ERK7","run_date":"2026-09-08T12:58:24","timeline":{"discoveries":[{"year":2020,"finding":"ERK7 localizes to the apical cap of the Toxoplasma IMC (just basal to the conoid) and is essential for conoid assembly; parasites depleted of ERK7 lose their conoids late during maturation, are immotile, and unable to invade new host cells.","method":"Auxin-inducible degron (AID) knockdown with loss-of-function phenotypic readout (conoid loss, motility, invasion assay); direct localization by fluorescence microscopy","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean conditional knockdown with multiple defined cellular phenotypes; localization directly determined; independently replicated in subsequent studies","pmids":["32073987"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2020,"finding":"AC9 (apical cap protein 9) directly binds ERK7 through a conserved C-terminal motif and is required for ERK7 localization and function at the apical cap; the crystal structure of the ERK7–AC9 complex reveals that AC9 acts as an unusual inhibitory scaffold that displaces nucleotide from the ERK7 kinase active site, keeping ERK7 in an 'off' state while concentrating it at its site of action.","method":"Proximity biotinylation (TurboID) to identify interaction network; crystal structure of ERK7–AC9 complex; direct binding demonstrated by structural and biochemical analysis; deletion/mutagenesis of the C-terminal AC9 motif","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure resolved, direct binding demonstrated, active-site nucleotide displacement mechanism defined in same study","pmids":["32409604"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2021,"finding":"Depletion of ERK7 leads to loss of the apical polar ring (APR), disorganization of subpellicular microtubules (SPMTs), severe impairment of microneme secretion, dilation of the apical plasma membrane, and altered rhoptry positioning — phenocopying depletion of AC9 or AC10; comparative proteomics confirmed disappearance of known apical complex proteins including APR markers.","method":"Conditional knockdown (AID system), ultrastructure expansion microscopy (U-ExM) with NHS-ester staining, electron microscopy, comparative proteomics of wild-type vs. ERK7-depleted parasites","journal":"mBio","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (U-ExM, EM, proteomics) in a single rigorous study, phenotype independently consistent with prior reports","pmids":["34607461"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2022,"finding":"AC9, AC10, and ERK7 form an essential trimeric complex in the apical cap organized by multivalent interactions; AC10 is a foundational component of the AC9:AC10:ERK7 complex, and multiple independent pairwise interaction regions between the three proteins drive oligomerization and tethering of ERK7 kinase activity in the apical cap cytoskeleton.","method":"Deletion analyses, yeast two-hybrid experiments, proximity biotinylation, conditional knockdown of individual components with apical complex stability readout","journal":"mBio","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal yeast two-hybrid with deletion mapping, multiple orthogonal methods, extends and refines the structurally validated AC9–ERK7 interaction from prior work","pmids":["35130732"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2023,"finding":"ERK7 protects the apical complex from premature degradation during parasite replication; ERK7 interacts with a putative E3 ubiquitin ligase CSAR1, and genetic disruption of CSAR1 fully suppresses apical complex loss caused by ERK7 knockdown; CSAR1 normally mediates turnover of maternal cytoskeleton from the residual body during cytokinesis, but in the absence of ERK7 it mislocalizes from the residual body to the apical complex, causing aberrant proteolysis of apical complex components.","method":"ERK7 interactome mapping (proximity biotinylation/mass spectrometry), genetic disruption of CSAR1, epistasis (CSAR1 KO suppresses ERK7 KD phenotype), localization studies by fluorescence microscopy","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — interactome mapping combined with genetic epistasis (suppressor) and localization experiments, multiple orthogonal approaches in one study","pmids":["37027006"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2016,"finding":"Knockout of TgERK7 (ΔTgERK7) significantly reduces intracellular proliferation of T. gondii tachyzoites in vitro and severely attenuates parasite virulence in mice.","method":"Double homologous recombination gene knockout; intracellular proliferation assay in vitro; mouse virulence assay","journal":"Parasitology research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — clean KO with defined proliferation phenotype, but mechanism of proliferation defect not resolved; results consistent with