{"gene":"TG_301440","gene_symbol":"CDPK1","run_date":"2026-09-08T18:09:52+00:00","timeline":{"discoveries":[{"year":2001,"finding":"TgCDPK1 protein is expressed in Toxoplasma tachyzoites, exhibits calcium-dependent kinase activity that does not require calmodulin or phospholipids, co-fractionates with KT5926-sensitive kinase activity, and phosphorylates three parasite proteins in vitro that correspond to the three KT5926-sensitive phosphoproteins detected in vivo, implicating it in regulating motility and host cell attachment.","method":"Biochemical fractionation, in vitro kinase assay, pharmacological inhibition with KT5926","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vitro kinase assay with substrate identification, co-fractionation, and in vivo phosphoprotein correlation; single lab but multiple orthogonal methods","pmids":["11154702"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2001,"finding":"Egress-triggering reduction in host cell [K+] activates a phospholipase C in Toxoplasma leading to increased cytoplasmic [Ca2+], which then activates TgCDPK1 as one of at least two downstream signaling pathways required for parasite egress; buffering cytoplasmic Ca2+ blocks egress and calcium ionophores bypass the need for low [K+].","method":"Pharmacological manipulation of Ca2+, potassium concentrations, and kinase inhibitors; egress assay","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — epistasis-level placement of CDPK1 downstream of Ca2+ signal in egress pathway, single lab, multiple pharmacological perturbations","pmids":["11526113"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2010,"finding":"TgCDPK1 is an essential regulator of calcium-dependent microneme exocytosis; conditional suppression of TgCDPK1 blocks calcium-dependent secretion from micronemes, abolishing parasite motility, host-cell invasion, and egress. A glycine gatekeeper mutant (resistant to pyrazolopyrimidine inhibitors) restored sensitivity reversal, confirming TgCDPK1 as the specific in vivo target.","method":"Conditional knockdown (promoter replacement), chemical-genetic inhibition with pyrazolopyrimidine compounds, mutant rescue experiment, microneme secretion assay, motility/invasion/egress assays","journal":"Nature","confidence":"High","confidence_rationale":"Tier 2 / Strong — conditional genetic knockdown combined with chemical-genetic approach and resistant-mutant rescue, multiple orthogonal methods, replicated across approaches in single study","pmids":["20485436"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2010,"finding":"Crystal structures of TgCDPK1 in autoinhibited (apo) and calcium-activated conformations reveal that the C-terminal CDPK activation domain (CAD) in the apo form contains a long N-terminal helix that inhibits the kinase domain analogously to CaMKII autoinhibition; calcium binding triggers major reorganization of the CAD to a position remote from the substrate-binding site, constituting a distinct mechanism of calcium-signal transduction.","method":"X-ray crystallography of apo and calcium-bound forms of TgCDPK1","journal":"Nature structural & molecular biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structures of two conformational states with functional interpretation, rigorous structural method","pmids":["20436473"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2010,"finding":"TgCDPK1 contains a unique glycine gatekeeper residue in its ATP-binding site that confers nanomolar sensitivity to bumped kinase inhibitors (BKIs); co-crystal structures of TgCDPK1 with BKIs confirm the structural basis for selectivity, and T. gondii expressing a glycine-to-methionine gatekeeper mutant shows significantly decreased BKI sensitivity in vivo, confirming TgCDPK1 as the BKI target mediating early infection inhibition.","method":"Co-crystal structure determination, in vitro kinase inhibition assays, gatekeeper mutant parasite line generation and growth inhibition assays","journal":"Nature structural & molecular biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — co-crystal structures plus in vivo genetic validation with gatekeeper mutant; two orthogonal methods in a single rigorous study","pmids":["20436472"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2010,"finding":"Inhibition of TgCDPK1 by 1NM-PP1 blocks gliding motility without inhibiting micronemal secretion, suggesting TgCDPK1 has a role in gliding motility downstream of or independent of microneme secretion; a G128M gatekeeper mutation in TgCDPK1 confers resistance to 1NM-PP1.","method":"Chemical-genetic inhibition with analog-sensitive kinase approach, motility assay, micronemal secretion assay, site-directed mutagenesis","journal":"Eukaryotic cell","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — chemical-genetic approach with gatekeeper mutant validation; single lab but two distinct phenotypic readouts; note partial contradiction with other work on microneme secretion","pmids":["20173034"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2012,"finding":"TgCDPK1 and TgCDPK3 control distinct calcium signaling pathways: TgCDPK1 is required universally for microneme secretion and for invasion, whereas TgCDPK3 is required for ionophore-induced egress but not for invasion; cGMP-dependent protein kinase (PKG) activation can partially compensate for TgCDPK3 inhibition during egress.","method":"Chemical-genetic inhibition of analog-sensitive CDPK1 and CDPK3 alleles; microneme secretion assay; invasion assay; egress assay; pharmacological activation of PKG","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 2 / Strong — chemical-genetic approach with orthogonal alleles for two kinases, multiple functional readouts, genetic epistasis established between CDPK1, CDPK3, and PKG","pmids":["23149386"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2013,"finding":"Using an analog-sensitive TgCDPK1 allele and a synthetic ATPγS analogue complementary to the expanded glycine-gatekeeper ATP pocket, 163 CDPK1 substrates were identified by selective thiophosphorylation; predicted phosphorylation sites were concordant with TgCDPK1 in vitro substrate preference, and phosphorylation of identified substrates was confirmed to be CDPK1-dependent both in vitro and in vivo.","method":"Chemical-genetic substrate identification (analog-sensitive kinase + thiophosphate labeling), phosphoproteomics, in vitro kinase assays, in vivo validation","journal":"ACS chemical biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — bio-orthogonal substrate labeling combined with in vitro and in vivo validation; multiple orthogonal methods in a single rigorous study","pmids":["23530747"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2015,"finding":"Removal of the autoinhibitory domain from TgCDPK1 alone is insufficient for kinase activation; the regulatory (CAD) domain plays a dual role, both inhibiting and allosterically activating the kinase domain in a Ca2+-dependent manner. A VHH antibody (1B7) that binds TgCDPK1 in a conformation-dependent manner potently inhibits it by disrupting this allosteric stabilization, as revealed by the crystal structure of the complex and molecular dynamics simulations.","method":"VHH antibody library screening, crystal structure of TgCDPK1-1B7 complex, molecular dynamics simulation, in vitro kinase activity assays with domain deletion mutants","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure plus molecular dynamics plus functional mutagenesis/domain deletion; multiple orthogonal methods revealing novel allosteric mechanism","pmids":["26305940"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2016,"finding":"CDPK1 is essential in T. gondii; CRISPR/Cas9-based complete gene deletion confirmed CDPK1 essentiality for parasite growth in vitro, as deletions could not be obtained without complementation.","method":"CRISPR/Cas9 gene deletion with Cre-loxP marker recycling, plaque assay","journal":"Infection and immunity","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — CRISPR/Cas9 gene deletion with clear essentiality phenotype; single lab, clean