{"gene":"TG_290170","gene_symbol":"PPKL","run_date":"2026-09-08T14:08:30","timeline":{"discoveries":[{"year":2023,"finding":"PPKL localizes dynamically during parasite division: in non-dividing parasites it is present in the cytoplasm, nucleus, and preconoidal region; as division begins, it enriches in the preconoidal region and cortical cytoskeleton of nascent daughter parasites; later it localizes to the basal complex ring.","method":"Live/fixed imaging with conditional knockdown system (AID), localization tracked across cell cycle stages","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct localization experiment with cell-cycle-stage resolution, single lab, preprint","pmids":["37398039"],"is_preprint":true,"attribution":[{"species":"Toxoplasma gondii","strain":"RH"}]},{"year":2024,"finding":"PPKL localizes to the apical region and nucleus and is partially distributed in the cytoplasm of the parasite.","method":"Fluorescence microscopy with organelle markers in AID-tagged parasites","journal":"Parasites & vectors","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct localization experiment with specific markers, independent lab from preprint, peer-reviewed","pmids":["38500196"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":"RH"},{"species":"Toxoplasma gondii","strain":"ME49"}]},{"year":2023,"finding":"Conditional depletion of PPKL is essential for parasite propagation and results in uncoupling of division: DNA duplication proceeds normally but daughter parasite formation is severely defective, with multiple nuclei accumulating in a single parasite.","method":"Auxin-inducible degron (AID) conditional knockdown with DNA/centrosome marker staining and quantitative phenotyping","journal":"bioRxiv / mBio","confidence":"High","confidence_rationale":"Tier 2 / Strong — AID knockdown replicated independently in two labs (Yang et al. and Wu et al.) with consistent phenotype, peer-reviewed publication plus preprint","pmids":["37398039","37877735","38500196"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":"RH"},{"species":"Toxoplasma gondii","strain":"ME49"}]},{"year":2023,"finding":"PPKL depletion does not impair centrosome duplication but affects the rigidity and arrangement of cortical microtubules, and is associated with elongated tubulin fibers in the parasite.","method":"AID-mediated depletion followed by immunofluorescence with centrosome and tubulin markers","journal":"bioRxiv / Parasites & vectors","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — two independent labs report consistent microtubule defect, single method (immunofluorescence) per study","pmids":["37398039","38500196"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":"RH"},{"species":"Toxoplasma gondii","strain":"ME49"}]},{"year":2023,"finding":"Both Co-Immunoprecipitation and proximity labeling identified the kinase DYRK1 as a potential functional partner of PPKL, and complete knockout of DYRK1 phenocopies loss of PPKL, indicating a functional relationship between these two signaling proteins.","method":"Co-immunoprecipitation, proximity biotin labeling (BioID), and DYRK1 knockout phenotypic analysis","journal":"bioRxiv / mBio","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP plus proximity labeling plus genetic epistasis (DYRK1 KO phenocopy), single lab","pmids":["37398039","37877735"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":"RH"}]},{"year":2023,"finding":"Global phosphoproteomics in PPKL-depleted parasites revealed a significant increase in phosphorylation of the microtubule-associated protein SPM1, suggesting PPKL regulates cortical microtubules by controlling the phosphorylation state of SPM1.","method":"Global phosphoproteomics (mass spectrometry) in AID-depleted parasites","journal":"bioRxiv / mBio","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — quantitative phosphoproteomics with defined substrate (SPM1), single lab, no direct dephosphorylation assay confirming PPKL acts directly on SPM1","pmids":["37398039","37877735"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":"RH"}]},{"year":2023,"finding":"Phosphoproteomics revealed that the phosphorylation of the cell cycle-associated kinase Crk1, a known regulator of daughter cell assembly, is altered in PPKL-depleted parasites, suggesting PPKL regulates daughter parasite development through the Crk1-dependent signaling pathway.","method":"Global phosphoproteomics (mass spectrometry) in AID-depleted parasites","journal":"bioRxiv / mBio","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — phosphoproteomics identifies Crk1 as downstream effector, single lab, pathway placement supported by known Crk1 function but no direct epistasis experiment shown","pmids":["37398039","37877735"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":"RH"}]},{"year":2024,"finding":"Proximity biotin labeling of PPKL identified diverse potential interacting proteins, with kelch domain-mediated protein interactions proposed to underlie PPKL's essential functions in parasite division.","method":"Proximity biotin labeling (BioID/TurboID) followed by mass spectrometry","journal":"Parasites & vectors","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single proximity labeling experiment, interactors not individually validated, domain attribution is inferential","pmids":["38500196"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":"RH"}]},{"year":2024,"finding":"Genetic complementation rescued the defects in parasite replication and morphology caused by PPKL depletion, confirming that the phenotypes are specifically attributable to loss of PPKL function.","method":"Genetic complementation of AID-depleted parasites with exogenous PPKL","journal":"Parasites & vectors","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — complementation rescue provides specificity control, single lab, peer-reviewed","pmids":["38500196"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":"RH"}]}],"current_model":"PPKL is an essential plant-like protein phosphatase in Toxoplasma gondii that localizes dynamically to the preconoidal region, cortical cytoskeleton, and basal complex during parasite division, where it regulates daughter parasite formation by controlling cortical microtubule rigidity (at least partly through dephosphorylation of the microtubule-associated protein SPM1), interacts functionally with the kinase DYRK1, and modulates the phosphorylation state of the cell cycle kinase Crk1 to coordinate daughter cell assembly."},"narrative":{"mechanistic_narrative":"PPKL is an essential protein phosphatase in Toxoplasma gondii that coordinates daughter parasite formation during the parasite's internal budding division [PMID:37398039, PMID:37877735, PMID:38500196]. The enzyme localizes dynamically across the cell cycle, moving from a diffuse cytoplasmic, nuclear, and preconoidal distribution in non-dividing parasites to enrichment at the preconoidal region and cortical cytoskeleton of nascent daughters and later to the basal complex ring [PMID:37398039, PMID:38500196]. Conditional depletion uncouples nuclear replication from cytokinesis: DNA duplication and centrosome duplication proceed normally, but daughter cell assembly fails, producing parasites with multiple accumulated nuclei [PMID:37398039, PMID:37877735, PMID:38500196]. PPKL controls the rigidity and arrangement of cortical microtubules, and its loss elongates tubulin fibers and raises phosphorylation of the microtubule-associated protein SPM1, placing SPM1 as a downstream target through which PPKL tunes microtubule behavior [PMID:37398039, PMID:38500196, PMID:37877735]. PPKL further modulates the phosphorylation state of the cell cycle kinase Crk1, a known regulator of daughter cell assembly, and acts in a functional relationship with the kinase DYRK1, whose knockout phenocopies PPKL loss [PMID:37398039, PMID:37877735]. Direct dephosphorylation of these targets by PPKL has not been demonstrated in the available corpus.","teleology":[{"year":2023,"claim":"Establishing where PPKL acts was the first question; its cell-cycle-staged relocation from cytoplasm/nucleus/preconoidal region to the cortical cytoskeleton and basal complex placed it at the machinery of daughter parasite construction.","evidence":"Live/fixed imaging with an AID conditional knockdown line tracked across division stages","pmids":["37398039"],"confidence":"Medium","gaps":["Preprint, single lab","Mechanism of recruitment to each compartment unknown","No structural or domain-level basis for localization dynamics"],"attribution":[{"species":"Toxoplasma gondii","strain":"RH"}]},{"year":2024,"claim":"An independent lab confirmed apical, nuclear, and partial cytoplasmic localization, corroborating the compartmental distribution in a second study.","evidence":"Fluorescence microscopy with organelle markers in AID-tagged parasites","pmids":["38500196"],"confidence":"Medium","gaps":["Basal complex enrichment not the focus of this study","No temporal resolution of