later studies","pmids":["27150970"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":"GT1"}]},{"year":2020,"finding":"Eradication of TgERK7 leads to significantly reduced host cell invasion (at 1–2 h post-infection) without affecting attachment (at 15 min), indicating ERK7 is required for the invasion step specifically, which is the primary cause of the downstream intracellular proliferation defect.","method":"Complementation strain (ΔTgERK7 re-expressing TgERK7), invasion assay and attachment assay at defined time points, compared with WT GT1 and complemented strain","journal":"Parasitology research","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — phenotypic readout with complementation control; invasion defect mechanistically consistent with later apical complex studies, but direct molecular mechanism not resolved in this paper","pmids":["32914221"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":"GT1"}]},{"year":2025,"finding":"The abundance and localization of ERK7 are not affected by deletion of the SPY O-fucosyltransferase, indicating O-fucosylation does not regulate ERK7 levels or localization.","method":"Proteomic analysis and immunofluorescence microscopy in SPYΔ vs. wild-type Toxoplasma","journal":"Glycobiology","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — direct experiment (proteomics + IFA) in a single study; this is a negative result for O-fucosylation regulation of ERK7","pmids":["40888172"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]}],"current_model":"Toxoplasma gondii ERK7 is an ancient autoactivating MAPK that localizes to the apical cap of the inner membrane complex (IMC) through multivalent interactions within an essential AC9:AC10:ERK7 trimeric scaffold complex; AC9 directly binds ERK7's active site to displace nucleotide and maintain it in an inhibited state at its site of action, while ERK7 activity is required to protect the apical complex (conoid, apical polar ring, subpellicular microtubules) from premature degradation by antagonizing the mislocalization of an E3 ligase (CSAR1), thereby ensuring proper apical complex biogenesis and the parasite's capacity for gliding motility, host cell invasion, and egress."},"narrative":{"mechanistic_narrative":"Toxoplasma gondii ERK7 is an autoactivating MAP kinase that localizes to the apical cap of the inner membrane complex, just basal to the conoid, where it governs biogenesis and stability of the parasite's apical complex [PMID:32073987, PMID:34607461]. ERK7 is tethered at this site through an essential trimeric scaffold: it binds AC9 directly via a conserved C-terminal motif, and AC9, AC10, and ERK7 assemble through multiple independent pairwise interactions into the AC9:AC10:ERK7 complex that concentrates the kinase in the apical cap cytoskeleton [PMID:32409604, PMID:35130732]. AC9 doubles as an inhibitory scaffold, engaging the ERK7 active site to displace nucleotide and hold the kinase in an 'off' state at its site of action [PMID:32409604]. Functionally, ERK7 activity protects the apical complex from premature degradation during replication by antagonizing the E3 ubiquitin ligase CSAR1: normally CSAR1 clears maternal cytoskeleton from the residual body, but upon ERK7 loss it mislocalizes to the apical complex and drives aberrant proteolysis of conoid, apical polar ring, and subpellicular microtubule components, with CSAR1 disruption fully suppressing the ERK7 knockdown phenotype [PMID:37027006, PMID:34607461]. Loss of ERK7 therefore abolishes conoid assembly, microneme secretion, gliding motility, host cell invasion, and downstream intracellular proliferation, and severely attenuates virulence in mice [PMID:32073987, PMID:27150970, PMID:32914221]. ERK7 abundance and localization are independent of SPY-mediated O-fucosylation [PMID:40888172].","teleology":[{"year":2016,"claim":"Establishing whether ERK7 matters for the parasite lifecycle, a clean knockout showed the kinase is required for tachyzoite proliferation and virulence, framing ERK7 as a functionally important target.","evidence":"Double homologous recombination knockout with in vitro proliferation and mouse virulence assays in strain GT1","pmids":["27150970"],"confidence":"Medium","gaps":["Mechanism of the proliferation defect not resolved","No subcellular localization or molecular partners defined"],"attribution":[{"species":"Toxoplasma gondii","strain":"GT1"}]},{"year":2020,"claim":"Localizing ERK7 to the apical cap and demonstrating conoid loss on depletion pinpointed the kinase as essential for apical complex biogenesis, and separately identified host cell invasion as the specific lifecycle step it enables.","evidence":"AID conditional knockdown with conoid/motility/invasion readouts and fluorescence localization; complementation-controlled invasion versus attachment timing assays in