genetic approach","pmids":["26755159"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":"RH; ME49"}]},{"year":2017,"finding":"TgDJ-1 directly binds to TgCDPK1 through an arginine dyad phospho-gatekeeper motif; the interaction is sensitive to oxidation and calcium, potentiates CDPK1 kinase activity, and is required for normal microneme exocytosis. Genetic deletion of TgDJ-1 upregulates CDPK1 expression, and disruption of the TgDJ-1/CDPK1 complex prevents microneme secretion in vivo.","method":"X-ray crystal structure of TgDJ-1, co-immunoprecipitation, in vitro kinase activity assays, site-directed mutagenesis of arginine dyad, genetic knockout of TgDJ-1, microneme secretion assay","journal":"mBio","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure of binding partner, direct binding assay, functional mutagenesis, genetic knockout with specific secretion phenotype; multiple orthogonal methods in single study","pmids":["28246362"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2017,"finding":"TgCDPK1 and TgCDPK3 contribute to signal quenching (Ca2+ signal dampening) before egress, as revealed by real-time Ca2+ imaging with the GCaMP6 biosensor; their inhibition alters the temporal dynamics of intracellular Ca2+ flux during egress.","method":"GCaMP6 genetically encoded Ca2+ biosensor live imaging, pharmacological inhibition of CDPK1 and CDPK3","journal":"Cellular microbiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — live Ca2+ imaging with genetically encoded biosensor, pharmacological inhibition; single lab, functional readout linked to signaling dynamics","pmids":["27781359"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2019,"finding":"TgCDPK1 controls F-actin flux required for motility, invasion, and egress; this flux depends on myosins A and H and is regulated by phosphorylation via PKG and CDPK1, and by methylation via AKMT. This F-actin flux is independent of microneme secretion and persists in the absence of the glideosome-associated connector (GAC).","method":"F-actin chromobody live imaging, formin gene disruption, epistasis with PKG and CDPK1 inhibitors, AKMT methyltransferase mutants","journal":"eLife","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — live F-actin imaging combined with genetic disruption and chemical-genetic epistasis; single lab, multiple orthogonal approaches","pmids":["30753127"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2019,"finding":"TgCDPK1 and TgCDPK3 mediate negative regulation of dense granule exocytosis by cytosolic Ca2+; elevated Ca2+ suppresses dense granule secretion in a manner contingent on CDPK1 and CDPK3 function, providing reciprocal control with microneme exocytosis.","method":"Dense granule secretion assays, Ca2+ manipulation, pharmacological inhibition of CDPK1 and CDPK3","journal":"Cellular microbiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — secretion assays with Ca2+ manipulation and kinase inhibition; single lab, two kinases tested; functional link established","pmids":["30673152"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2020,"finding":"Thermal proteome profiling of Toxoplasma identified CDPK1 as a target of the antiparasitic compound ENH1; CDPK1 inhibition by ENH1 was confirmed in vitro and in parasites by comparison of sensitive vs. resistant CDPK1 alleles. CDPK1 inhibition explained the egress block, but ENH1 effects on calcium homeostasis and parasite viability were CDPK1-independent, implicating additional targets.","method":"Thermal proteome profiling (in situ target identification), in vitro kinase assay, resistant CDPK1 allele comparison, egress assay","journal":"ACS chemical biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — thermal proteome profiling plus allele-specific validation; single lab, novel target identification method with functional validation","pmids":["32597628"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2023,"finding":"Sub-minute resolution phosphoproteomics and bio-orthogonal substrate labeling identified 163 proteins phosphorylated in a CDPK1-dependent manner in T. gondii. Among these, a HOOK activating adaptor is a CDPK1 substrate; CDPK1 kinase activity and HOOK are required for rapid apical trafficking of micronemes as parasites initiate motility. A partially conserved FHF complex (HOOK, FTS homolog, and two parasite-specific proteins) mediates this trafficking, and parasites lacking HOOK or FTS show impaired microneme secretion and a block in host cell invasion.","method":"Sub-minute resolution phosphoproteomics, bio-orthogonal kinase substrate labeling (analog-sensitive CDPK1), conditional genetic depletion of HOOK and FTS, microneme secretion assay, invasion assay, live imaging","journal":"eLife","confidence":"High","confidence_rationale":"Tier 1 / Strong — phosphoproteomics combined with bio-orthogonal substrate labeling and genetic validation of downstream complex; multiple orthogonal methods revealing mechanistic pathway","pmids":["37933960"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2023,"finding":"CDPK2A and CDPK1 function in a signaling module upstream of Toxoplasma motility with partial functional redundancy; CDPK2A contributes to microneme discharge, gliding motility, and egress, and epistasis analysis using analog-sensitive alleles and conditional depletion revealed that CDPK2A acts upstream of or in parallel with CDPK1 in the egress/motility pathway.","method":"Analog-sensitive kinase alleles (chemical-genetic), conditional protein depletion (auxin-inducible degron), microneme secretion assay, motility assay, egress assay, epistasis analysis","journal":"mBio","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — chemical-genetic and conditional depletion approaches with epistasis; single lab, multiple orthogonal methods","pmids":["37610220"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2006,"finding":"TgCDPK1 binds a radiolabeled imidazopyridine ligand with sub-nanomolar affinity; mutational analysis of the catalytic site hydrophobic binding pocket (analogous to the gatekeeper residue in PKG) blocks ligand binding, and a compound-refractory CDPK1 mutant (G128Q) overexpressed in transgenic parasites does not reduce susceptibility to the imidazopyridine compound in vivo, indicating that selective inhibition of CDPK1 alone is not responsible for the enhanced anti-parasitic potency of this compound.","method":"Radiolabeled ligand binding assay with parasite extracts, site-directed mutagenesis, transgenic overexpression of mutant CDPK1, in vivo parasite growth assays","journal":"Molecular and biochemical parasitology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — binding assay with mutagenesis and transgenic validation; single lab, multiple approaches; negative result for selective CDPK1 inhibition as sole mechanism is itself mechanistically informative","pmids":["16765465"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2025,"finding":"Forward genetic screen using chemical mutagenesis and BKI-1748 resistance selection identified TgMAPKL1 (in addition to TgCDPK1) as a target of BKIs; single mutations in TgMAPKL1 ATP binding site (L162Q) or TgCDPK1 gatekeeper (G128M) each confer 2–6-fold resistance, while combining both mutations yields up to 157-fold resistance, indicating that BKIs dually target both kinases.","method":"Chemical mutagenesis forward genetic screen, whole-genome sequencing of resistant clones, site-directed mutagenesis to introduce individual resistance mutations, in vitro kinase inhibition, parasite growth inhibition assays","journal":"ACS infectious diseases","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — forward genetic screen with genome sequencing and site-directed mutagenesis validation; single lab, orthogonal combination of genetics and biochemistry","pmids":["40407244"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":"RH"}]}],"current_model":"TgCDPK1 is an essential calcium-dependent serine/threonine kinase in Toxoplasma gondii that transduces intracellular Ca2+ signals into microneme exocytosis, gliding motility, host-cell invasion, and egress; its activation requires Ca2+-induced reorganization of the C-terminal CAD domain plus allosteric stabilization of the kinase domain (not mere derepression), it is potentiated by direct interaction with TgDJ-1, it phosphorylates at least 163 substrates including a HOOK adaptor that drives apical microneme trafficking via a parasite FHF-like dynein-activating complex, it negatively regulates dense granule secretion reciprocally with microneme discharge, it acts in a partially redundant module with CDPK2A upstream of motility, and its unique glycine gatekeeper residue makes it the primary cellular target of bumped kinase inhibitors (with TgMAPKL1 as a secondary co-target)."