{"gene":"TG_305860","gene_symbol":"CDPK3","run_date":"2026-09-08T18:09:53+00:00","timeline":{"discoveries":[{"year":2012,"finding":"TgCDPK3 is required for ionophore-induced egress but not for invasion; microneme secretion depends on TgCDPK3 only when triggered by certain stimuli (not universally), demonstrating that egress and invasion are controlled by distinct kinase-mediated signalling pathways.","method":"Chemical-genetic inhibition of TgCDPK1 and TgCDPK3 using analog-sensitive kinase alleles; ionophore-induced egress and invasion assays","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 2 / Strong — chemical-genetic approach with specific inhibition, replicated across multiple assay types; independently corroborated by two additional papers published the same year","pmids":["23149386"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2012,"finding":"TgCDPK3 localizes to the inner side of the plasma membrane via dual acylation (N-myristoylation and palmitoylation), and this membrane targeting is required for its activity in promoting Ca2+-dependent egress.","method":"Genetic knockout; complementation with acylation-site mutants; localization by fluorescence microscopy; ionophore- and DTT-induced egress assays","journal":"PLoS pathogens","confidence":"High","confidence_rationale":"Tier 2 / Moderate — clean knockout, complementation with acylation mutants, and localization imaging in one study; independently corroborated by concurrent papers","pmids":["23226109"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2012,"finding":"TgCDPK3 regulates microneme secretion specifically when parasites are intracellular, and the high K+ concentration of the host cell environment appears to determine the requirement for TgCDPK3 in this context.","method":"Genetic knockout; microneme secretion assays in intracellular vs. extracellular parasites; ionic environment manipulation","journal":"PLoS pathogens","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — single lab, clean knockout with defined assay readout, but ionic-environment mechanism is inferred rather than directly demonstrated","pmids":["23226109"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2012,"finding":"Forward genetic screen identified four independent loss-of-function missense mutations in TgCDPK3 that confer defects in ionophore-induced egress; biochemical analysis of recombinant mutant proteins confirmed these mutations alter key functional regions. Genetic knockout confirmed TgCDPK3 is required for rapid egress induction and for formation of latent cysts in mouse brain.","method":"Forward genetic screen; whole-genome sequencing; biochemical analysis of recombinant proteins with missense mutations; complementation; genetic knockout; mouse infection model","journal":"PLoS pathogens","confidence":"High","confidence_rationale":"Tier 2 / Strong — forward genetics converging on same gene (4 independent alleles), complementation, and biochemical validation; independently replicated by two concurrent studies","pmids":["23209419"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2009,"finding":"TgCDPKif3 (TgCDPK3) is an active kinase that auto-phosphorylates and phosphorylates substrates (Histone IIas, TgALD1) in a Ca2+-dependent manner; the catalytic lysine is essential for activity. It localizes to the apical ends of tachyzoites and relocalizes between intra- and extracellular stages. It interacts with and phosphorylates T. gondii aldolase 1 (TgALD1) in vitro, and this interaction was confirmed by co-immunoprecipitation in mammalian cells.","method":"Recombinant protein purification; [γ-32P]ATP labeling; phosphatase treatment; catalytic lysine-to-alanine mutagenesis; in vitro kinase assay with Ca2+ titration; immunofluorescence localization; co-immunoprecipitation","journal":"Parasitology international","confidence":"Medium","confidence_rationale":"Tier 1–2 / Weak — in vitro kinase assay with mutagenesis and co-IP, but single lab and interaction confirmed in mammalian overexpression system, not endogenously in Toxoplasma","pmids":["19699312"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2015,"finding":"TgCDPK3 phosphorylates Myosin A (TgMyoA) at serines 21 and 743 in vitro, and this phosphorylation is required for rapid initiation of motility and egress. Phosphomimetic mutations at S21+S743 in TgMyoA rescue the egress and motility defects of Tgcdpk3 mutant parasites, and TgCDPK3 was identified as a near-neighbor/direct interactor of TgMyoA by BioID proximity labeling.","method":"BioID proximity-based protein interaction trap; peptide array phosphorylation profiling; in vitro kinase assay; complementation of TgmyoA null with phosphosite alanine and phosphomimetic mutants; egress assays; motility assays","journal":"PLoS pathogens","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro phosphorylation, BioID interaction, and genetic rescue with phosphomimetic mutants in one study; multiple orthogonal methods establishing substrate-kinase relationship","pmids":["26544049"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2014,"finding":"PfCDPK1 (the Plasmodium falciparum ortholog of TgCDPK3) can functionally complement the egress defect of a TgCDPK3 mutant strain, but only when PfCDPK1 is localized to the plasma membrane and retains kinase activity, demonstrating that membrane localization and catalytic activity are essential for the egress function of this kinase family.","method":"Heterologous complementation of TgCDPK3 mutant with PfCDPK1; localization mutants; kinase-dead mutants; ionophore-induced egress assays; pharmacological inhibition","journal":"Antimicrobial agents and chemotherapy","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic complementation with domain/activity mutants across species, two orthogonal approaches (localization mutant + kinase-dead mutant), single lab","pmids":["24550330"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2017,"finding":"A forward genetic suppressor screen in a cdpk3 knockout strain identified SCE1 (Suppressor of Ca2+-dependent Egress 1) as a negative regulator of egress that is phosphorylated in a CDPK3-dependent manner; CDPK3-dependent phosphorylation of SCE1 is required to relieve its suppressive activity and allow egress. Loss of SCE1 restores many Δcdpk3-dependent phosphorylation events to near wild-type levels.","method":"Forward genetic suppressor screen in cdpk3 KO background; whole-genome sequencing; global phosphoproteomics; genetic knockout of SCE1","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — epistasis established by suppressor screen, phosphoproteomic analysis, and genetic knockout, multiple orthogonal methods in one study","pmids":["28258212"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2017,"finding":"TgCDPK1 and TgCDPK3 play a role in signal quenching of Ca2+ flux before egress, as revealed by live imaging with a genetically encoded Ca2+ biosensor (GCaMP6), placing both kinases within the temporal dynamics of the Ca2+ signalling cascade.","method":"Live-cell Ca2+ imaging with GCaMP6 biosensor; pharmacological and genetic perturbation of CDPK1 and CDPK3","journal":"Cellular microbiology","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — live biosensor imaging is a direct functional readout, but pathway placement based on a single study","pmids":["27781359"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2018,"finding":"TgCDPK3 phosphorylates the tyrosine transporter ApiAT5-3 at serine 56 following induction of Ca2+ signalling, as identified by phosphoproteomics comparing WT and Δcdpk3 parasites. Mutating S56 to non-phosphorylatable