GT1","pmids":["32073987","32914221"],"confidence":"High","gaps":["Kinase substrates in apical complex assembly not identified","How ERK7 activity is coupled to conoid maturation unresolved"],"attribution":[{"species":"Toxoplasma gondii","strain":""},{"species":"Toxoplasma gondii","strain":"GT1"}]},{"year":2020,"claim":"Asking how ERK7 is delivered and controlled at the apical cap, structural and biochemical work showed AC9 is a direct binding partner that both tethers the kinase and inhibits it by displacing active-site nucleotide, defining an unusual scaffold-as-inhibitor mechanism.","evidence":"TurboID interactome, ERK7–AC9 crystal structure, and C-terminal motif deletion/mutagenesis","pmids":["32409604"],"confidence":"High","gaps":["How and when ERK7 is released into an active state in vivo not defined","Physiological substrates of the active kinase unknown"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2021,"claim":"Defining the full structural consequence of ERK7 loss, high-resolution imaging and proteomics showed depletion collapses the apical polar ring and subpellicular microtubules and impairs secretion, phenocopying AC9/AC10 loss and cementing ERK7 as a keystone of apical cytoskeleton integrity.","evidence":"AID knockdown with U-ExM, electron microscopy, and comparative proteomics","pmids":["34607461"],"confidence":"High","gaps":["Direct effectors linking ERK7 activity to microtubule and APR stability not identified"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2022,"claim":"Resolving the architecture that anchors the kinase, interaction mapping showed AC9, AC10, and ERK7 form an essential trimeric complex organized by multivalent pairwise contacts, with AC10 a foundational scaffold component.","evidence":"Yeast two-hybrid with deletion mapping, proximity biotinylation, and conditional knockdown of individual components","pmids":["35130732"],"confidence":"High","gaps":["Stoichiometry and higher-order assembly of the complex not defined","How the scaffold gates kinase activity temporally unresolved"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2023,"claim":"Explaining why apical structures are lost, interactome and epistasis experiments revealed ERK7 protects the apical complex by preventing mislocalization of the E3 ligase CSAR1, whose disruption fully suppresses the ERK7 depletion phenotype.","evidence":"Proximity biotinylation/mass spectrometry, CSAR1 genetic disruption with suppressor epistasis, and localization microscopy","pmids":["37027006"],"confidence":"High","gaps":["Whether CSAR1 is a direct ERK7 phosphorylation substrate not established","Molecular basis of CSAR1 spatial restriction to the residual body unknown"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2025,"claim":"Testing a candidate post-translational regulator, deletion of the SPY O-fucosyltransferase left ERK7 abundance and localization unchanged, excluding O-fucosylation as a determinant of ERK7 levels or targeting.","evidence":"Proteomics and immunofluorescence in SPYΔ versus wild-type parasites","pmids":["40888172"],"confidence":"Medium","gaps":["Other post-translational modifications regulating ERK7 not tested"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":null,"claim":"The direct in vivo substrates of ERK7 kinase activity, and the trigger that relieves AC9-mediated inhibition to activate the kinase, remain unidentified.","evidence":"","pmids":[],"confidence":"High","gaps":["No phosphorylation substrates mapped","Activation trigger and timing of ERK7 catalysis in situ unknown"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]}],"mechanism_profile":{"molecular_activity":[],"localization":[],"pathway":[],"complexes":["AC9:AC10:ERK7 trimeric apical cap scaffold complex"],"partners":["AC9 (APICAL CAP PROTEIN 9; DIRECT INHIBITORY SCAFFOLD BINDER)","AC10 (APICAL CAP PROTEIN 10; FOUNDATIONAL SCAFFOLD COMPONENT)","CSAR1 (PUTATIVE E3 UBIQUITIN LIGASE ANTAGONIZED BY ERK7)"],"other_free_text":["AC9 binds the ERK7 active site and displaces nucleotide, holding the kinase inactive while concentrating it at the apical cap (idx 1).","ERK7 depletion causes loss of conoid, apical polar ring, and subpellicular microtubules, impaired microneme secretion, and altered rhoptry positioning, phenocopying AC9/AC10 loss (idx 2).","In the absence of ERK7, CSAR1 mislocalizes from the residual body to the apical complex and drives aberrant proteolysis; CSAR1 knockout suppresses the ERK7 phenotype (idx 4).","SPY-mediated O-fucosylation does not regulate ERK7 abundance or localization (idx 7)."]