},"narrative":{"mechanistic_narrative":"TgCDPK1 is an essential calcium-dependent serine/threonine protein kinase in Toxoplasma gondii that transduces intracellular Ca2+ signals into microneme exocytosis, gliding motility, host-cell invasion, and egress [PMID:20485436, PMID:26755159]. Its activity is calcium-dependent and independent of calmodulin or phospholipids [PMID:11154702], and calcium is delivered to the kinase upstream via a phospholipase C-generated signal that couples egress triggers to Ca2+-activated CDPK1 [PMID:11526113]. Activation is structurally governed by the C-terminal CDPK activation (CAD) domain: in the apo state an N-terminal CAD helix autoinhibits the kinase domain, and Ca2+ binding drives a large CAD reorganization that repositions it away from the substrate site [PMID:20436473]; this CAD serves a dual role, both inhibiting and allosterically stabilizing the active kinase, so that derepression alone is insufficient for activation [PMID:26305940]. Direct binding of TgDJ-1 through an arginine-dyad phospho-gatekeeper motif further potentiates kinase activity in a calcium- and oxidation-sensitive manner and is required for normal microneme secretion [PMID:28246362]. Phosphoproteomic and bio-orthogonal substrate labeling identified 163 CDPK1-dependent substrates, including a HOOK activating adaptor that, together with an FHF-like complex, drives rapid apical microneme trafficking as parasites initiate motility [PMID:23530747, PMID:37933960]. Functionally, CDPK1 controls F-actin flux underlying motility [PMID:30753127], acts with partial redundancy in a signaling module with CDPK2A upstream of motility and egress [PMID:37610220], operates in a pathway distinct from CDPK3 [PMID:23149386], and negatively regulates dense granule secretion reciprocally with microneme discharge [PMID:30673152]. A unique glycine gatekeeper residue in the ATP-binding site makes TgCDPK1 exquisitely sensitive to bumped kinase inhibitors and analog-sensitive/chemical-genetic approaches [PMID:20436472, PMID:20173034], with TgMAPKL1 identified as a secondary BKI co-target [PMID:40407244].","teleology":[{"year":2001,"claim":"Established that a calcium-dependent kinase activity in Toxoplasma corresponds to TgCDPK1 and phosphorylates specific parasite substrates, linking it to motility and attachment.","evidence":"Biochemical fractionation, in vitro kinase assays, and KT5926 inhibition correlating in vitro and in vivo phosphoproteins in tachyzoites","pmids":["11154702"],"confidence":"Medium","gaps":["Substrate identities not molecularly defined","No genetic evidence for essentiality or in vivo function"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2001,"claim":"Placed CDPK1 downstream of a Ca2+ signal in the egress pathway, defining how an environmental trigger (low host [K+]) is transduced into parasite exit.","evidence":"Pharmacological manipulation of Ca2+, K+, and kinase inhibitors with egress assays","pmids":["11526113"],"confidence":"Medium","gaps":["The identity of the second parallel egress pathway not resolved","Inhibitor selectivity for CDPK1 not genetically confirmed at this stage"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2006,"claim":"Tested whether selective CDPK1 inhibition alone accounts for an imidazopyridine compound's antiparasitic potency, showing it does not and pointing to additional targets.","evidence":"Radiolabeled ligand binding, gatekeeper-pocket mutagenesis, and transgenic G128Q mutant growth assays","pmids":["16765465"],"confidence":"Medium","gaps":["Additional relevant targets of the compound not identified","Overexpression rather than allelic replacement limits interpretation"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2010,"claim":"Demonstrated genetically and chemical-genetically that CDPK1 is essential for calcium-dependent microneme secretion and hence motility, invasion, and egress.","evidence":"Conditional knockdown plus pyrazolopyrimidine inhibition with resistant gatekeeper-mutant rescue and secretion/motility/invasion/egress assays","pmids":["20485436"],"confidence":"High","gaps":["Direct substrates driving secretion not yet identified","Molecular link between kinase activity and vesicle discharge unknown"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2010,"claim":"Resolved the structural basis of Ca2+-signal transduction by CDPK1, showing CAD-mediated autoinhibition relieved by calcium-driven domain reorganization.","evidence":"X-ray crystallography of apo and Ca2+-bound TgCDPK1","pmids":["20436473"],"confidence":"High","gaps":["Whether derepression alone suffices for activation not addressed","Dynamics in solution not captured by static structures"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2010,"claim":"Defined the unique glycine gatekeeper as the structural determinant of BKI selectivity and validated CDPK1 as the in vivo BKI target.","evidence":"Co-crystal structures with BKIs, in vitro inhibition, and glycine-to-methionine gatekeeper mutant parasites","pmids":["20436472"],"confidence":"High","gaps":["Whether other kinases share BKI sensitivity not excluded","Relationship to microneme vs motility phenotypes not dissected here"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2010,"claim":"Provided evidence that CDPK1 acts in gliding motility independently of, or downstream of, microneme secretion.","evidence":"Analog-sensitive 1NM-PP1 inhibition with G128M mutant, motility and micronemal secretion assays","pmids":["20173034"],"confidence":"Medium","gaps":["Partial contradiction with other work reporting a secretion block","Molecular effectors of the motility role not defined"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2012,"claim":"Distinguished CDPK1 from CDPK3 as controllers of separate calcium pathways, with CDPK1 universally required for microneme secretion and invasion.","evidence":"Chemical-genetic inhibition of analog-sensitive CDPK1 and CDPK3 alleles with secretion, invasion, and egress assays and PKG activation","pmids":["23149386"],"confidence":"High","gaps":["Molecular basis for pathway divergence not defined","How PKG compensates for CDPK3 not mechanistically resolved"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2013,"claim":"Defined the CDPK1 substrate repertoire by identifying 163 substrates via bio-orthogonal thiophosphorylation, mapping the kinase's downstream reach.","evidence":"Analog-sensitive kinase with ATPgammaS analogue, phosphoproteomics, and in vitro/in vivo validation","pmids":["23530747"],"confidence":"High","gaps":["Functional roles of most substrates untested","Temporal order of phosphorylation events not resolved"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2015,"claim":"Revised the activation model, showing the CAD domain both inhibits and allosterically activates the kinase, and that derepression alone is insufficient.","evidence":"VHH antibody screening, crystal structure of the TgCDPK1-1B7 complex, molecular dynamics, and domain-deletion kinase assays","pmids":["26305940"],"confidence":"High","gaps":["Physiological modulators of allosteric stabilization not identified here","Therapeutic exploitation of the conformational epitope untested in vivo"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2016,"claim":"Confirmed CDPK1 essentiality by complete gene deletion, establishing it as a non-redundant requirement for parasite growth.","evidence":"CRISPR/Cas9 deletion with