relocation"],"attribution":[{"species":"Toxoplasma gondii","strain":"RH"},{"species":"Toxoplasma gondii","strain":"ME49"}]},{"year":2023,"claim":"Whether PPKL is required for division and at which step was resolved by conditional depletion, showing it is essential and specifically uncouples DNA/centrosome duplication from daughter cell formation.","evidence":"AID conditional knockdown with DNA and centrosome marker staining and quantitative phenotyping, replicated in two labs","pmids":["37398039","37877735","38500196"],"confidence":"High","gaps":["Molecular step at which budding fails not defined","Does not distinguish direct phosphatase activity from scaffolding role"],"attribution":[{"species":"Toxoplasma gondii","strain":"RH"},{"species":"Toxoplasma gondii","strain":"ME49"}]},{"year":2023,"claim":"The cellular basis of the division defect was linked to the cortical cytoskeleton by showing depletion disrupts cortical microtubule rigidity and arrangement while sparing centrosome duplication.","evidence":"AID depletion with centrosome and tubulin immunofluorescence in two independent labs","pmids":["37398039","38500196"],"confidence":"Medium","gaps":["Single method (immunofluorescence) per study","Direct effect on microtubule dynamics not measured"],"attribution":[{"species":"Toxoplasma gondii","strain":"RH"},{"species":"Toxoplasma gondii","strain":"ME49"}]},{"year":2023,"claim":"Candidate substrates and effectors were identified by phosphoproteomics, nominating SPM1 as a microtubule-associated target and Crk1 as a downstream cell cycle effector of PPKL.","evidence":"Global phosphoproteomics (mass spectrometry) in AID-depleted parasites","pmids":["37398039","37877735"],"confidence":"Medium","gaps":["No direct dephosphorylation assay confirming PPKL acts on SPM1 or Crk1","Direct versus indirect phosphorylation changes not distinguished","Single lab"],"attribution":[{"species":"Toxoplasma gondii","strain":"RH"}]},{"year":2023,"claim":"A functional partner kinase was defined by combining interaction mapping with genetic epistasis, showing DYRK1 associates with PPKL and its knockout phenocopies PPKL loss.","evidence":"Co-immunoprecipitation, BioID proximity labeling, and DYRK1 knockout phenotyping","pmids":["37398039","37877735"],"confidence":"Medium","gaps":["Directionality of the PPKL-DYRK1 relationship unresolved","Whether DYRK1 is a PPKL substrate or upstream regulator unknown","Single lab"],"attribution":[{"species":"Toxoplasma gondii","strain":"RH"}]},{"year":2024,"claim":"Phenotype specificity was established by genetic complementation, confirming the replication and morphology defects arise from loss of PPKL itself.","evidence":"Genetic complementation of AID-depleted parasites with exogenous PPKL","pmids":["38500196"],"confidence":"Medium","gaps":["Does not address which domain or activity is required for rescue","Single lab"],"attribution":[{"species":"Toxoplasma gondii","strain":"RH"}]},{"year":2024,"claim":"The broader interactome was surveyed to explain how PPKL executes its function, proposing kelch-domain-mediated protein interactions as central to its essential role.","evidence":"Proximity biotin labeling (BioID/TurboID) with mass spectrometry","pmids":["38500196"],"confidence":"Low","gaps":["Interactors not individually validated","Kelch-domain attribution is inferential","Single proximity labeling experiment"],"attribution":[{"species":"Toxoplasma gondii","strain":"RH"}]},{"year":null,"claim":"It remains unknown whether PPKL directly dephosphorylates SPM1, Crk1, or any identified interactor, and how its localization-dependent activity is molecularly regulated across the cell cycle.","evidence":"No direct enzymatic or reconstitution assay in the available corpus","pmids":[],"confidence":"Low","gaps":["No in vitro phosphatase-substrate assay","Catalytic mechanism and substrate recognition undefined","Regulation of dynamic relocation unknown"],"attribution":[{"species":"Toxoplasma gondii","strain":"RH"}]}],"mechanism_profile":{"molecular_activity":[],"localization":[],"pathway":[],"complexes":[],"partners":["DYRK1","SPM1","CRK1"],"other_free_text":["Essential for parasite propagation; depletion uncouples DNA/centrosome duplication from daughter cell formation, causing multinucleated parasites [#2, #3].","Controls cortical microtubule rigidity and arrangement; SPM1 phosphorylation rises upon depletion [#3, #5].","DYRK1 knockout phenocopies PPKL loss, indicating a functional partnership [#4].","Kelch-domain-mediated interactions proposed to underlie function, based on proximity labeling [#7]."]