alanine incurs a fitness cost, suggesting S56 phosphorylation is beneficial but not essential for tyrosine import function.","method":"Phosphoproteomic comparison of WT vs. Δcdpk3 parasites; conditional knockout of apiAT5-3; phosphosite mutagenesis (S56A); metabolomics; heterologous expression for transporter characterization","journal":"Molecular microbiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — phosphoproteomics identifies substrate, phosphosite mutagenesis establishes functional consequence, multiple methods in one study; but direct kinase-substrate relationship relies on phosphoproteomic inference","pmids":["30402958"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2019,"finding":"TgCDPK3 function is required for Ca2+-mediated negative regulation of dense granule exocytosis; in the absence of TgCDPK3, this Ca2+-mediated suppression of dense granule secretion is lost, demonstrating that TgCDPK3 participates in the reciprocal Ca2+-dependent control of microneme vs. dense granule secretion.","method":"Genetic knockout of TgCDPK3; dense granule secretion assays under Ca2+-elevated conditions; comparison with TgCDPK1 knockout","journal":"Cellular microbiology","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — clean KO with defined secretion assay readout, but single lab and single method for CDPK3-specific contribution","pmids":["30673152"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2022,"finding":"Using WT and conditional Δcdpk3 parasites in sub-minute phosphoproteomic timecourses after Ca2+ release, CDPK3 was shown to be partly responsible for a positive feedback loop linking calcium and cyclic nucleotide (cAMP) signalling during egress; CDPK3 activity influences cAMP and lipid metabolism pathways that accelerate the egress response.","method":"Conditional knockout; sub-minute phosphoproteomics timecourse after Ca2+ ionophore; lipidomics; pharmacological inhibition of PDEs","journal":"PLoS pathogens","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — phosphoproteomics with temporal resolution and conditional KO, multiple readouts; pathway placement is partial as the feedback loop is inferred from phosphoproteomic correlations","pmids":["36265000"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2023,"finding":"Under extracellular stress conditions, TgCDPK3 functions as an eEF2 (elongation factor 2) kinase: it phosphorylates eEF2, thereby inhibiting translational elongation. Loss of CDPK3 in stressed extracellular parasites leads to decreased eEF2 phosphorylation and enhanced elongation rates. CDPK3 and eEF2 interact by co-immunoprecipitation specifically under stress conditions, coinciding with CDPK3 relocalization from the plasma membrane to the cytosol due to loss of N-myristoylation under stress.","method":"BLAST analysis identifying CDPK3 as homolog of yeast eEF2 kinase RCK2; co-immunoprecipitation of CDPK3 and eEF2 in stressed parasites; eEF2 phosphorylation assay in cdpk3 KO parasites; translational elongation rate assay; localization by imaging under stress vs. normal conditions; N-myristoylation analysis","journal":"mSphere","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP of endogenous interaction, KO with defined phosphorylation and elongation phenotype, localization shift; single lab but multiple orthogonal methods","pmids":["37272703"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2022,"finding":"TgCDPK3 (from type II ME49 strain) interacts with host autophagy proteins Atg3 and Atg5, and this interaction promotes localization of IRGs (immunity-related GTPases) and GBPs (guanylate-binding proteins) around the parasitophorous vacuole membrane, thereby inhibiting intracellular parasite proliferation and facilitating long-term latency of the less virulent strain.","method":"Co-immunoprecipitation of TgCDPK3 with host Atg3 and Atg5; CDPK3 knockout in ME49 strain; IRG/GBP localization assay by fluorescence microscopy; intracellular proliferation and plaque assays","journal":"Frontiers in immunology","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — Co-IP and KO with defined localization and proliferation readouts, but single lab and functional mechanism (how CDPK3-Atg interaction promotes IRG/GBP targeting) not fully resolved","pmids":["35757711"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":"ME49"}]},{"year":2025,"finding":"TgCDPK3 interacts with host Arginase-1 (Arg1) as identified by mass spectrometry and confirmed by GST pull-down, co-immunoprecipitation, and co-localization assays. This interaction reduces Arginase-1 enzymatic activity, promoting M1-biased macrophage polarization (increased iNOS, decreased Arg1), which restricts intracellular parasite proliferation.","method":"Mass spectrometry of TgCDPK3-binding proteins; GST pull-down; co-immunoprecipitation; immunofluorescence co-localization; Arginase activity assay; Western blotting for Arg1 and iNOS; FACS for M1/M2 macrophage markers; ELISA for cytokines; intracellular proliferation and plaque assays","journal":"Parasites & vectors","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal methods (MS, pull-down, co-IP, activity assay, cellular phenotype), single lab","pmids":["40394721"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":"ME49"}]}],"current_model":"TgCDPK3 is a Ca2+-dependent protein kinase anchored to the inner face of the plasma membrane via dual acylation (N-myristoylation/palmitoylation) that acts as a central transducer of calcium signals during host-cell egress: it directly phosphorylates Myosin A (TgMyoA) at S21 and S743 to initiate gliding motility, phosphorylates and inactivates the negative egress regulator SCE1, participates in a positive cAMP-Ca2+ feedback loop that accelerates the egress response, and controls microneme but not dense granule secretion in the high-K+ intracellular environment; under extracellular stress CDPK3 loses N-myristoylation, relocalizes to the cytosol, and functions as the first identified protozoan eEF2 kinase to regulate translational elongation, while in macrophages it interacts with host Arginase-1 to promote M1 polarization and restrict parasite proliferation."},"narrative":{"mechanistic_narrative":"TgCDPK3 is a Ca2+-dependent protein kinase that serves as a central transducer of calcium signals governing Toxoplasma gondii egress from host cells [PMID:23149386, PMID:23209419]. It is an active autophosphorylating kinase whose catalytic lysine is essential, and it phosphorylates substrates in a Ca2+-dependent manner [PMID:19699312]. The enzyme is anchored to the inner face of the plasma membrane through dual acylation (N-myristoylation and palmitoylation), and both this membrane targeting and its catalytic activity are required for its role in Ca2+-dependent egress [PMID:23226109, PMID:24550330]. During egress, TgCDPK3 directly phosphorylates Myosin A (TgMyoA) at serines 21 and 743 to initiate rapid gliding motility, with phosphomimetic substitutions rescuing the motility and egress defects of cdpk3 mutants [PMID:26544049], and it drives egress in part by phosphorylating and thereby relieving the suppressive activity of the negative regulator SCE1 [PMID:28258212]. It further shapes the egress response by controlling microneme secretion in an intracellular, high-K+-dependent manner, negatively regulating dense granule exocytosis, quenching the Ca2+ flux, and participating in a positive cAMP–Ca2+ feedback loop that accelerates the response [PMID:23226109, PMID:30673152, PMID:27781359, PMID:36265000]. Beyond egress, under extracellular stress TgCDPK3 loses N-myristoylation, relocalizes to the cytosol, and acts as an eEF2 