},"strain_conflicts":[]},"prefetch_data":{},"corpus_meta":[{"pmid":"32409604","id":"PMC_32409604","title":"Ancient MAPK ERK7 is regulated by an unusual inhibitory scaffold required for Toxoplasma apical complex biogenesis.","date":"2020","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/32409604","citation_count":44,"is_preprint":false},{"pmid":"32073987","id":"PMC_32073987","title":"Loss of a conserved MAPK causes catastrophic failure in assembly of a specialized cilium-like structure in Toxoplasma gondii.","date":"2020","source":"Molecular biology of the cell","url":"https://pubmed.ncbi.nlm.nih.gov/32073987","citation_count":30,"is_preprint":false},{"pmid":"34607461","id":"PMC_34607461","title":"Revisiting the Role of Toxoplasma gondii ERK7 in the Maintenance and Stability of the Apical Complex.","date":"2021","source":"mBio","url":"https://pubmed.ncbi.nlm.nih.gov/34607461","citation_count":28,"is_preprint":false},{"pmid":"35130732","id":"PMC_35130732","title":"Multivalent Interactions Drive the Toxoplasma AC9:AC10:ERK7 Complex To Concentrate ERK7 in the Apical Cap.","date":"2022","source":"mBio","url":"https://pubmed.ncbi.nlm.nih.gov/35130732","citation_count":21,"is_preprint":false},{"pmid":"33173004","id":"PMC_33173004","title":"Loss of the Conserved Alveolate Kinase MAPK2 Decouples Toxoplasma Cell Growth from Cell Division.","date":"2020","source":"mBio","url":"https://pubmed.ncbi.nlm.nih.gov/33173004","citation_count":21,"is_preprint":false},{"pmid":"37027006","id":"PMC_37027006","title":"Toxoplasma ERK7 protects the apical complex from premature degradation.","date":"2023","source":"The Journal of cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/37027006","citation_count":19,"is_preprint":false},{"pmid":"27150970","id":"PMC_27150970","title":"TgERK7 is involved in the intracellular proliferation of Toxoplasma gondii.","date":"2016","source":"Parasitology research","url":"https://pubmed.ncbi.nlm.nih.gov/27150970","citation_count":12,"is_preprint":false},{"pmid":"32654774","id":"PMC_32654774","title":"Immunostimulatory efficacy and protective potential of putative TgERK7 protein in mice experimentally infected by Toxoplasma gondii.","date":"2020","source":"International journal of medical microbiology : IJMM","url":"https://pubmed.ncbi.nlm.nih.gov/32654774","citation_count":5,"is_preprint":false},{"pmid":"32099558","id":"PMC_32099558","title":"Immune Responses Induced by pVAX/TgERK7 against Toxoplasma gondii Infection in BALB/c Mice.","date":"2019","source":"Iranian journal of parasitology","url":"https://pubmed.ncbi.nlm.nih.gov/32099558","citation_count":3,"is_preprint":false},{"pmid":"32914221","id":"PMC_32914221","title":"Toxoplasma invasion delayed by TgERK7 eradication.","date":"2020","source":"Parasitology research","url":"https://pubmed.ncbi.nlm.nih.gov/32914221","citation_count":2,"is_preprint":false},{"pmid":"40888172","id":"PMC_40888172","title":"O-fucosylation affects abundance but not localization of select nucleocytoplasmic proteins in toxoplasma gondii.","date":"2025","source":"Glycobiology","url":"https://pubmed.ncbi.nlm.nih.gov/40888172","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":8375,"output_tokens":2349,"usd":0.03018,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":7852,"output_tokens":3474,"usd":0.063055,"stage2_stop_reason":"end_turn"},"total_usd":0.093235,"stage1_batch_id":"msgbatch_01TEPNZ4MCzJ7xtsNwosiDRk","stage2_batch_id":"msgbatch_01FQShM7n1sfCr4pWTDShmki","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2020,\n      \"finding\": \"ERK7 localizes to the apical cap of the Toxoplasma IMC (just basal to the conoid) and is essential for conoid assembly; parasites depleted of ERK7 lose their conoids late during maturation, are immotile, and unable to invade new host cells.\",\n      \"method\": \"Auxin-inducible degron (AID) knockdown with loss-of-function phenotypic readout (conoid loss, motility, invasion assay); direct localization by fluorescence microscopy\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean conditional knockdown with multiple defined cellular phenotypes; localization directly determined; independently replicated in subsequent studies\",\n      \"pmids\": [\"32073987\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"AC9 (apical cap protein 9) directly binds ERK7 through a conserved C-terminal motif and is required for ERK7 localization and function at the apical cap; the crystal structure of the ERK7–AC9 complex reveals that AC9 acts as an unusual inhibitory scaffold that displaces nucleotide from the ERK7 kinase active site, keeping ERK7 in an 'off' state while concentrating it at its site of action.\",\n      \"method\": \"Proximity biotinylation (TurboID) to identify interaction network; crystal structure of ERK7–AC9 complex; direct binding demonstrated by structural and biochemical analysis; deletion/mutagenesis of the C-terminal AC9 motif\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure resolved, direct binding demonstrated, active-site nucleotide displacement mechanism defined in same study\",\n      \"pmids\": [\"32409604\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Depletion of ERK7 leads to loss of the apical polar ring (APR), disorganization of subpellicular microtubules (SPMTs), severe impairment of microneme secretion, dilation of the apical plasma membrane, and altered rhoptry positioning — phenocopying depletion of AC9 or AC10; comparative proteomics confirmed disappearance of known apical complex proteins including APR markers.