},"strain_conflicts":[]},"prefetch_data":{},"corpus_meta":[{"pmid":"37877735","id":"PMC_37877735","title":"The protein phosphatase PPKL is a key regulator of daughter parasite development in Toxoplasma gondii.","date":"2023","source":"mBio","url":"https://pubmed.ncbi.nlm.nih.gov/37877735","citation_count":15,"is_preprint":false},{"pmid":"38500196","id":"PMC_38500196","title":"The plant-like protein phosphatase PPKL regulates parasite replication and morphology in Toxoplasma gondii.","date":"2024","source":"Parasites & vectors","url":"https://pubmed.ncbi.nlm.nih.gov/38500196","citation_count":5,"is_preprint":false},{"pmid":"37398039","id":"PMC_37398039","title":"The protein phosphatase PPKL is a key regulator of daughter parasite development in Toxoplasma gondii.","date":"2023","source":"bioRxiv : the preprint server for biology","url":"https://pubmed.ncbi.nlm.nih.gov/37398039","citation_count":2,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":4059,"output_tokens":2230,"usd":0.022814,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":7686,"output_tokens":3418,"usd":0.06194,"stage2_stop_reason":"end_turn"},"total_usd":0.084754,"stage1_batch_id":"msgbatch_015xmQKDRqnTh9anE4m7LiD6","stage2_batch_id":"msgbatch_01JLpkFTVhoQBNgqHCKFrVVU","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2023,\n      \"finding\": \"PPKL localizes dynamically during parasite division: in non-dividing parasites it is present in the cytoplasm, nucleus, and preconoidal region; as division begins, it enriches in the preconoidal region and cortical cytoskeleton of nascent daughter parasites; later it localizes to the basal complex ring.\",\n      \"method\": \"Live/fixed imaging with conditional knockdown system (AID), localization tracked across cell cycle stages\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct localization experiment with cell-cycle-stage resolution, single lab, preprint\",\n      \"pmids\": [\"37398039\"],\n      \"is_preprint\": true,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH\"}]\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"PPKL localizes to the apical region and nucleus and is partially distributed in the cytoplasm of the parasite.\",\n      \"method\": \"Fluorescence microscopy with organelle markers in AID-tagged parasites\",\n      \"journal\": \"Parasites & vectors\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct localization experiment with specific markers, independent lab from preprint, peer-reviewed\",\n      \"pmids\": [\"38500196\"],\n      \"is_preprint\": false,\n      \"attribution\": [\n        {\"species\": \"Toxoplasma gondii\", \"strain\": \"RH\"},\n        {\"species\": \"Toxoplasma gondii\", \"strain\": \"ME49\"}\n      ]\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Conditional depletion of PPKL is essential for parasite propagation and results in uncoupling of division: DNA duplication proceeds normally but daughter parasite formation is severely defective, with multiple nuclei accumulating in a single parasite.\",\n      \"method\": \"Auxin-inducible degron (AID) conditional knockdown with DNA/centrosome marker staining and quantitative phenotyping\",\n      \"journal\": \"bioRxiv / mBio\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — AID knockdown replicated independently in two labs (Yang et al. and Wu et al.) with consistent phenotype, peer-reviewed publication plus preprint\",\n      \"pmids\": [\"37398039\", \"37877735\", \"38500196\"],\n      \"is_preprint\": false,\n      \"attribution\": [\n        {\"species\": \"Toxoplasma gondii\", \"strain\": \"RH\"},\n        {\"species\": \"Toxoplasma gondii\", \"strain\": \"ME49\"}\n      ]\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"PPKL depletion does not impair centrosome duplication but affects the rigidity and arrangement of cortical microtubules, and is associated with elongated tubulin fibers in the parasite.\",\n      \"method\": \"AID-mediated depletion followed by immunofluorescence with centrosome and tubulin markers\",\n      \"journal\": \"bioRxiv / Parasites & vectors\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — two independent labs report consistent microtubule defect, single method (immunofluorescence) per study\",\n      \"pmids\": [\"37398039\", \"38500196\"],\n      \"is_preprint\": false,\n      \"attribution\": [\n        {\"species\": \"Toxoplasma gondii\", \"strain\": \"RH\"},\n        {\"species\": \"Toxoplasma gondii\", \"strain\": \"ME49\"}\n      ]\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Both Co-Immunoprecipitation and proximity labeling identified the kinase DYRK1 as a potential functional partner of PPKL, and complete knockout of DYRK1 phenocopies loss of PPKL, indicating a functional relationship between these two signaling proteins.