kinase that phosphorylates elongation factor 2 to inhibit translational elongation [PMID:37272703]. In the type II ME49 strain, TgCDPK3 also engages host factors — interacting with host Arginase-1 to bias macrophages toward M1 polarization and with autophagy proteins Atg3/Atg5 to promote IRG/GBP recruitment to the parasitophorous vacuole — both of which restrict intracellular proliferation [PMID:40394721, PMID:35757711].","teleology":[{"year":2009,"claim":"Establishing that TgCDPK3 is a genuine Ca2+-dependent kinase was the prerequisite for any signalling role, showing it autophosphorylates and phosphorylates substrates only when Ca2+ is present and only with an intact catalytic lysine.","evidence":"Recombinant kinase assays with [γ-32P]ATP, Ca2+ titration, catalytic lysine mutagenesis, and co-IP of the aldolase TgALD1","pmids":["19699312"],"confidence":"Medium","gaps":["TgALD1 interaction confirmed only in a mammalian overexpression system, not endogenously in Toxoplasma","physiological substrates in the parasite not identified at this stage"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2012,"claim":"Multiple 2012 studies converged to define TgCDPK3's cellular role, showing it is specifically required for ionophore-induced egress but not invasion, that four independent loss-of-function alleles reproduce the egress defect, and that its egress function depends on plasma-membrane targeting via dual acylation.","evidence":"Chemical-genetic analog-sensitive kinase inhibition, forward genetic screening with whole-genome sequencing, knockout/complementation with acylation-site mutants, and egress/invasion assays","pmids":["23149386","23226109","23209419"],"confidence":"High","gaps":["direct kinase substrates driving egress not yet identified","mechanism linking membrane localization to activity unresolved"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2012,"claim":"The requirement for TgCDPK3 in microneme secretion was shown to be context-dependent, tied to the high-K+ intracellular environment rather than a universal secretory step.","evidence":"Knockout with microneme secretion assays comparing intracellular vs. extracellular parasites and ionic-environment manipulation","pmids":["23226109"],"confidence":"Medium","gaps":["ionic-environment mechanism inferred rather than directly demonstrated","molecular sensor of K+ not identified"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2014,"claim":"Heterologous complementation demonstrated that the egress function is a conserved property of this kinase family, requiring both membrane localization and catalytic activity.","evidence":"Complementation of a TgCDPK3 mutant with Plasmodium falciparum PfCDPK1 using localization and kinase-dead mutants plus egress assays","pmids":["24550330"],"confidence":"Medium","gaps":["substrates of the complementing kinase not defined","single-lab result"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2015,"claim":"Identification of TgMyoA as a direct substrate provided the mechanistic link between CDPK3 activity and the motility that drives egress, resolving how the kinase triggers movement.","evidence":"BioID proximity labeling, peptide-array and in vitro kinase assays mapping S21/S743, and genetic rescue with phosphomimetic TgMyoA mutants","pmids":["26544049"],"confidence":"High","gaps":["whether MyoA phosphorylation is the sole egress-relevant substrate not established","in vivo phosphorylation kinetics not resolved"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2016,"claim":"Live Ca2+ imaging placed CDPK3 within the temporal dynamics of the egress signalling cascade, implicating it in quenching the Ca2+ flux prior to egress.","evidence":"GCaMP6 Ca2+ biosensor live imaging with genetic and pharmacological perturbation of CDPK1 and CDPK3","pmids":["27781359"],"confidence":"Medium","gaps":["molecular basis of signal quenching not defined","CDPK1 vs CDPK3 contributions not fully separated"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2017,"claim":"A suppressor screen revealed that CDPK3 promotes egress in part by inactivating a brake, identifying SCE1 as a negative egress regulator whose CDPK3-dependent phosphorylation relieves suppression.","evidence":"Forward genetic suppressor screen in a cdpk3 KO background, whole-genome sequencing, global phosphoproteomics, and SCE1 knockout","pmids":["28258212"],"confidence":"High","gaps":["direct CDPK3-SCE1 phosphorylation not shown in vitro","downstream effectors of SCE1 unidentified"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2018,"claim":"Phosphoproteomics extended the CDPK3 substrate repertoire beyond the motor to metabolic control, identifying the tyrosine transporter ApiAT5-3 S56 as a Ca2+-induced CDPK3-dependent phosphosite.","evidence":"WT vs Δcdpk3 phosphoproteomics, apiAT5-3 conditional knockout, S56A phosphosite mutagenesis, metabolomics, and heterologous transporter characterization","pmids":["30402958"],"confidence":"Medium","gaps":["direct kinase-substrate relationship rests on phosphoproteomic inference","S56 phosphorylation is beneficial but not essential, leaving its precise role open"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2019,"claim":"CDPK3 was shown to mediate the reciprocal Ca2+-dependent control of secretory organelles, being required for Ca2+-mediated suppression of dense granule exocytosis.","evidence":"CDPK3 knockout with dense granule secretion assays under Ca2+-elevated conditions, compared to CDPK1 knockout","pmids":["30673152"],"confidence":"Medium","gaps":["substrates mediating dense granule suppression not identified","single method for CDPK3-specific contribution"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2022,"claim":"Sub-minute phosphoproteomics revealed CDPK3 as a partial driver of a positive cAMP-Ca2+ feedback loop that accelerates the egress response, linking calcium and cyclic-nucleotide signalling.","evidence":"Conditional knockout, sub-minute phosphoproteomics timecourse after Ca2+ ionophore, lipidomics, and PDE inhibition","pmids":["36265000"],"confidence":"Medium","gaps":["feedback loop inferred from phosphoproteomic correlations","direct enzymatic targets in the cAMP pathway not identified"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2022,"claim":"CDPK3 was shown to have a host-directed function during latency, with the ME49 kinase engaging host autophagy proteins to recruit anti-parasitic effectors to the vacuole.","evidence":"Co-IP of TgCDPK3 with host Atg3/Atg5, ME49 CDPK3 knockout, IRG/GBP localization imaging, and proliferation/plaque assays","pmids":["35757711"],"confidence":"Medium","gaps":["mechanism by which the CDPK3-Atg interaction promotes IRG/GBP targeting not resolved","single lab"],"attribution":[{"species":"Toxoplasma gondii","strain":"ME49"}]},{"year":2023,"claim":"A distinct stress-conditional role was uncovered: loss of N-myristoylation relocalizes CDPK3 to the cytosol where it acts as the first protozoan eEF2 kinase to inhibit translational elongation.","evidence":"Homology analysis to yeast RCK2, stress-conditional co-IP of CDPK3 and eEF2, eEF2 phosphorylation and elongation-rate assays in cdpk3 KO, and localization/myristoylation analysis","pmids":["37272703"],"confidence":"Medium","gaps":["signal driving loss of N-myristoylation under stress not defined","single lab"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2025,"claim":"The host-directed repertoire was extended to immune reprogramming, with CDPK3 interacting with host Arginase-1 to reduce its activity and bias macrophages toward M1 polarization.","evidence":"Mass spectrometry, GST