\",\n      \"method\": \"Conditional knockdown (AID system), ultrastructure expansion microscopy (U-ExM) with NHS-ester staining, electron microscopy, comparative proteomics of wild-type vs. ERK7-depleted parasites\",\n      \"journal\": \"mBio\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (U-ExM, EM, proteomics) in a single rigorous study, phenotype independently consistent with prior reports\",\n      \"pmids\": [\"34607461\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"AC9, AC10, and ERK7 form an essential trimeric complex in the apical cap organized by multivalent interactions; AC10 is a foundational component of the AC9:AC10:ERK7 complex, and multiple independent pairwise interaction regions between the three proteins drive oligomerization and tethering of ERK7 kinase activity in the apical cap cytoskeleton.\",\n      \"method\": \"Deletion analyses, yeast two-hybrid experiments, proximity biotinylation, conditional knockdown of individual components with apical complex stability readout\",\n      \"journal\": \"mBio\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal yeast two-hybrid with deletion mapping, multiple orthogonal methods, extends and refines the structurally validated AC9–ERK7 interaction from prior work\",\n      \"pmids\": [\"35130732\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"ERK7 protects the apical complex from premature degradation during parasite replication; ERK7 interacts with a putative E3 ubiquitin ligase CSAR1, and genetic disruption of CSAR1 fully suppresses apical complex loss caused by ERK7 knockdown; CSAR1 normally mediates turnover of maternal cytoskeleton from the residual body during cytokinesis, but in the absence of ERK7 it mislocalizes from the residual body to the apical complex, causing aberrant proteolysis of apical complex components.\",\n      \"method\": \"ERK7 interactome mapping (proximity biotinylation/mass spectrometry), genetic disruption of CSAR1, epistasis (CSAR1 KO suppresses ERK7 KD phenotype), localization studies by fluorescence microscopy\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — interactome mapping combined with genetic epistasis (suppressor) and localization experiments, multiple orthogonal approaches in one study\",\n      \"pmids\": [\"37027006\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Knockout of TgERK7 (ΔTgERK7) significantly reduces intracellular proliferation of T. gondii tachyzoites in vitro and severely attenuates parasite virulence in mice.\",\n      \"method\": \"Double homologous recombination gene knockout; intracellular proliferation assay in vitro; mouse virulence assay\",\n      \"journal\": \"Parasitology research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean KO with defined proliferation phenotype, but mechanism of proliferation defect not resolved; results consistent with later studies\",\n      \"pmids\": [\"27150970\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"GT1\"}]\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Eradication of TgERK7 leads to significantly reduced host cell invasion (at 1–2 h post-infection) without affecting attachment (at 15 min), indicating ERK7 is required for the invasion step specifically, which is the primary cause of the downstream intracellular proliferation defect.\",\n      \"method\": \"Complementation strain (ΔTgERK7 re-expressing TgERK7), invasion assay and attachment assay at defined time points, compared with WT GT1 and complemented strain\",\n      \"journal\": \"Parasitology research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — phenotypic readout with complementation control; invasion defect mechanistically consistent with later apical complex studies, but direct molecular mechanism not resolved in this paper\",\n      \"pmids\": [\"32914221\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"GT1\"}]\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"The abundance and localization of ERK7 are not affected by deletion of the SPY O-fucosyltransferase, indicating O-fucosylation does not regulate ERK7 levels or localization.