Cre-loxP marker recycling and plaque assays, obtainable only with complementation","pmids":["26755159"],"confidence":"Medium","gaps":["Stage-specific requirements not dissected","Essential substrate(s) not pinpointed"],"attribution":[{"species":"Toxoplasma gondii","strain":"RH; ME49"}]},{"year":2017,"claim":"Identified TgDJ-1 as a direct binding partner that potentiates CDPK1 activity in a redox- and calcium-sensitive manner required for microneme secretion.","evidence":"Crystal structure of TgDJ-1, co-immunoprecipitation, arginine-dyad mutagenesis, in vitro kinase assays, and TgDJ-1 knockout secretion assays","pmids":["28246362"],"confidence":"High","gaps":["How oxidation modulates the interaction mechanistically not fully resolved","Physiological trigger integrating redox and Ca2+ signals unknown"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2017,"claim":"Showed CDPK1 (with CDPK3) participates in dampening the cytosolic Ca2+ signal before egress, expanding its role beyond effector output to signal shaping.","evidence":"GCaMP6 live Ca2+ imaging with pharmacological CDPK1/CDPK3 inhibition","pmids":["27781359"],"confidence":"Medium","gaps":["Substrates mediating signal quenching not identified","Direct vs indirect contribution of CDPK1 not separated from CDPK3"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2019,"claim":"Placed CDPK1 as a regulator of F-actin flux underlying motility, invasion, and egress, acting alongside PKG and AKMT independently of microneme secretion.","evidence":"F-actin chromobody live imaging, formin disruption, and PKG/CDPK1 inhibitor epistasis with AKMT mutants","pmids":["30753127"],"confidence":"Medium","gaps":["Direct CDPK1 substrates controlling actin flux not identified","How phosphorylation and methylation inputs are integrated unclear"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2019,"claim":"Established that CDPK1 negatively regulates dense granule exocytosis in response to Ca2+, providing reciprocal control with microneme discharge.","evidence":"Dense granule secretion assays with Ca2+ manipulation and CDPK1/CDPK3 inhibition","pmids":["30673152"],"confidence":"Medium","gaps":["Substrates mediating dense granule suppression unknown","CDPK1-specific contribution not fully separated from CDPK3"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2020,"claim":"Identified CDPK1 as a cellular target of the compound ENH1 responsible for its egress block, while showing calcium-homeostasis and viability effects are CDPK1-independent.","evidence":"Thermal proteome profiling, in vitro kinase assay, sensitive vs resistant CDPK1 allele comparison, and egress assays","pmids":["32597628"],"confidence":"Medium","gaps":["Additional ENH1 targets not identified","Mechanistic basis of CDPK1-independent effects unknown"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2023,"claim":"Connected CDPK1 kinase activity to a concrete trafficking machine by identifying a HOOK adaptor substrate and an FHF-like complex driving rapid apical microneme delivery for motility initiation.","evidence":"Sub-minute phosphoproteomics, bio-orthogonal substrate labeling, conditional depletion of HOOK and FTS, and secretion/invasion/live imaging","pmids":["37933960"],"confidence":"High","gaps":["Motor/dynein activation steps downstream of HOOK phosphorylation not fully resolved","Roles of the two parasite-specific FHF components undefined"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2023,"claim":"Defined CDPK2A as functioning with partial redundancy upstream of or in parallel with CDPK1 in the motility/egress module.","evidence":"Analog-sensitive alleles, auxin-inducible degron depletion, and epistasis with secretion, motility, and egress assays","pmids":["37610220"],"confidence":"Medium","gaps":["Shared vs distinct substrates of CDPK1 and CDPK2A not defined","Precise epistatic order not fully resolved"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2025,"claim":"Revealed that BKIs dually target CDPK1 and TgMAPKL1, refining the mechanism of BKI action and resistance.","evidence":"Chemical mutagenesis forward screen, whole-genome sequencing, and site-directed CDPK1 (G128M)/TgMAPKL1 (L162Q) resistance mutation validation with inhibition assays","pmids":["40407244"],"confidence":"Medium","gaps":["Relative contribution of each kinase to killing not quantified","Whether TgMAPKL1 acts in the CDPK1 pathway unknown"],"attribution":[{"species":"Toxoplasma gondii","strain":"RH"}]},{"year":null,"claim":"The direct substrates linking CDPK1 to F-actin flux, dense granule suppression, and Ca2+ signal quenching, and how CDPK1 integrates with CDPK2A/CDPK3/PKG signaling, remain to be mechanistically defined.","evidence":"","pmids":[],"confidence":"Medium","gaps":["Effector substrates for actin regulation and secretion suppression unidentified","Integration among the CDPK1/CDPK2A/CDPK3/PKG modules not resolved","Physiological inputs controlling the TgDJ-1/redox potentiation unknown"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]}],"mechanism_profile":{"molecular_activity":[],"localization":[],"pathway":[],"complexes":["TgCDPK1-TgDJ-1 complex (direct binding via arginine-dyad phospho-gatekeeper motif)","TgCDPK1-1B7 VHH antibody inhibitory complex (experimental)","FHF-like complex (HOOK, FTS homolog, two parasite-specific proteins) acting downstream of CDPK1"],"partners":["TGDJ-1 (DIRECT BINDER, POTENTIATES KINASE ACTIVITY)","HOOK ACTIVATING ADAPTOR (SUBSTRATE)","CDPK2A (PARTIALLY REDUNDANT MODULE PARTNER)","CDPK3 (PARALLEL CALCIUM PATHWAY)","PKG (FUNCTIONAL EPISTASIS IN MOTILITY/EGRESS)","AKMT (CO-REGULATOR OF F-ACTIN FLUX)","TGMAPKL1 (SECONDARY BKI CO-TARGET)"],"other_free_text":["Essential kinase in T. gondii; complete gene deletion is not viable without complementation (RH; ME49).","Calcium-dependent activity independent of calmodulin and phospholipids.","Activation requires Ca2+-induced CAD reorganization plus allosteric stabilization, not mere derepression.","Unique glycine gatekeeper (G128) confers nanomolar BKI/analog-sensitive-inhibitor sensitivity; G128M/G128Q mutants confer resistance.","Identified as cellular target of antiparasitic compounds ENH1 and BKI-1748.","163 CDPK1-dependent phosphosubstrates identified."]},"strain_conflicts":[]},"prefetch_data":{},"corpus_meta":[{"pmid":"20485436","id":"PMC_20485436","title":"Calcium-dependent protein kinase 1 is an essential regulator of exocytosis in Toxoplasma.","date":"2010","source":"Nature","url":"https://pubmed.ncbi.nlm.nih.gov/20485436","citation_count":285,"is_preprint":false},{"pmid":"20436473","id":"PMC_20436473","title":"Structures of apicomplexan calcium-dependent protein kinases reveal mechanism of activation by calcium.","date":"2010","source":"Nature structural & molecular biology","url":"https://pubmed.ncbi.nlm.nih.gov/20436473","citation_count":178,"is_preprint":false},{"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":"20436472","id":"PMC_20436472","title":"Toxoplasma gondii calcium-dependent protein kinase 1 is a target for selective kinase inhibitors.","date":"2010","source":"Nature structural & molecular biology","url":"https://pubmed.ncbi.nlm.nih.gov/20436472","citation_count":161,"is_preprint":false},{"pmid":"11526113","id":"PMC_11526113","title":"The loss of cytoplasmic potassium upon host cell breakdown triggers egress of Toxoplasma gondii.","date":"2001","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/11526113","citation_count":161,"is_preprint":false},{"pmid":"11154702","id":"PMC_11154702","title":"Toxoplasma gondii attachment to host cells is regulated by a calmodulin-like domain protein kinase.","date":"2001","source":"The Journal of biological 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single lab but multiple orthogonal methods\",\n      \"pmids\": [\"11154702\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"Egress-triggering reduction in host cell [K+] activates a phospholipase C in Toxoplasma leading to increased cytoplasmic [Ca2+], which then activates TgCDPK1 as one of at least two downstream signaling pathways required for parasite egress; buffering cytoplasmic Ca2+ blocks egress and calcium ionophores bypass the need for low [K+].