pull-down, co-IP, co-localization, arginase activity assay, FACS/ELISA for macrophage markers, and proliferation/plaque assays","pmids":["40394721"],"confidence":"Medium","gaps":["structural basis of the CDPK3-Arg1 interaction not defined","whether CDPK3 phosphorylates Arg1 not established"],"attribution":[{"species":"Toxoplasma gondii","strain":"ME49"}]},{"year":null,"claim":"How CDPK3 is spatially and temporally switched between its membrane-anchored egress function and its cytosolic eEF2-kinase and host-directed functions, and the full physiological substrate set, remain open.","evidence":"","pmids":[],"confidence":"Low","gaps":["the molecular signal controlling stress-induced demyristoylation and relocalization is unknown","no comprehensive validated in vivo substrate catalog","relationship between egress substrates and host-directed functions unresolved"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]}],"mechanism_profile":{"molecular_activity":[],"localization":[],"pathway":[],"complexes":[],"partners":["TGMYOA (MYOSIN A) — DIRECT SUBSTRATE, PHOSPHORYLATED AT S21/S743 [#5]","SCE1 — CDPK3-DEPENDENT PHOSPHORYLATION RELIEVES EGRESS SUPPRESSION [#7]","TGALD1 (ALDOLASE 1) — INTERACTOR/SUBSTRATE [#4]","APIAT5-3 TYROSINE TRANSPORTER — PHOSPHORYLATED AT S56 [#9]","EEF2 (ELONGATION FACTOR 2) — SUBSTRATE UNDER STRESS [#12]","HOST ARGINASE-1 — INTERACTOR, ACTIVITY REDUCED [#14]","HOST ATG3 AND ATG5 — INTERACTORS [#13]"],"other_free_text":["Membrane targeting via dual acylation (N-myristoylation + palmitoylation) required for egress function; stress-induced loss of N-myristoylation drives cytosolic relocalization [#1, #12]","Requirement for microneme secretion is intracellular- and high-K+-dependent [#2]","Negatively regulates dense granule exocytosis in a Ca2+-dependent manner [#10]","Participates in a positive cAMP-Ca2+ feedback loop accelerating egress [#11]","Required for formation of latent cysts in mouse brain [#3]"]},"strain_conflicts":[]},"prefetch_data":{},"corpus_meta":[{"pmid":"23149386","id":"PMC_23149386","title":"Distinct signalling pathways control Toxoplasma egress and host-cell invasion.","date":"2012","source":"The EMBO journal","url":"https://pubmed.ncbi.nlm.nih.gov/23149386","citation_count":167,"is_preprint":false},{"pmid":"23226109","id":"PMC_23226109","title":"TgCDPK3 regulates calcium-dependent egress of Toxoplasma gondii from host cells.","date":"2012","source":"PLoS pathogens","url":"https://pubmed.ncbi.nlm.nih.gov/23226109","citation_count":120,"is_preprint":false},{"pmid":"23209419","id":"PMC_23209419","title":"A forward genetic screen reveals that calcium-dependent protein kinase 3 regulates egress in Toxoplasma.","date":"2012","source":"PLoS pathogens","url":"https://pubmed.ncbi.nlm.nih.gov/23209419","citation_count":107,"is_preprint":false},{"pmid":"26544049","id":"PMC_26544049","title":"Phosphorylation of a Myosin Motor by TgCDPK3 Facilitates Rapid Initiation of Motility during Toxoplasma gondii egress.","date":"2015","source":"PLoS pathogens","url":"https://pubmed.ncbi.nlm.nih.gov/26544049","citation_count":70,"is_preprint":false},{"pmid":"23530747","id":"PMC_23530747","title":"Exploiting the unique ATP-binding pocket of toxoplasma calcium-dependent protein kinase 1 to identify its substrates.","date":"2013","source":"ACS chemical biology","url":"https://pubmed.ncbi.nlm.nih.gov/23530747","citation_count":47,"is_preprint":false},{"pmid":"26305940","id":"PMC_26305940","title":"Allosteric activation of apicomplexan calcium-dependent protein kinases.","date":"2015","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/26305940","citation_count":43,"is_preprint":false},{"pmid":"27781359","id":"PMC_27781359","title":"Analysis of Ca2+ mediated signaling regulating Toxoplasma infectivity reveals complex relationships between key molecules.","date":"2017","source":"Cellular microbiology","url":"https://pubmed.ncbi.nlm.nih.gov/27781359","citation_count":36,"is_preprint":false},{"pmid":"24176018","id":"PMC_24176018","title":"Protective immunity against Toxoplasma gondii induced by DNA immunization with the gene encoding a novel vaccine candidate: calcium-dependent protein kinase 3.","date":"2013","source":"BMC infectious diseases","url":"https://pubmed.ncbi.nlm.nih.gov/24176018","citation_count":33,"is_preprint":false},{"pmid":"30008721","id":"PMC_30008721","title":"Immunization With a DNA Vaccine Cocktail Encoding TgPF, TgROP16, TgROP18, TgMIC6, and TgCDPK3 Genes Protects Mice Against Chronic Toxoplasmosis.","date":"2018","source":"Frontiers in immunology","url":"https://pubmed.ncbi.nlm.nih.gov/30008721","citation_count":32,"is_preprint":false},{"pmid":"35986017","id":"PMC_35986017","title":"Live-attenuated ME49Δcdpk3 strain of Toxoplasma gondii protects against acute and chronic toxoplasmosis.","date":"2022","source":"NPJ vaccines","url":"https://pubmed.ncbi.nlm.nih.gov/35986017","citation_count":25,"is_preprint":false},{"pmid":"19699312","id":"PMC_19699312","title":"Molecular analyses of Toxoplasma gondii calmodulin-like domain protein kinase isoform 3.","date":"2009","source":"Parasitology international","url":"https://pubmed.ncbi.nlm.nih.gov/19699312","citation_count":22,"is_preprint":false},{"pmid":"28258212","id":"PMC_28258212","title":"A forward genetic screen identifies a negative regulator of rapid Ca2+-dependent cell egress (MS1) in the intracellular parasite Toxoplasma gondii.","date":"2017","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/28258212","citation_count":21,"is_preprint":false},{"pmid":"36265000","id":"PMC_36265000","title":"A positive feedback loop mediates crosstalk between calcium, cyclic nucleotide and lipid signalling in calcium-induced Toxoplasma gondii egress.","date":"2022","source":"PLoS pathogens","url":"https://pubmed.ncbi.nlm.nih.gov/36265000","citation_count":19,"is_preprint":false},{"pmid":"31425673","id":"PMC_31425673","title":"Vaccination with recombinant Toxoplasma gondii CDPK3 induces protective immunity against experimental toxoplasmosis.","date":"2019","source":"Acta tropica","url":"https://pubmed.ncbi.nlm.nih.gov/31425673","citation_count":17,"is_preprint":false},{"pmid":"24550330","id":"PMC_24550330","title":"Expression of the essential Kinase PfCDPK1 from Plasmodium falciparum in Toxoplasma gondii facilitates the discovery of novel antimalarial drugs.","date":"2014","source":"Antimicrobial agents and chemotherapy","url":"https://pubmed.ncbi.nlm.nih.gov/24550330","citation_count":17,"is_preprint":false},{"pmid":"30402958","id":"PMC_30402958","title":"Characterisation of the Toxoplasma gondii tyrosine transporter and its phosphorylation by the calcium-dependent protein kinase 3.","date":"2018","source":"Molecular microbiology","url":"https://pubmed.ncbi.nlm.nih.gov/30402958","citation_count":16,"is_preprint":false},{"pmid":"32864371","id":"PMC_32864371","title":"In-depth computational analysis of calcium-dependent protein kinase 3 of Toxoplasma gondii provides promising targets for vaccination.","date":"2020","source":"Clinical and experimental vaccine research","url":"https://pubmed.ncbi.nlm.nih.gov/32864371","citation_count":14,"is_preprint":false},{"pmid":"30673152","id":"PMC_30673152","title":"Calcium negatively regulates secretion from dense granules in Toxoplasma gondii.","date":"2019","source":"Cellular microbiology","url":"https://pubmed.ncbi.nlm.nih.gov/30673152","citation_count":14,"is_preprint":false},{"pmid":"35003067","id":"PMC_35003067","title":"Protective Effect Against Toxoplasmosis in BALB/c Mice Vaccinated With Recombinant Toxoplasma gondii MIF, CDPK3, and 14-3-3 Protein Cocktail Vaccine.","date":"2021","source":"Frontiers