\",\n      \"method\": \"Co-immunoprecipitation, proximity biotin labeling (BioID), and DYRK1 knockout phenotypic analysis\",\n      \"journal\": \"bioRxiv / mBio\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP plus proximity labeling plus genetic epistasis (DYRK1 KO phenocopy), single lab\",\n      \"pmids\": [\"37398039\", \"37877735\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH\"}]\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Global phosphoproteomics in PPKL-depleted parasites revealed a significant increase in phosphorylation of the microtubule-associated protein SPM1, suggesting PPKL regulates cortical microtubules by controlling the phosphorylation state of SPM1.\",\n      \"method\": \"Global phosphoproteomics (mass spectrometry) in AID-depleted parasites\",\n      \"journal\": \"bioRxiv / mBio\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — quantitative phosphoproteomics with defined substrate (SPM1), single lab, no direct dephosphorylation assay confirming PPKL acts directly on SPM1\",\n      \"pmids\": [\"37398039\", \"37877735\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH\"}]\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Phosphoproteomics revealed that the phosphorylation of the cell cycle-associated kinase Crk1, a known regulator of daughter cell assembly, is altered in PPKL-depleted parasites, suggesting PPKL regulates daughter parasite development through the Crk1-dependent signaling pathway.\",\n      \"method\": \"Global phosphoproteomics (mass spectrometry) in AID-depleted parasites\",\n      \"journal\": \"bioRxiv / mBio\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — phosphoproteomics identifies Crk1 as downstream effector, single lab, pathway placement supported by known Crk1 function but no direct epistasis experiment shown\",\n      \"pmids\": [\"37398039\", \"37877735\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH\"}]\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Proximity biotin labeling of PPKL identified diverse potential interacting proteins, with kelch domain-mediated protein interactions proposed to underlie PPKL's essential functions in parasite division.\",\n      \"method\": \"Proximity biotin labeling (BioID/TurboID) followed by mass spectrometry\",\n      \"journal\": \"Parasites & vectors\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single proximity labeling experiment, interactors not individually validated, domain attribution is inferential\",\n      \"pmids\": [\"38500196\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH\"}]\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Genetic complementation rescued the defects in parasite replication and morphology caused by PPKL depletion, confirming that the phenotypes are specifically attributable to loss of PPKL function.\",\n      \"method\": \"Genetic complementation of AID-depleted parasites with exogenous PPKL\",\n      \"journal\": \"Parasites & vectors\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — complementation rescue provides specificity control, single lab, peer-reviewed\",\n      \"pmids\": [\"38500196\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH\"}]\n    }\n  ],\n  \"current_model\": \"PPKL is an essential plant-like protein phosphatase in Toxoplasma gondii that localizes dynamically to the preconoidal region, cortical cytoskeleton, and basal complex during parasite division, where it regulates daughter parasite formation by controlling cortical microtubule rigidity (at least partly through dephosphorylation of the microtubule-associated protein SPM1), interacts functionally with the kinase DYRK1, and modulates the phosphorylation state of the cell cycle kinase Crk1 to coordinate daughter cell assembly.