\",\n      \"method\": \"Proteomic analysis and immunofluorescence microscopy in SPYΔ vs. wild-type Toxoplasma\",\n      \"journal\": \"Glycobiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — direct experiment (proteomics + IFA) in a single study; this is a negative result for O-fucosylation regulation of ERK7\",\n      \"pmids\": [\"40888172\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    }\n  ],\n  \"current_model\": \"Toxoplasma gondii ERK7 is an ancient autoactivating MAPK that localizes to the apical cap of the inner membrane complex (IMC) through multivalent interactions within an essential AC9:AC10:ERK7 trimeric scaffold complex; AC9 directly binds ERK7's active site to displace nucleotide and maintain it in an inhibited state at its site of action, while ERK7 activity is required to protect the apical complex (conoid, apical polar ring, subpellicular microtubules) from premature degradation by antagonizing the mislocalization of an E3 ligase (CSAR1), thereby ensuring proper apical complex biogenesis and the parasite's capacity for gliding motility, host cell invasion, and egress.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"Toxoplasma gondii ERK7 is an autoactivating MAP kinase that localizes to the apical cap of the inner membrane complex, just basal to the conoid, where it governs biogenesis and stability of the parasite's apical complex [#0, #2]. ERK7 is tethered at this site through an essential trimeric scaffold: it binds AC9 directly via a conserved C-terminal motif, and AC9, AC10, and ERK7 assemble through multiple independent pairwise interactions into the AC9:AC10:ERK7 complex that concentrates the kinase in the apical cap cytoskeleton [#1, #3]. AC9 doubles as an inhibitory scaffold, engaging the ERK7 active site to displace nucleotide and hold the kinase in an 'off' state at its site of action [#1]. Functionally, ERK7 activity protects the apical complex from premature degradation during replication by antagonizing the E3 ubiquitin ligase CSAR1: normally CSAR1 clears maternal cytoskeleton from the residual body, but upon ERK7 loss it mislocalizes to the apical complex and drives aberrant proteolysis of conoid, apical polar ring, and subpellicular microtubule components, with CSAR1 disruption fully suppressing the ERK7 knockdown phenotype [#4, #2]. Loss of ERK7 therefore abolishes conoid assembly, microneme secretion, gliding motility, host cell invasion, and downstream intracellular proliferation, and severely attenuates virulence in mice [#0, #5, #6]. ERK7 abundance and localization are independent of SPY-mediated O-fucosylation [#7].\"\n  ,\n  \"teleology\": [\n    {\n      \"year\": 2016,\n      \"claim\": \"Establishing whether ERK7 matters for the parasite lifecycle, a clean knockout showed the kinase is required for tachyzoite proliferation and virulence, framing ERK7 as a functionally important target.\",\n      \"evidence\": \"Double homologous recombination knockout with in vitro proliferation and mouse virulence assays in strain GT1\",\n      \"pmids\": [\"27150970\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism of the proliferation defect not resolved\", \"No subcellular localization or molecular partners defined\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"GT1\"}]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Localizing ERK7 to the apical cap and demonstrating conoid loss on depletion pinpointed the kinase as essential for apical complex biogenesis, and separately identified host cell invasion as the specific lifecycle step it enables.\",\n      \"evidence\": \"AID conditional knockdown with conoid/motility/invasion readouts and fluorescence localization; complementation-controlled invasion versus attachment timing assays in GT1\",\n      \"pmids\": [\"32073987\", \"32914221\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Kinase substrates in apical complex assembly not identified\", \"How ERK7 activity is coupled to conoid maturation unresolved\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}, {\"species\": \"Toxoplasma gondii\", \"strain\": \"GT1\"}]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Asking how ERK7 is delivered and controlled at the apical cap, structural and biochemical work showed AC9 is a direct binding partner that both tethers the kinase and inhibits it by displacing active-site nucleotide, defining an unusual scaffold-as-inhibitor mechanism.\",\n      \"evidence\": \"TurboID interactome, ERK7\\u2013AC9 crystal structure, and C-terminal motif deletion/mutagenesis\",\n      \"pmids\": [\"32409604\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How and when ERK7 is released into an active state in vivo not defined\", \"Physiological substrates of the active kinase unknown\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Defining the full structural consequence of ERK7 loss, high-resolution imaging and proteomics showed depletion collapses the apical polar ring and subpellicular microtubules and impairs secretion, phenocopying AC9/AC10 loss and cementing ERK7 as a keystone of apical cytoskeleton integrity.