in immunology","url":"https://pubmed.ncbi.nlm.nih.gov/35003067","citation_count":13,"is_preprint":false},{"pmid":"35142606","id":"PMC_35142606","title":"Anti-Toxoplasma gondii effect of lupane-type triterpenes from the bark of black alder (Alnus glutinosa) and identification of a potential target by reverse docking.","date":"2022","source":"Parasite (Paris, France)","url":"https://pubmed.ncbi.nlm.nih.gov/35142606","citation_count":10,"is_preprint":false},{"pmid":"35757711","id":"PMC_35757711","title":"Toxoplasma gondii CDPK3 Controls the Intracellular Proliferation of Parasites in Macrophages.","date":"2022","source":"Frontiers in immunology","url":"https://pubmed.ncbi.nlm.nih.gov/35757711","citation_count":8,"is_preprint":false},{"pmid":"25526188","id":"PMC_25526188","title":"Identification and bioinformatic analysis of a putative calcium-dependent protein kinase (CDPK6) from Toxoplasma gondii.","date":"2014","source":"Genetics and molecular research : GMR","url":"https://pubmed.ncbi.nlm.nih.gov/25526188","citation_count":8,"is_preprint":false},{"pmid":"37272703","id":"PMC_37272703","title":"Identification of Toxoplasma calcium-dependent protein kinase 3 as a stress-activated elongation factor 2 kinase.","date":"2023","source":"mSphere","url":"https://pubmed.ncbi.nlm.nih.gov/37272703","citation_count":5,"is_preprint":false},{"pmid":"38752002","id":"PMC_38752002","title":"Immunoinformatics studies and design of a novel multi-epitope peptide vaccine against Toxoplasma gondii based on calcium-dependent protein kinases antigens through an in-silico analysis.","date":"2024","source":"Clinical and experimental vaccine research","url":"https://pubmed.ncbi.nlm.nih.gov/38752002","citation_count":5,"is_preprint":false},{"pmid":"38310017","id":"PMC_38310017","title":"Protective effect against toxoplasmosis in BALB/C mice vaccinated with recombinant Toxoplasma gondii CDPK3, GRA35, and ROP46 protein cocktail vaccine.","date":"2024","source":"Vaccine","url":"https://pubmed.ncbi.nlm.nih.gov/38310017","citation_count":4,"is_preprint":false},{"pmid":"40394721","id":"PMC_40394721","title":"Toxoplasma calcium-dependent protein kinases 3 mediates M1 macrophage polarization by targeting host Arginase-1.","date":"2025","source":"Parasites & vectors","url":"https://pubmed.ncbi.nlm.nih.gov/40394721","citation_count":0,"is_preprint":false},{"pmid":"42252477","id":"PMC_42252477","title":"Gene-edited live-attenuated vaccines against Toxoplasma gondii: recent advances and future frontiers.","date":"2026","source":"Parasites & vectors","url":"https://pubmed.ncbi.nlm.nih.gov/42252477","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":16241,"output_tokens":4721,"usd":0.059769,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":11139,"output_tokens":5625,"usd":0.09816,"stage2_stop_reason":"end_turn"},"total_usd":0.0,"stage1_batch_id":"msgbatch_01XFXJD6iZecXyWn9uU5Qom9","stage2_batch_id":"msgbatch_013GToKgs4AKSCRE4yvueviH","note":"batch pricing = 50% of standard","skipped":true},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2012,\n      \"finding\": \"TgCDPK3 is required for ionophore-induced egress but not for invasion; microneme secretion depends on TgCDPK3 only when triggered by certain stimuli (not universally), demonstrating that egress and invasion are controlled by distinct kinase-mediated signalling pathways.\",\n      \"method\": \"Chemical-genetic inhibition of TgCDPK1 and TgCDPK3 using analog-sensitive kinase alleles; ionophore-induced egress and invasion assays\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — chemical-genetic approach with specific inhibition, replicated across multiple assay types; independently corroborated by two additional papers published the same year\",\n      \"pmids\": [\"23149386\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"TgCDPK3 localizes to the inner side of the plasma membrane via dual acylation (N-myristoylation and palmitoylation), and this membrane targeting is required for its activity in promoting Ca2+-dependent egress.\",\n      \"method\": \"Genetic knockout; complementation with acylation-site mutants; localization by fluorescence microscopy; ionophore- and DTT-induced egress assays\",\n      \"journal\": \"PLoS pathogens\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean knockout, complementation with acylation mutants, and localization imaging in one study; independently corroborated by concurrent papers\",\n      \"pmids\": [\"23226109\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"TgCDPK3 regulates microneme secretion specifically when parasites are intracellular, and the high K+ concentration of the host cell environment appears to determine the requirement for TgCDPK3 in this context.\",\n      \"method\": \"Genetic knockout; microneme secretion assays in intracellular vs. extracellular parasites; ionic environment manipulation\",\n      \"journal\": \"PLoS pathogens\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — single lab, clean knockout with defined assay readout, but ionic-environment mechanism is inferred rather than directly demonstrated\",\n      \"pmids\": [\"23226109\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Forward genetic screen identified four independent loss-of-function missense mutations in TgCDPK3 that confer defects in ionophore-induced egress; biochemical analysis of recombinant mutant proteins confirmed these mutations alter key functional regions. Genetic knockout confirmed TgCDPK3 is required for rapid egress induction and for formation of latent cysts in mouse brain.\",\n      \"method\": \"Forward genetic screen; whole-genome sequencing; biochemical analysis of recombinant proteins with missense mutations; complementation; genetic knockout; mouse infection model\",\n      \"journal\": \"PLoS pathogens\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — forward genetics converging on same gene (4 independent alleles), complementation, and biochemical validation; independently replicated by two concurrent studies\",\n      \"pmids\": [\"23209419\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"TgCDPKif3 (TgCDPK3) is an active kinase that auto-phosphorylates and phosphorylates substrates (Histone IIas, TgALD1) in a Ca2+-dependent manner; the catalytic lysine is essential for activity. It localizes to the apical ends of tachyzoites and relocalizes between intra- and extracellular stages. It interacts with and phosphorylates T. gondii aldolase 1 (TgALD1) in vitro, and this interaction was confirmed by co-immunoprecipitation in mammalian cells.\",\n      \"method\": \"Recombinant protein purification; [γ-32P]ATP labeling; phosphatase treatment; catalytic lysine-to-alanine mutagenesis; in vitro kinase assay with Ca2+ titration; immunofluorescence localization; co-immunoprecipitation\",\n      \"journal\": \"Parasitology international\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1–2 / Weak — in vitro kinase assay with mutagenesis and co-IP, but single lab and interaction confirmed in mammalian overexpression system, not endogenously in Toxoplasma\",\n      \"pmids\": [\"19699312\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"TgCDPK3 phosphorylates Myosin A (TgMyoA) at serines 21 and 743 in vitro, and this phosphorylation is required for rapid initiation of motility and egress. Phosphomimetic mutations at S21+S743 in TgMyoA rescue the egress and motility defects of Tgcdpk3 mutant parasites, and TgCDPK3 was identified as a near-neighbor/direct interactor of TgMyoA by BioID proximity labeling.