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"PPKL is an essential protein phosphatase in Toxoplasma gondii that coordinates daughter parasite formation during the parasite's internal budding division [#2, #8]. The enzyme localizes dynamically across the cell cycle, moving from a diffuse cytoplasmic, nuclear, and preconoidal distribution in non-dividing parasites to enrichment at the preconoidal region and cortical cytoskeleton of nascent daughters and later to the basal complex ring [#0, #1]. Conditional depletion uncouples nuclear replication from cytokinesis: DNA duplication and centrosome duplication proceed normally, but daughter cell assembly fails, producing parasites with multiple accumulated nuclei [#2, #3]. PPKL controls the rigidity and arrangement of cortical microtubules, and its loss elongates tubulin fibers and raises phosphorylation of the microtubule-associated protein SPM1, placing SPM1 as a downstream target through which PPKL tunes microtubule behavior [#3, #5]. PPKL further modulates the phosphorylation state of the cell cycle kinase Crk1, a known regulator of daughter cell assembly, and acts in a functional relationship with the kinase DYRK1, whose knockout phenocopies PPKL loss [#4, #6]. Direct dephosphorylation of these targets by PPKL has not been demonstrated in the available corpus.\",\n  \"teleology\": [\n    {\n      \"year\": 2023,\n      \"claim\": \"Establishing where PPKL acts was the first question; its cell-cycle-staged relocation from cytoplasm/nucleus/preconoidal region to the cortical cytoskeleton and basal complex placed it at the machinery of daughter parasite construction.\",\n      \"evidence\": \"Live/fixed imaging with an AID conditional knockdown line tracked across division stages\",\n      \"pmids\": [\"37398039\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Preprint, single lab\", \"Mechanism of recruitment to each compartment unknown\", \"No structural or domain-level basis for localization dynamics\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH\"}]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"An independent lab confirmed apical, nuclear, and partial cytoplasmic localization, corroborating the compartmental distribution in a second study.\",\n      \"evidence\": \"Fluorescence microscopy with organelle markers in AID-tagged parasites\",\n      \"pmids\": [\"38500196\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Basal complex enrichment not the focus of this study\", \"No temporal resolution of relocation\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH\"}, {\"species\": \"Toxoplasma gondii\", \"strain\": \"ME49\"}]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Whether PPKL is required for division and at which step was resolved by conditional depletion, showing it is essential and specifically uncouples DNA/centrosome duplication from daughter cell formation.\",\n      \"evidence\": \"AID conditional knockdown with DNA and centrosome marker staining and quantitative phenotyping, replicated in two labs\",\n      \"pmids\": [\"37398039\", \"37877735\", \"38500196\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular step at which budding fails not defined\", \"Does not distinguish direct phosphatase activity from scaffolding role\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH\"}, {\"species\": \"Toxoplasma gondii\", \"strain\": \"ME49\"}]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"The cellular basis of the division defect was linked to the cortical cytoskeleton by showing depletion disrupts cortical microtubule rigidity and arrangement while sparing centrosome duplication.\",\n      \"evidence\": \"AID depletion with centrosome and tubulin immunofluorescence in two independent labs\",\n      \"pmids\": [\"37398039\", \"38500196\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single method (immunofluorescence) per study\", \"Direct effect on microtubule dynamics not measured\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH\"}, {\"species\": \"Toxoplasma gondii\", \"strain\": \"ME49\"}]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Candidate substrates and effectors were identified by phosphoproteomics, nominating SPM1 as a microtubule-associated target and Crk1 as a downstream cell cycle effector of PPKL.\",\n      \"evidence\": \"Global phosphoproteomics (mass spectrometry) in AID-depleted parasites\",\n      \"pmids\": [\"37398039\", \"37877735\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No direct dephosphorylation assay confirming PPKL acts on SPM1 or Crk1\", \"Direct versus indirect phosphorylation changes not distinguished\", \"Single lab\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH\"}]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"A functional partner kinase was defined by combining interaction mapping with genetic epistasis, showing DYRK1 associates with PPKL and its knockout phenocopies PPKL loss.