\",\n      \"method\": \"Pharmacological manipulation of Ca2+, potassium concentrations, and kinase inhibitors; egress assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — epistasis-level placement of CDPK1 downstream of Ca2+ signal in egress pathway, single lab, multiple pharmacological perturbations\",\n      \"pmids\": [\"11526113\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"TgCDPK1 is an essential regulator of calcium-dependent microneme exocytosis; conditional suppression of TgCDPK1 blocks calcium-dependent secretion from micronemes, abolishing parasite motility, host-cell invasion, and egress. A glycine gatekeeper mutant (resistant to pyrazolopyrimidine inhibitors) restored sensitivity reversal, confirming TgCDPK1 as the specific in vivo target.\",\n      \"method\": \"Conditional knockdown (promoter replacement), chemical-genetic inhibition with pyrazolopyrimidine compounds, mutant rescue experiment, microneme secretion assay, motility/invasion/egress assays\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — conditional genetic knockdown combined with chemical-genetic approach and resistant-mutant rescue, multiple orthogonal methods, replicated across approaches in single study\",\n      \"pmids\": [\"20485436\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Crystal structures of TgCDPK1 in autoinhibited (apo) and calcium-activated conformations reveal that the C-terminal CDPK activation domain (CAD) in the apo form contains a long N-terminal helix that inhibits the kinase domain analogously to CaMKII autoinhibition; calcium binding triggers major reorganization of the CAD to a position remote from the substrate-binding site, constituting a distinct mechanism of calcium-signal transduction.\",\n      \"method\": \"X-ray crystallography of apo and calcium-bound forms of TgCDPK1\",\n      \"journal\": \"Nature structural & molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structures of two conformational states with functional interpretation, rigorous structural method\",\n      \"pmids\": [\"20436473\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"TgCDPK1 contains a unique glycine gatekeeper residue in its ATP-binding site that confers nanomolar sensitivity to bumped kinase inhibitors (BKIs); co-crystal structures of TgCDPK1 with BKIs confirm the structural basis for selectivity, and T. gondii expressing a glycine-to-methionine gatekeeper mutant shows significantly decreased BKI sensitivity in vivo, confirming TgCDPK1 as the BKI target mediating early infection inhibition.\",\n      \"method\": \"Co-crystal structure determination, in vitro kinase inhibition assays, gatekeeper mutant parasite line generation and growth inhibition assays\",\n      \"journal\": \"Nature structural & molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — co-crystal structures plus in vivo genetic validation with gatekeeper mutant; two orthogonal methods in a single rigorous study\",\n      \"pmids\": [\"20436472\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Inhibition of TgCDPK1 by 1NM-PP1 blocks gliding motility without inhibiting micronemal secretion, suggesting TgCDPK1 has a role in gliding motility downstream of or independent of microneme secretion; a G128M gatekeeper mutation in TgCDPK1 confers resistance to 1NM-PP1.\",\n      \"method\": \"Chemical-genetic inhibition with analog-sensitive kinase approach, motility assay, micronemal secretion assay, site-directed mutagenesis\",\n      \"journal\": \"Eukaryotic cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — chemical-genetic approach with gatekeeper mutant validation; single lab but two distinct phenotypic readouts; note partial contradiction with other work on microneme secretion\",\n      \"pmids\": [\"20173034\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"TgCDPK1 and TgCDPK3 control distinct calcium signaling pathways: TgCDPK1 is required universally for microneme secretion and for invasion, whereas TgCDPK3 is required for ionophore-induced egress but not for invasion; cGMP-dependent protein kinase (PKG) activation can partially compensate for TgCDPK3 inhibition during egress.\",\n      \"method\": \"Chemical-genetic inhibition of analog-sensitive CDPK1 and CDPK3 alleles; microneme secretion assay; invasion assay; egress assay; pharmacological activation of PKG\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — chemical-genetic approach with orthogonal alleles for two kinases, multiple functional readouts, genetic epistasis established between CDPK1, CDPK3, and PKG\",\n      \"pmids\": [\"23149386\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Using an analog-sensitive TgCDPK1 allele and a synthetic ATPγS analogue complementary to the expanded glycine-gatekeeper ATP pocket, 163 CDPK1 substrates were identified by selective thiophosphorylation; predicted phosphorylation sites were concordant with TgCDPK1 in vitro substrate preference, and phosphorylation of identified substrates was confirmed to be CDPK1-dependent both in vitro and in vivo.\",\n      \"method\": \"Chemical-genetic substrate identification (analog-sensitive kinase + thiophosphate labeling), phosphoproteomics, in vitro kinase assays, in vivo validation\",\n      \"journal\": \"ACS chemical biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — bio-orthogonal substrate labeling combined with in vitro and in vivo validation; multiple orthogonal methods in a single rigorous study\",\n      \"pmids\": [\"23530747\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Removal of the autoinhibitory domain from TgCDPK1 alone is insufficient for kinase activation; the regulatory (CAD) domain plays a dual role, both inhibiting and allosterically activating the kinase domain in a Ca2+-dependent manner. A VHH antibody (1B7) that binds TgCDPK1 in a conformation-dependent manner potently inhibits it by disrupting this allosteric stabilization, as revealed by the crystal structure of the complex and molecular dynamics simulations.\",\n      \"method\": \"VHH antibody library screening, crystal structure of TgCDPK1-1B7 complex, molecular dynamics simulation, in vitro kinase activity assays with domain deletion mutants\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure plus molecular dynamics plus functional mutagenesis/domain deletion; multiple orthogonal methods revealing novel allosteric mechanism\",\n      \"pmids\": [\"26305940\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"CDPK1 is essential in T. gondii; CRISPR/Cas9-based complete gene deletion confirmed CDPK1 essentiality for parasite growth in vitro, as deletions could not be obtained without complementation.\",\n      \"method\": \"CRISPR/Cas9 gene deletion with Cre-loxP marker recycling, plaque assay\",\n      \"journal\": \"Infection and immunity\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — CRISPR/Cas9 gene deletion with clear essentiality phenotype; single lab, clean genetic approach\",\n      \"pmids\": [\"26755159\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH; ME49\"}]\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"TgDJ-1 directly binds to TgCDPK1 through an arginine dyad phospho-gatekeeper motif; the interaction is sensitive to oxidation and calcium, potentiates CDPK1 kinase activity, and is required for normal microneme exocytosis. Genetic deletion of TgDJ-1 upregulates CDPK1 expression, and disruption of the TgDJ-1/CDPK1 complex prevents microneme secretion in vivo.\",\n      \"method\": \"X-ray crystal structure of TgDJ-1, co-immunoprecipitation, in vitro kinase activity assays, site-directed mutagenesis of arginine dyad, genetic knockout of TgDJ-1, microneme secretion assay\",\n      \"journal\": \"mBio\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure of binding partner, direct binding assay, functional mutagenesis, genetic knockout with specific secretion phenotype; multiple orthogonal methods in single study\",\n      \"pmids\": [\"28246362\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"TgCDPK1 and TgCDPK3 contribute to signal quenching (Ca2+ signal dampening) before egress, as revealed by real-time Ca2+ imaging with the GCaMP6 biosensor; their inhibition alters the temporal dynamics of intracellular Ca2+ flux during egress.