\",\n      \"method\": \"BioID proximity-based protein interaction trap; peptide array phosphorylation profiling; in vitro kinase assay; complementation of TgmyoA null with phosphosite alanine and phosphomimetic mutants; egress assays; motility assays\",\n      \"journal\": \"PLoS pathogens\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro phosphorylation, BioID interaction, and genetic rescue with phosphomimetic mutants in one study; multiple orthogonal methods establishing substrate-kinase relationship\",\n      \"pmids\": [\"26544049\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"PfCDPK1 (the Plasmodium falciparum ortholog of TgCDPK3) can functionally complement the egress defect of a TgCDPK3 mutant strain, but only when PfCDPK1 is localized to the plasma membrane and retains kinase activity, demonstrating that membrane localization and catalytic activity are essential for the egress function of this kinase family.\",\n      \"method\": \"Heterologous complementation of TgCDPK3 mutant with PfCDPK1; localization mutants; kinase-dead mutants; ionophore-induced egress assays; pharmacological inhibition\",\n      \"journal\": \"Antimicrobial agents and chemotherapy\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic complementation with domain/activity mutants across species, two orthogonal approaches (localization mutant + kinase-dead mutant), single lab\",\n      \"pmids\": [\"24550330\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"A forward genetic suppressor screen in a cdpk3 knockout strain identified SCE1 (Suppressor of Ca2+-dependent Egress 1) as a negative regulator of egress that is phosphorylated in a CDPK3-dependent manner; CDPK3-dependent phosphorylation of SCE1 is required to relieve its suppressive activity and allow egress. Loss of SCE1 restores many Δcdpk3-dependent phosphorylation events to near wild-type levels.\",\n      \"method\": \"Forward genetic suppressor screen in cdpk3 KO background; whole-genome sequencing; global phosphoproteomics; genetic knockout of SCE1\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — epistasis established by suppressor screen, phosphoproteomic analysis, and genetic knockout, multiple orthogonal methods in one study\",\n      \"pmids\": [\"28258212\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"TgCDPK1 and TgCDPK3 play a role in signal quenching of Ca2+ flux before egress, as revealed by live imaging with a genetically encoded Ca2+ biosensor (GCaMP6), placing both kinases within the temporal dynamics of the Ca2+ signalling cascade.\",\n      \"method\": \"Live-cell Ca2+ imaging with GCaMP6 biosensor; pharmacological and genetic perturbation of CDPK1 and CDPK3\",\n      \"journal\": \"Cellular microbiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — live biosensor imaging is a direct functional readout, but pathway placement based on a single study\",\n      \"pmids\": [\"27781359\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"TgCDPK3 phosphorylates the tyrosine transporter ApiAT5-3 at serine 56 following induction of Ca2+ signalling, as identified by phosphoproteomics comparing WT and Δcdpk3 parasites. Mutating S56 to non-phosphorylatable alanine incurs a fitness cost, suggesting S56 phosphorylation is beneficial but not essential for tyrosine import function.\",\n      \"method\": \"Phosphoproteomic comparison of WT vs. Δcdpk3 parasites; conditional knockout of apiAT5-3; phosphosite mutagenesis (S56A); metabolomics; heterologous expression for transporter characterization\",\n      \"journal\": \"Molecular microbiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — phosphoproteomics identifies substrate, phosphosite mutagenesis establishes functional consequence, multiple methods in one study; but direct kinase-substrate relationship relies on phosphoproteomic inference\",\n      \"pmids\": [\"30402958\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"TgCDPK3 function is required for Ca2+-mediated negative regulation of dense granule exocytosis; in the absence of TgCDPK3, this Ca2+-mediated suppression of dense granule secretion is lost, demonstrating that TgCDPK3 participates in the reciprocal Ca2+-dependent control of microneme vs. dense granule secretion.\",\n      \"method\": \"Genetic knockout of TgCDPK3; dense granule secretion assays under Ca2+-elevated conditions; comparison with TgCDPK1 knockout\",\n      \"journal\": \"Cellular microbiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — clean KO with defined secretion assay readout, but single lab and single method for CDPK3-specific contribution\",\n      \"pmids\": [\"30673152\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Using WT and conditional Δcdpk3 parasites in sub-minute phosphoproteomic timecourses after Ca2+ release, CDPK3 was shown to be partly responsible for a positive feedback loop linking calcium and cyclic nucleotide (cAMP) signalling during egress; CDPK3 activity influences cAMP and lipid metabolism pathways that accelerate the egress response.\",\n      \"method\": \"Conditional knockout; sub-minute phosphoproteomics timecourse after Ca2+ ionophore; lipidomics; pharmacological inhibition of PDEs\",\n      \"journal\": \"PLoS pathogens\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — phosphoproteomics with temporal resolution and conditional KO, multiple readouts; pathway placement is partial as the feedback loop is inferred from phosphoproteomic correlations\",\n      \"pmids\": [\"36265000\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Under extracellular stress conditions, TgCDPK3 functions as an eEF2 (elongation factor 2) kinase: it phosphorylates eEF2, thereby inhibiting translational elongation. Loss of CDPK3 in stressed extracellular parasites leads to decreased eEF2 phosphorylation and enhanced elongation rates. CDPK3 and eEF2 interact by co-immunoprecipitation specifically under stress conditions, coinciding with CDPK3 relocalization from the plasma membrane to the cytosol due to loss of N-myristoylation under stress.\",\n      \"method\": \"BLAST analysis identifying CDPK3 as homolog of yeast eEF2 kinase RCK2; co-immunoprecipitation of CDPK3 and eEF2 in stressed parasites; eEF2 phosphorylation assay in cdpk3 KO parasites; translational elongation rate assay; localization by imaging under stress vs. normal conditions; N-myristoylation analysis\",\n      \"journal\": \"mSphere\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP of endogenous interaction, KO with defined phosphorylation and elongation phenotype, localization shift; single lab but multiple orthogonal methods\",\n      \"pmids\": [\"37272703\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"TgCDPK3 (from type II ME49 strain) interacts with host autophagy proteins Atg3 and Atg5, and this interaction promotes localization of IRGs (immunity-related GTPases) and GBPs (guanylate-binding proteins) around the parasitophorous vacuole membrane, thereby inhibiting intracellular parasite proliferation and facilitating long-term latency of the less virulent strain.\",\n      \"method\": \"Co-immunoprecipitation of TgCDPK3 with host Atg3 and Atg5; CDPK3 knockout in ME49 strain; IRG/GBP localization assay by fluorescence microscopy; intracellular proliferation and plaque assays\",\n      \"journal\": \"Frontiers in immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — Co-IP and KO with defined localization and proliferation readouts, but single lab and functional mechanism (how CDPK3-Atg interaction promotes IRG/GBP targeting) not fully resolved\",\n      \"pmids\": [\"35757711\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"ME49\"}]\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"TgCDPK3 interacts with host Arginase-1 (Arg1) as identified by mass spectrometry and confirmed by GST pull-down, co-immunoprecipitation, and co-localization assays. This interaction reduces Arginase-1 enzymatic activity, promoting M1-biased macrophage polarization (increased iNOS, decreased Arg1), which restricts intracellular parasite proliferation.