\",\n      \"evidence\": \"AID knockdown with U-ExM, electron microscopy, and comparative proteomics\",\n      \"pmids\": [\"34607461\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct effectors linking ERK7 activity to microtubule and APR stability not identified\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Resolving the architecture that anchors the kinase, interaction mapping showed AC9, AC10, and ERK7 form an essential trimeric complex organized by multivalent pairwise contacts, with AC10 a foundational scaffold component.\",\n      \"evidence\": \"Yeast two-hybrid with deletion mapping, proximity biotinylation, and conditional knockdown of individual components\",\n      \"pmids\": [\"35130732\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Stoichiometry and higher-order assembly of the complex not defined\", \"How the scaffold gates kinase activity temporally unresolved\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Explaining why apical structures are lost, interactome and epistasis experiments revealed ERK7 protects the apical complex by preventing mislocalization of the E3 ligase CSAR1, whose disruption fully suppresses the ERK7 depletion phenotype.\",\n      \"evidence\": \"Proximity biotinylation/mass spectrometry, CSAR1 genetic disruption with suppressor epistasis, and localization microscopy\",\n      \"pmids\": [\"37027006\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether CSAR1 is a direct ERK7 phosphorylation substrate not established\", \"Molecular basis of CSAR1 spatial restriction to the residual body unknown\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Testing a candidate post-translational regulator, deletion of the SPY O-fucosyltransferase left ERK7 abundance and localization unchanged, excluding O-fucosylation as a determinant of ERK7 levels or targeting.\",\n      \"evidence\": \"Proteomics and immunofluorescence in SPY\\u0394 versus wild-type parasites\",\n      \"pmids\": [\"40888172\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Other post-translational modifications regulating ERK7 not tested\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The direct in vivo substrates of ERK7 kinase activity, and the trigger that relieves AC9-mediated inhibition to activate the kinase, remain unidentified.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No phosphorylation substrates mapped\", \"Activation trigger and timing of ERK7 catalysis in situ unknown\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0004707\", \"supporting_discovery_ids\": [0, 1], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]},\n      {\"term_id\": \"GO:0004672\", \"supporting_discovery_ids\": [1], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0120227\", \"supporting_discovery_ids\": [0, 1, 3], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"GO:0044409\", \"supporting_discovery_ids\": [0, 6], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]},\n      {\"term_id\": \"GO:0007163\", \"supporting_discovery_ids\": [2, 4], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]}\n    ],\n    \"complexes\": [\n      \"AC9:AC10:ERK7 trimeric apical cap scaffold complex\"\n    ],\n    \"partners\": [\n      \"AC9 (apical cap protein 9; direct inhibitory scaffold binder)\",\n      \"AC10 (apical cap protein 10; foundational scaffold component)\",\n      \"CSAR1 (putative E3 ubiquitin ligase antagonized by ERK7)\"\n    ],\n    \"other_free_text\": [\n      \"AC9 binds the ERK7 active site and displaces nucleotide, holding the kinase inactive while concentrating it at the apical cap (idx 1).\",\n      \"ERK7 depletion causes loss of conoid, apical polar ring, and subpellicular microtubules, impaired microneme secretion, and altered rhoptry positioning, phenocopying AC9/AC10 loss (idx 2).\",\n      \"In the absence of ERK7, CSAR1 mislocalizes from the residual body to the apical complex and drives aberrant proteolysis; CSAR1 knockout suppresses the ERK7 phenotype (idx 4).\",\n      \"SPY-mediated O-fucosylation does not regulate ERK7 abundance or localization (idx 7).\"\n    ]\n  },\n  \"strain_conflicts\": []\n}\n```","audit_flag":null,"strains":[{"strain":"ME49","gene_id":"TGME49_233010","name":"ERK7","product":"mitogen-activated protein kinase ERK7","uniprot_acc":"S8GK09","chromosome":"TGME49_chrVIII","start_pos":2336150},{"strain":"GT1","gene_id":"TGGT1_233010","name":null,"product":"putative cell-cycle-assocaited protein kinase ERK7","uniprot_acc":null,"chromosome":"TGGT1_chrVIII","start_pos":2333548},{"strain":"RH88","gene_id":"TGRH88_076990","name":null,"product":"putative cell-cycle-assocaited protein kinase ERK7","uniprot_acc":null,"chromosome":"CM023089","start_pos":7443817},{"strain":"VEG","gene_id":"TGVEG_233010","name":null,"product":"putative cell-cycle-assocaited protein kinase ERK7","uniprot_acc":"V4Z6R8","chromosome":"TGVEG_chrVIII","start_pos":2346707}]}