\",\n      \"evidence\": \"Co-immunoprecipitation, BioID proximity labeling, and DYRK1 knockout phenotyping\",\n      \"pmids\": [\"37398039\", \"37877735\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Directionality of the PPKL-DYRK1 relationship unresolved\", \"Whether DYRK1 is a PPKL substrate or upstream regulator unknown\", \"Single lab\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH\"}]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Phenotype specificity was established by genetic complementation, confirming the replication and morphology defects arise from loss of PPKL itself.\",\n      \"evidence\": \"Genetic complementation of AID-depleted parasites with exogenous PPKL\",\n      \"pmids\": [\"38500196\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Does not address which domain or activity is required for rescue\", \"Single lab\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH\"}]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"The broader interactome was surveyed to explain how PPKL executes its function, proposing kelch-domain-mediated protein interactions as central to its essential role.\",\n      \"evidence\": \"Proximity biotin labeling (BioID/TurboID) with mass spectrometry\",\n      \"pmids\": [\"38500196\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Interactors not individually validated\", \"Kelch-domain attribution is inferential\", \"Single proximity labeling experiment\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH\"}]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"It remains unknown whether PPKL directly dephosphorylates SPM1, Crk1, or any identified interactor, and how its localization-dependent activity is molecularly regulated across the cell cycle.\",\n      \"evidence\": \"No direct enzymatic or reconstitution assay in the available corpus\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No in vitro phosphatase-substrate assay\", \"Catalytic mechanism and substrate recognition undefined\", \"Regulation of dynamic relocation unknown\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH\"}]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0004721\", \"supporting_discovery_ids\": [5, 6], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH\"}]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005737\", \"supporting_discovery_ids\": [0, 1], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH\"}]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [0, 1], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH\"}]},\n      {\"term_id\": \"GO:0072687\", \"supporting_discovery_ids\": [0, 3], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH\"}]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"GO:0000281\", \"supporting_discovery_ids\": [2, 6], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH\"}]},\n      {\"term_id\": \"GO:0000226\", \"supporting_discovery_ids\": [3, 5], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH\"}]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"DYRK1\", \"SPM1\", \"Crk1\"],\n    \"other_free_text\": [\n      \"Essential for parasite propagation; depletion uncouples DNA/centrosome duplication from daughter cell formation, causing multinucleated parasites [#2, #3].\",\n      \"Controls cortical microtubule rigidity and arrangement; SPM1 phosphorylation rises upon depletion [#3, #5].\",\n      \"DYRK1 knockout phenocopies PPKL loss, indicating a functional partnership [#4].\",\n      \"Kelch-domain-mediated interactions proposed to underlie function, based on proximity labeling [#7].\"\n    ]\n  },\n  \"strain_conflicts\": []\n}","audit_flag":null,"strains":[{"strain":"ME49","gene_id":"TGME49_290170","name":"PPKL","product":"protein phosphatase PPKL","uniprot_acc":"S8GHS2","chromosome":"TGME49_chrIX","start_pos":3615756},{"strain":"GT1","gene_id":"TGGT1_290170","name":null,"product":"kelch repeat domain containing/Serine/threonine protein phosphatase protein","uniprot_acc":null,"chromosome":"TGGT1_chrIX","start_pos":3424119},{"strain":"RH88","gene_id":"TGRH88_014840","name":null,"product":"kelch repeat domain containing/Serine/threonine protein phosphatase protein","uniprot_acc":null,"chromosome":"CM023090","start_pos":3679956},{"strain":"VEG","gene_id":"TGVEG_290170","name":null,"product":"kelch repeat domain containing/Serine/threonine protein phosphatase protein","uniprot_acc":"B9QE64","chromosome":"TGVEG_chrIX","start_pos":3651091}]}