\",\n      \"method\": \"Mass spectrometry of TgCDPK3-binding proteins; GST pull-down; co-immunoprecipitation; immunofluorescence co-localization; Arginase activity assay; Western blotting for Arg1 and iNOS; FACS for M1/M2 macrophage markers; ELISA for cytokines; intracellular proliferation and plaque assays\",\n      \"journal\": \"Parasites & vectors\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal methods (MS, pull-down, co-IP, activity assay, cellular phenotype), single lab\",\n      \"pmids\": [\"40394721\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"ME49\"}]\n    }\n  ],\n  \"current_model\": \"TgCDPK3 is a Ca2+-dependent protein kinase anchored to the inner face of the plasma membrane via dual acylation (N-myristoylation/palmitoylation) that acts as a central transducer of calcium signals during host-cell egress: it directly phosphorylates Myosin A (TgMyoA) at S21 and S743 to initiate gliding motility, phosphorylates and inactivates the negative egress regulator SCE1, participates in a positive cAMP-Ca2+ feedback loop that accelerates the egress response, and controls microneme but not dense granule secretion in the high-K+ intracellular environment; under extracellular stress CDPK3 loses N-myristoylation, relocalizes to the cytosol, and functions as the first identified protozoan eEF2 kinase to regulate translational elongation, while in macrophages it interacts with host Arginase-1 to promote M1 polarization and restrict parasite proliferation.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"TgCDPK3 is a Ca2+-dependent protein kinase that serves as a central transducer of calcium signals governing Toxoplasma gondii egress from host cells [#0, #3]. It is an active autophosphorylating kinase whose catalytic lysine is essential, and it phosphorylates substrates in a Ca2+-dependent manner [#4]. The enzyme is anchored to the inner face of the plasma membrane through dual acylation (N-myristoylation and palmitoylation), and both this membrane targeting and its catalytic activity are required for its role in Ca2+-dependent egress [#1, #6]. During egress, TgCDPK3 directly phosphorylates Myosin A (TgMyoA) at serines 21 and 743 to initiate rapid gliding motility, with phosphomimetic substitutions rescuing the motility and egress defects of cdpk3 mutants [#5], and it drives egress in part by phosphorylating and thereby relieving the suppressive activity of the negative regulator SCE1 [#7]. It further shapes the egress response by controlling microneme secretion in an intracellular, high-K+-dependent manner, negatively regulating dense granule exocytosis, quenching the Ca2+ flux, and participating in a positive cAMP–Ca2+ feedback loop that accelerates the response [#2, #10, #8, #11]. Beyond egress, under extracellular stress TgCDPK3 loses N-myristoylation, relocalizes to the cytosol, and acts as an eEF2 kinase that phosphorylates elongation factor 2 to inhibit translational elongation [#12]. In the type II ME49 strain, TgCDPK3 also engages host factors — interacting with host Arginase-1 to bias macrophages toward M1 polarization and with autophagy proteins Atg3/Atg5 to promote IRG/GBP recruitment to the parasitophorous vacuole — both of which restrict intracellular proliferation [#14, #13].\",\n  \"teleology\": [\n    {\n      \"year\": 2009,\n      \"claim\": \"Establishing that TgCDPK3 is a genuine Ca2+-dependent kinase was the prerequisite for any signalling role, showing it autophosphorylates and phosphorylates substrates only when Ca2+ is present and only with an intact catalytic lysine.\",\n      \"evidence\": \"Recombinant kinase assays with [\\u03b3-32P]ATP, Ca2+ titration, catalytic lysine mutagenesis, and co-IP of the aldolase TgALD1\",\n      \"pmids\": [\"19699312\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"TgALD1 interaction confirmed only in a mammalian overexpression system, not endogenously in Toxoplasma\", \"physiological substrates in the parasite not identified at this stage\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Multiple 2012 studies converged to define TgCDPK3's cellular role, showing it is specifically required for ionophore-induced egress but not invasion, that four independent loss-of-function alleles reproduce the egress defect, and that its egress function depends on plasma-membrane targeting via dual acylation.\",\n      \"evidence\": \"Chemical-genetic analog-sensitive kinase inhibition, forward genetic screening with whole-genome sequencing, knockout/complementation with acylation-site mutants, and egress/invasion assays\",\n      \"pmids\": [\"23149386\", \"23226109\", \"23209419\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"direct kinase substrates driving egress not yet identified\", \"mechanism linking membrane localization to activity unresolved\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"The requirement for TgCDPK3 in microneme secretion was shown to be context-dependent, tied to the high-K+ intracellular environment rather than a universal secretory step.\",\n      \"evidence\": \"Knockout with microneme secretion assays comparing intracellular vs. extracellular parasites and ionic-environment manipulation\",\n      \"pmids\": [\"23226109\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"ionic-environment mechanism inferred rather than directly demonstrated\", \"molecular sensor of K+ not identified\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Heterologous complementation demonstrated that the egress function is a conserved property of this kinase family, requiring both membrane localization and catalytic activity.\",\n      \"evidence\": \"Complementation of a TgCDPK3 mutant with Plasmodium falciparum PfCDPK1 using localization and kinase-dead mutants plus egress assays\",\n      \"pmids\": [\"24550330\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"substrates of the complementing kinase not defined\", \"single-lab result\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Identification of TgMyoA as a direct substrate provided the mechanistic link between CDPK3 activity and the motility that drives egress, resolving how the kinase triggers movement.\",\n      \"evidence\": \"BioID proximity labeling, peptide-array and in vitro kinase assays mapping S21/S743, and genetic rescue with phosphomimetic TgMyoA mutants\",\n      \"pmids\": [\"26544049\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"whether MyoA phosphorylation is the sole egress-relevant substrate not established\", \"in vivo phosphorylation kinetics not resolved\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Live Ca2+ imaging placed CDPK3 within the temporal dynamics of the egress signalling cascade, implicating it in quenching the Ca2+ flux prior to egress.\",\n      \"evidence\": \"GCaMP6 Ca2+ biosensor live imaging with genetic and pharmacological perturbation of CDPK1 and CDPK3\",\n      \"pmids\": [\"27781359\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"molecular basis of signal quenching not defined\", \"CDPK1 vs CDPK3 contributions not fully separated\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"A suppressor screen revealed that CDPK3 promotes egress in part by inactivating a brake, identifying SCE1 as a negative egress regulator whose CDPK3-dependent phosphorylation relieves suppression.