\",\n      \"method\": \"GCaMP6 genetically encoded Ca2+ biosensor live imaging, pharmacological inhibition of CDPK1 and CDPK3\",\n      \"journal\": \"Cellular microbiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — live Ca2+ imaging with genetically encoded biosensor, pharmacological inhibition; single lab, functional readout linked to signaling dynamics\",\n      \"pmids\": [\"27781359\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"TgCDPK1 controls F-actin flux required for motility, invasion, and egress; this flux depends on myosins A and H and is regulated by phosphorylation via PKG and CDPK1, and by methylation via AKMT. This F-actin flux is independent of microneme secretion and persists in the absence of the glideosome-associated connector (GAC).\",\n      \"method\": \"F-actin chromobody live imaging, formin gene disruption, epistasis with PKG and CDPK1 inhibitors, AKMT methyltransferase mutants\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — live F-actin imaging combined with genetic disruption and chemical-genetic epistasis; single lab, multiple orthogonal approaches\",\n      \"pmids\": [\"30753127\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"TgCDPK1 and TgCDPK3 mediate negative regulation of dense granule exocytosis by cytosolic Ca2+; elevated Ca2+ suppresses dense granule secretion in a manner contingent on CDPK1 and CDPK3 function, providing reciprocal control with microneme exocytosis.\",\n      \"method\": \"Dense granule secretion assays, Ca2+ manipulation, pharmacological inhibition of CDPK1 and CDPK3\",\n      \"journal\": \"Cellular microbiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — secretion assays with Ca2+ manipulation and kinase inhibition; single lab, two kinases tested; functional link established\",\n      \"pmids\": [\"30673152\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Thermal proteome profiling of Toxoplasma identified CDPK1 as a target of the antiparasitic compound ENH1; CDPK1 inhibition by ENH1 was confirmed in vitro and in parasites by comparison of sensitive vs. resistant CDPK1 alleles. CDPK1 inhibition explained the egress block, but ENH1 effects on calcium homeostasis and parasite viability were CDPK1-independent, implicating additional targets.\",\n      \"method\": \"Thermal proteome profiling (in situ target identification), in vitro kinase assay, resistant CDPK1 allele comparison, egress assay\",\n      \"journal\": \"ACS chemical biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — thermal proteome profiling plus allele-specific validation; single lab, novel target identification method with functional validation\",\n      \"pmids\": [\"32597628\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Sub-minute resolution phosphoproteomics and bio-orthogonal substrate labeling identified 163 proteins phosphorylated in a CDPK1-dependent manner in T. gondii. Among these, a HOOK activating adaptor is a CDPK1 substrate; CDPK1 kinase activity and HOOK are required for rapid apical trafficking of micronemes as parasites initiate motility. A partially conserved FHF complex (HOOK, FTS homolog, and two parasite-specific proteins) mediates this trafficking, and parasites lacking HOOK or FTS show impaired microneme secretion and a block in host cell invasion.\",\n      \"method\": \"Sub-minute resolution phosphoproteomics, bio-orthogonal kinase substrate labeling (analog-sensitive CDPK1), conditional genetic depletion of HOOK and FTS, microneme secretion assay, invasion assay, live imaging\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — phosphoproteomics combined with bio-orthogonal substrate labeling and genetic validation of downstream complex; multiple orthogonal methods revealing mechanistic pathway\",\n      \"pmids\": [\"37933960\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"CDPK2A and CDPK1 function in a signaling module upstream of Toxoplasma motility with partial functional redundancy; CDPK2A contributes to microneme discharge, gliding motility, and egress, and epistasis analysis using analog-sensitive alleles and conditional depletion revealed that CDPK2A acts upstream of or in parallel with CDPK1 in the egress/motility pathway.\",\n      \"method\": \"Analog-sensitive kinase alleles (chemical-genetic), conditional protein depletion (auxin-inducible degron), microneme secretion assay, motility assay, egress assay, epistasis analysis\",\n      \"journal\": \"mBio\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — chemical-genetic and conditional depletion approaches with epistasis; single lab, multiple orthogonal methods\",\n      \"pmids\": [\"37610220\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"TgCDPK1 binds a radiolabeled imidazopyridine ligand with sub-nanomolar affinity; mutational analysis of the catalytic site hydrophobic binding pocket (analogous to the gatekeeper residue in PKG) blocks ligand binding, and a compound-refractory CDPK1 mutant (G128Q) overexpressed in transgenic parasites does not reduce susceptibility to the imidazopyridine compound in vivo, indicating that selective inhibition of CDPK1 alone is not responsible for the enhanced anti-parasitic potency of this compound.\",\n      \"method\": \"Radiolabeled ligand binding assay with parasite extracts, site-directed mutagenesis, transgenic overexpression of mutant CDPK1, in vivo parasite growth assays\",\n      \"journal\": \"Molecular and biochemical parasitology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — binding assay with mutagenesis and transgenic validation; single lab, multiple approaches; negative result for selective CDPK1 inhibition as sole mechanism is itself mechanistically informative\",\n      \"pmids\": [\"16765465\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Forward genetic screen using chemical mutagenesis and BKI-1748 resistance selection identified TgMAPKL1 (in addition to TgCDPK1) as a target of BKIs; single mutations in TgMAPKL1 ATP binding site (L162Q) or TgCDPK1 gatekeeper (G128M) each confer 2–6-fold resistance, while combining both mutations yields up to 157-fold resistance, indicating that BKIs dually target both kinases.\",\n      \"method\": \"Chemical mutagenesis forward genetic screen, whole-genome sequencing of resistant clones, site-directed mutagenesis to introduce individual resistance mutations, in vitro kinase inhibition, parasite growth inhibition assays\",\n      \"journal\": \"ACS infectious diseases\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — forward genetic screen with genome sequencing and site-directed mutagenesis validation; single lab, orthogonal combination of genetics and biochemistry\",\n      \"pmids\": [\"40407244\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH\"}]\n    }\n  ],\n  \"current_model\": \"TgCDPK1 is an essential calcium-dependent serine/threonine kinase in Toxoplasma gondii that transduces intracellular Ca2+ signals into microneme exocytosis, gliding motility, host-cell invasion, and egress; its activation requires Ca2+-induced reorganization of the C-terminal CAD domain plus allosteric stabilization of the kinase domain (not mere derepression), it is potentiated by direct interaction with TgDJ-1, it phosphorylates at least 163 substrates including a HOOK adaptor that drives apical microneme trafficking via a parasite FHF-like dynein-activating complex, it negatively regulates dense granule secretion reciprocally with microneme discharge, it acts in a partially redundant module with CDPK2A upstream of motility, and its unique glycine gatekeeper residue makes it the primary cellular target of bumped kinase inhibitors (with TgMAPKL1 as a secondary co-target).