\",\n      \"evidence\": \"Forward genetic suppressor screen in a cdpk3 KO background, whole-genome sequencing, global phosphoproteomics, and SCE1 knockout\",\n      \"pmids\": [\"28258212\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"direct CDPK3-SCE1 phosphorylation not shown in vitro\", \"downstream effectors of SCE1 unidentified\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Phosphoproteomics extended the CDPK3 substrate repertoire beyond the motor to metabolic control, identifying the tyrosine transporter ApiAT5-3 S56 as a Ca2+-induced CDPK3-dependent phosphosite.\",\n      \"evidence\": \"WT vs \\u0394cdpk3 phosphoproteomics, apiAT5-3 conditional knockout, S56A phosphosite mutagenesis, metabolomics, and heterologous transporter characterization\",\n      \"pmids\": [\"30402958\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"direct kinase-substrate relationship rests on phosphoproteomic inference\", \"S56 phosphorylation is beneficial but not essential, leaving its precise role open\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"CDPK3 was shown to mediate the reciprocal Ca2+-dependent control of secretory organelles, being required for Ca2+-mediated suppression of dense granule exocytosis.\",\n      \"evidence\": \"CDPK3 knockout with dense granule secretion assays under Ca2+-elevated conditions, compared to CDPK1 knockout\",\n      \"pmids\": [\"30673152\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"substrates mediating dense granule suppression not identified\", \"single method for CDPK3-specific contribution\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Sub-minute phosphoproteomics revealed CDPK3 as a partial driver of a positive cAMP-Ca2+ feedback loop that accelerates the egress response, linking calcium and cyclic-nucleotide signalling.\",\n      \"evidence\": \"Conditional knockout, sub-minute phosphoproteomics timecourse after Ca2+ ionophore, lipidomics, and PDE inhibition\",\n      \"pmids\": [\"36265000\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"feedback loop inferred from phosphoproteomic correlations\", \"direct enzymatic targets in the cAMP pathway not identified\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"CDPK3 was shown to have a host-directed function during latency, with the ME49 kinase engaging host autophagy proteins to recruit anti-parasitic effectors to the vacuole.\",\n      \"evidence\": \"Co-IP of TgCDPK3 with host Atg3/Atg5, ME49 CDPK3 knockout, IRG/GBP localization imaging, and proliferation/plaque assays\",\n      \"pmids\": [\"35757711\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"mechanism by which the CDPK3-Atg interaction promotes IRG/GBP targeting not resolved\", \"single lab\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"ME49\"}]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"A distinct stress-conditional role was uncovered: loss of N-myristoylation relocalizes CDPK3 to the cytosol where it acts as the first protozoan eEF2 kinase to inhibit translational elongation.\",\n      \"evidence\": \"Homology analysis to yeast RCK2, stress-conditional co-IP of CDPK3 and eEF2, eEF2 phosphorylation and elongation-rate assays in cdpk3 KO, and localization/myristoylation analysis\",\n      \"pmids\": [\"37272703\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"signal driving loss of N-myristoylation under stress not defined\", \"single lab\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"The host-directed repertoire was extended to immune reprogramming, with CDPK3 interacting with host Arginase-1 to reduce its activity and bias macrophages toward M1 polarization.\",\n      \"evidence\": \"Mass spectrometry, GST pull-down, co-IP, co-localization, arginase activity assay, FACS/ELISA for macrophage markers, and proliferation/plaque assays\",\n      \"pmids\": [\"40394721\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"structural basis of the CDPK3-Arg1 interaction not defined\", \"whether CDPK3 phosphorylates Arg1 not established\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"ME49\"}]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How CDPK3 is spatially and temporally switched between its membrane-anchored egress function and its cytosolic eEF2-kinase and host-directed functions, and the full physiological substrate set, remain open.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"the molecular signal controlling stress-induced demyristoylation and relocalization is unknown\", \"no comprehensive validated in vivo substrate catalog\", \"relationship between egress substrates and host-directed functions unresolved\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0004683\", \"supporting_discovery_ids\": [4, 5, 7], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]},\n      {\"term_id\": \"GO:0004674\", \"supporting_discovery_ids\": [4, 5, 9, 12], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]},\n      {\"term_id\": \"GO:0005509\", \"supporting_discovery_ids\": [4], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [1, 6], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]},\n      {\"term_id\": \"GO:0020009\", \"supporting_discovery_ids\": [4], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [12], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"GO:0051649\", \"supporting_discovery_ids\": [0, 3], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]},\n      {\"term_id\": \"GO:0002250\", \"supporting_discovery_ids\": [13, 14], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"ME49\"}]},\n      {\"term_id\": \"GO:0006417\", \"supporting_discovery_ids\": [12], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]},\n      {\"term_id\": \"GO:0019722\", \"supporting_discovery_ids\": [8, 11], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\n      \"TgMyoA (Myosin A) — direct substrate, phosphorylated at S21/S743 [#5]\",\n      \"SCE1 — CDPK3-dependent phosphorylation relieves egress suppression [#7]\",\n      \"TgALD1 (aldolase 1) — interactor/substrate [#4]\",\n      \"ApiAT5-3 tyrosine transporter — phosphorylated at S56 [#9]\",\n      \"eEF2 (elongation factor 2) — substrate under stress [#12]\",\n      \"host Arginase-1 — interactor, activity reduced [#14]\",\n      \"host Atg3 and Atg5 — interactors [#13]\"\n    ],\n    \"other_free_text\": [\n      \"Membrane targeting via dual acylation (N-myristoylation + palmitoylation) required for egress function; stress-induced loss of N-myristoylation drives cytosolic relocalization [#1, #12]\",\n      \"Requirement for microneme secretion is intracellular- and high-K+-dependent [#2]\",\n      \"Negatively regulates dense granule exocytosis in a Ca2+-dependent manner [#10]\",\n      \"Participates in a positive cAMP-Ca2+ feedback loop accelerating egress [#11]\",\n      \"Required for formation of latent cysts in mouse brain [#3]\"\n    ]\n  },\n  \"strain_conflicts\": []\n}","audit_flag":null,"strains":[{"strain":"ME49","gene_id":"TGME49_305860","name":"CDPK3","product":"calcium-dependent protein kinase CDPK3","uniprot_acc":"A0A125YQG4","chromosome":"TGME49_chrIX","start_pos":5628418},{"strain":"GT1","gene_id":"TGGT1_305860","name":null,"product":"calcium-dependent protein kinase CDPK3","uniprot_acc":null,"chromosome":"TGGT1_chrIX","start_pos":5409701},{"strain":"RH88","gene_id":"TGRH88_017420","name":null,"product":"calcium-dependent protein kinase CDPK3","uniprot_acc":null,"chromosome":"CM023090","start_pos":5752163},{"strain":"VEG","gene_id":"TGVEG_305860","name":null,"product":"calcium-dependent protein kinase CDPK3","uniprot_acc":"A0A0F7V4V1","chromosome":"TGVEG_chrIX","start_pos":5667331}]}