\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"TgCDPK1 is an essential calcium-dependent serine/threonine protein kinase in Toxoplasma gondii that transduces intracellular Ca2+ signals into microneme exocytosis, gliding motility, host-cell invasion, and egress [#2, #9]. Its activity is calcium-dependent and independent of calmodulin or phospholipids [#0], and calcium is delivered to the kinase upstream via a phospholipase C-generated signal that couples egress triggers to Ca2+-activated CDPK1 [#1]. Activation is structurally governed by the C-terminal CDPK activation (CAD) domain: in the apo state an N-terminal CAD helix autoinhibits the kinase domain, and Ca2+ binding drives a large CAD reorganization that repositions it away from the substrate site [#3]; this CAD serves a dual role, both inhibiting and allosterically stabilizing the active kinase, so that derepression alone is insufficient for activation [#8]. Direct binding of TgDJ-1 through an arginine-dyad phospho-gatekeeper motif further potentiates kinase activity in a calcium- and oxidation-sensitive manner and is required for normal microneme secretion [#10]. Phosphoproteomic and bio-orthogonal substrate labeling identified 163 CDPK1-dependent substrates, including a HOOK activating adaptor that, together with an FHF-like complex, drives rapid apical microneme trafficking as parasites initiate motility [#7, #15]. Functionally, CDPK1 controls F-actin flux underlying motility [#12], acts with partial redundancy in a signaling module with CDPK2A upstream of motility and egress [#16], operates in a pathway distinct from CDPK3 [#6], and negatively regulates dense granule secretion reciprocally with microneme discharge [#13]. A unique glycine gatekeeper residue in the ATP-binding site makes TgCDPK1 exquisitely sensitive to bumped kinase inhibitors and analog-sensitive/chemical-genetic approaches [#4, #5], with TgMAPKL1 identified as a secondary BKI co-target [#18].\",\n  \"teleology\": [\n    {\n      \"year\": 2001,\n      \"claim\": \"Established that a calcium-dependent kinase activity in Toxoplasma corresponds to TgCDPK1 and phosphorylates specific parasite substrates, linking it to motility and attachment.\",\n      \"evidence\": \"Biochemical fractionation, in vitro kinase assays, and KT5926 inhibition correlating in vitro and in vivo phosphoproteins in tachyzoites\",\n      \"pmids\": [\"11154702\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Substrate identities not molecularly defined\", \"No genetic evidence for essentiality or in vivo function\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Placed CDPK1 downstream of a Ca2+ signal in the egress pathway, defining how an environmental trigger (low host [K+]) is transduced into parasite exit.\",\n      \"evidence\": \"Pharmacological manipulation of Ca2+, K+, and kinase inhibitors with egress assays\",\n      \"pmids\": [\"11526113\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"The identity of the second parallel egress pathway not resolved\", \"Inhibitor selectivity for CDPK1 not genetically confirmed at this stage\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Tested whether selective CDPK1 inhibition alone accounts for an imidazopyridine compound's antiparasitic potency, showing it does not and pointing to additional targets.\",\n      \"evidence\": \"Radiolabeled ligand binding, gatekeeper-pocket mutagenesis, and transgenic G128Q mutant growth assays\",\n      \"pmids\": [\"16765465\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Additional relevant targets of the compound not identified\", \"Overexpression rather than allelic replacement limits interpretation\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Demonstrated genetically and chemical-genetically that CDPK1 is essential for calcium-dependent microneme secretion and hence motility, invasion, and egress.\",\n      \"evidence\": \"Conditional knockdown plus pyrazolopyrimidine inhibition with resistant gatekeeper-mutant rescue and secretion/motility/invasion/egress assays\",\n      \"pmids\": [\"20485436\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct substrates driving secretion not yet identified\", \"Molecular link between kinase activity and vesicle discharge unknown\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Resolved the structural basis of Ca2+-signal transduction by CDPK1, showing CAD-mediated autoinhibition relieved by calcium-driven domain reorganization.\",\n      \"evidence\": \"X-ray crystallography of apo and Ca2+-bound TgCDPK1\",\n      \"pmids\": [\"20436473\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether derepression alone suffices for activation not addressed\", \"Dynamics in solution not captured by static structures\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Defined the unique glycine gatekeeper as the structural determinant of BKI selectivity and validated CDPK1 as the in vivo BKI target.\",\n      \"evidence\": \"Co-crystal structures with BKIs, in vitro inhibition, and glycine-to-methionine gatekeeper mutant parasites\",\n      \"pmids\": [\"20436472\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether other kinases share BKI sensitivity not excluded\", \"Relationship to microneme vs motility phenotypes not dissected here\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Provided evidence that CDPK1 acts in gliding motility independently of, or downstream of, microneme secretion.\",\n      \"evidence\": \"Analog-sensitive 1NM-PP1 inhibition with G128M mutant, motility and micronemal secretion assays\",\n      \"pmids\": [\"20173034\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Partial contradiction with other work reporting a secretion block\", \"Molecular effectors of the motility role not defined\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Distinguished CDPK1 from CDPK3 as controllers of separate calcium pathways, with CDPK1 universally required for microneme secretion and invasion.\",\n      \"evidence\": \"Chemical-genetic inhibition of analog-sensitive CDPK1 and CDPK3 alleles with secretion, invasion, and egress assays and PKG activation\",\n      \"pmids\": [\"23149386\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular basis for pathway divergence not defined\", \"How PKG compensates for CDPK3 not mechanistically resolved\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Defined the CDPK1 substrate repertoire by identifying 163 substrates via bio-orthogonal thiophosphorylation, mapping the kinase's downstream reach.\",\n      \"evidence\": \"Analog-sensitive kinase with ATPgammaS analogue, phosphoproteomics, and in vitro/in vivo validation\",\n      \"pmids\": [\"23530747\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional roles of most substrates untested\", \"Temporal order of phosphorylation events not resolved\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Revised the activation model, showing the CAD domain both inhibits and allosterically activates the kinase, and that derepression alone is insufficient.\",\n      \"evidence\": \"VHH antibody screening, crystal structure of the TgCDPK1-1B7 complex, molecular dynamics, and domain-deletion kinase assays\",\n      \"pmids\": [\"26305940\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Physiological modulators of allosteric stabilization not identified here\", \"Therapeutic exploitation of the conformational epitope untested in vivo\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Confirmed CDPK1 essentiality by complete gene deletion, establishing it as a non-redundant requirement for parasite growth.\",\n      \"evidence\": \"CRISPR/Cas9 deletion with Cre-loxP marker recycling and plaque assays, obtainable only with complementation\",\n      \"pmids\": [\"26755159\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Stage-specific requirements not dissected\", \"Essential substrate(s) not pinpointed\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH; ME49\"}]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Identified TgDJ-1 as a direct binding partner that potentiates CDPK1 activity in a redox- and calcium-sensitive manner required for microneme secretion.\",\n      \"evidence\": \"Crystal structure of TgDJ-1, co-immunoprecipitation, arginine-dyad mutagenesis, in vitro kinase assays, and TgDJ-1 knockout secretion assays\",\n      \"pmids\": [\"28246362\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How oxidation modulates the interaction mechanistically not fully resolved\", \"Physiological trigger integrating redox and Ca2+ signals unknown\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Showed CDPK1 (with CDPK3) participates in dampening the cytosolic Ca2+ signal before egress, expanding its role beyond effector output to signal shaping.\",\n      \"evidence\": \"GCaMP6 live Ca2+ imaging with pharmacological CDPK1/CDPK3 inhibition\",\n      \"pmids\": [\"27781359\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Substrates mediating signal quenching not identified\", \"Direct vs indirect contribution of CDPK1 not separated from CDPK3\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Placed CDPK1 as a regulator of F-actin flux underlying motility, invasion, and egress, acting alongside PKG and AKMT independently of microneme secretion.\",\n      \"evidence\": \"F-actin chromobody live imaging, formin disruption, and PKG/CDPK1 inhibitor epistasis with AKMT mutants\",\n      \"pmids\": [\"30753127\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct CDPK1 substrates controlling actin flux not identified\", \"How phosphorylation and methylation inputs are integrated unclear\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Established that CDPK1 negatively regulates dense granule exocytosis in response to Ca2+, providing reciprocal control with microneme discharge.\",\n      \"evidence\": \"Dense granule secretion assays with Ca2+ manipulation and CDPK1/CDPK3 inhibition\",\n      \"pmids\": [\"30673152\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Substrates mediating dense granule suppression unknown\", \"CDPK1-specific contribution not fully separated from CDPK3\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Identified CDPK1 as a cellular target of the compound ENH1 responsible for its egress block, while showing calcium-homeostasis and viability effects are CDPK1-independent.\",\n      \"evidence\": \"Thermal proteome profiling, in vitro kinase assay, sensitive vs resistant CDPK1 allele comparison, and egress assays\",\n      \"pmids\": [\"32597628\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Additional ENH1 targets not identified\", \"Mechanistic basis of CDPK1-independent effects unknown\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Connected CDPK1 kinase activity to a concrete trafficking machine by identifying a HOOK adaptor substrate and an FHF-like complex driving rapid apical microneme delivery for motility initiation.\",\n      \"evidence\": \"Sub-minute phosphoproteomics, bio-orthogonal substrate labeling, conditional depletion of HOOK and FTS, and secretion/invasion/live imaging\",\n      \"pmids\": [\"37933960\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Motor/dynein activation steps downstream of HOOK phosphorylation not fully resolved\", \"Roles of the two parasite-specific FHF components undefined\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Defined CDPK2A as functioning with partial redundancy upstream of or in parallel with CDPK1 in the motility/egress module.\",\n      \"evidence\": \"Analog-sensitive alleles, auxin-inducible degron depletion, and epistasis with secretion, motility, and egress assays\",\n      \"pmids\": [\"37610220\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Shared vs distinct substrates of CDPK1 and CDPK2A not defined\", \"Precise epistatic order not fully resolved\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Revealed that BKIs dually target CDPK1 and TgMAPKL1, refining the mechanism of BKI action and resistance.\",\n      \"evidence\": \"Chemical mutagenesis forward screen, whole-genome sequencing, and site-directed CDPK1 (G128M)/TgMAPKL1 (L162Q) resistance mutation validation with inhibition assays\",\n      \"pmids\": [\"40407244\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Relative contribution of each kinase to killing not quantified\", \"Whether TgMAPKL1 acts in the CDPK1 pathway unknown\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH\"}]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The direct substrates linking CDPK1 to F-actin flux, dense granule suppression, and Ca2+ signal quenching, and how CDPK1 integrates with CDPK2A/CDPK3/PKG signaling, remain to be mechanistically defined.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Effector substrates for actin regulation and secretion suppression unidentified\", \"Integration among the CDPK1/CDPK2A/CDPK3/PKG modules not resolved\", \"Physiological inputs controlling the TgDJ-1/redox potentiation unknown\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0004683\", \"supporting_discovery_ids\": [0, 2, 7], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]},\n      {\"term_id\": \"GO:0005509\", \"supporting_discovery_ids\": [3, 8], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]},\n      {\"term_id\": \"GO:0004674\", \"supporting_discovery_ids\": [7, 15], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]},\n      {\"term_id\": \"GO:0005524\", \"supporting_discovery_ids\": [4, 17], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]}\n    ],\n    \"localization\": [],\n    \"pathway\": [\n      {\"term_id\": \"GO:0060402\", \"supporting_discovery_ids\": [1, 2, 11], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]},\n      {\"term_id\": \"GO:0006887\", \"supporting_discovery_ids\": [2, 10, 13, 15], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]},\n      {\"term_id\": \"GO:0071702\", \"supporting_discovery_ids\": [15], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]},\n      {\"term_id\": \"GO:0030048\", \"supporting_discovery_ids\": [12], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]},\n      {\"term_id\": \"GO:0052126\", \"supporting_discovery_ids\": [2, 6], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]}\n    ],\n    \"complexes\": [\n      \"TgCDPK1-TgDJ-1 complex (direct binding via arginine-dyad phospho-gatekeeper motif)\",\n      \"TgCDPK1-1B7 VHH antibody inhibitory complex (experimental)\",\n      \"FHF-like complex (HOOK, FTS homolog, two parasite-specific proteins) acting downstream of CDPK1\"\n    ],\n    \"partners\": [\n      \"TgDJ-1 (direct binder, potentiates kinase activity)\",\n      \"HOOK activating adaptor (substrate)\",\n      \"CDPK2A (partially redundant module partner)\",\n      \"CDPK3 (parallel calcium pathway)\",\n      \"PKG (functional epistasis in motility/egress)\",\n      \"AKMT (co-regulator of F-actin flux)\",\n      \"TgMAPKL1 (secondary BKI co-target)\"\n    ],\n    \"other_free_text\": [\n      \"Essential kinase in T. gondii; complete gene deletion is not viable without complementation (RH; ME49).\",\n      \"Calcium-dependent activity independent of calmodulin and phospholipids.\",\n      \"Activation requires Ca2+-induced CAD reorganization plus allosteric stabilization, not mere derepression.\",\n      \"Unique glycine gatekeeper (G128) confers nanomolar BKI/analog-sensitive-inhibitor sensitivity; G128M/G128Q mutants confer resistance.\",\n      \"Identified as cellular target of antiparasitic compounds ENH1 and BKI-1748.\",\n      \"163 CDPK1-dependent phosphosubstrates identified.\"\n    ]\n  },\n  \"strain_conflicts\": []\n}","audit_flag":null,"strains":[{"strain":"ME49","gene_id":"TGME49_301440","name":"CDPK1","product":"calcium-dependent protein kinase CDPK1","uniprot_acc":"A0A125YH30","chromosome":"TGME49_chrIV","start_pos":2479620},{"strain":"GT1","gene_id":"TGGT1_301440","name":null,"product":"calcium-dependent protein kinase CDPK1","uniprot_acc":null,"chromosome":"TGGT1_chrIV","start_pos":2366836},{"strain":"RH88","gene_id":"TGRH88_009540","name":null,"product":"calcium-dependent protein kinase CDPK1","uniprot_acc":null,"chromosome":"CM023085","start_pos":2426034},{"strain":"VEG","gene_id":"TGVEG_301440","name":null,"product":"calcium-dependent protein kinase CDPK1","uniprot_acc":"A0A125YH31","chromosome":"TGVEG_chrIV","start_pos":2515865}]}