{"gene":"TG_311360","gene_symbol":"PKG","run_date":"2026-09-08T15:15:13","timeline":{"discoveries":[{"year":2002,"finding":"T. gondii PKG is an essential protein; genetic disruption of the PKG locus is only achievable when a complementing copy is provided in trans. A catalytic site substitution (T761Q or T761M) renders PKG refractory to compound 1 (trisubstituted pyrrole inhibitor) without affecting other kinase properties, confirming PKG as the primary molecular target of this antiparasitic compound.","method":"Genetic disruption with trans-complementation; active-site mutagenesis; recombinant expression; in vitro kinase assay; mouse infection model","journal":"Eukaryotic cell","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — active-site mutagenesis combined with genetic complementation, recombinant enzyme characterization, and in vivo validation in mouse model","pmids":["12455981"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2002,"finding":"T. gondii PKG exists as two isoforms: isoform-I (~110 kDa, membrane-associated) and isoform-II (slightly smaller, cytosolic). Isoform-II arises from alternative translational initiation at an internal methionine codon. Membrane association of isoform-I is mediated by N-terminal myristoylation at G2 and palmitoylation at C4. A putative third allosteric cGMP-binding site unique to apicomplexan PKGs is functionally significant; mutation of this site markedly reduces cGMP-dependent kinase activity in transiently transfected parasites.","method":"Targeted mutagenesis of translational initiation sites; metabolic labeling; immunofluorescence; in vitro kinase activity assays with transiently transfected parasites","journal":"Molecular and biochemical parasitology","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — multiple orthogonal methods (mutagenesis, metabolic labeling, IFA, kinase assay) in a single rigorous study","pmids":["11897122"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""},{"species":"Eimeria tenella","strain":""}]},{"year":2017,"finding":"PKGI (plasma membrane-associated isoform) is necessary and fully sufficient for all PKG-dependent cellular processes (microneme secretion, motility, invasion, egress), whereas PKGII (cytosolic isoform) is functionally insufficient and dispensable when PKGI is present. The first 15 residues of PKGI, including a myristoylated Gly2, are critical for membrane association and PKG function; swapping this N-terminal region abolishes function.","method":"Auxin-inducible degron (AID) conditional depletion; CRISPR-Cas9 genome editing; isoform-specific rescue constructs; N-terminal mutagenesis; invasion/egress assays","journal":"mBio","confidence":"High","confidence_rationale":"Tier 2 / Strong — conditional genetic depletion with domain-swap rescue and mutagenesis, multiple cellular phenotype readouts, single lab with multiple orthogonal methods","pmids":["28465425"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2016,"finding":"Microneme secretion in T. gondii is centrally controlled by PKG in a cGMP-dependent manner. Serum albumin, a host protein, triggers microneme secretion by elevating cGMP and activating PKG independently of elevated intracellular Ca2+; Ca2+ elevation augments but is not required for PKG-dependent secretion. PKG also controls a downstream Ca2+ release from a neutral intracellular store distinct from the ER.","method":"Genetically encoded Ca2+ indicators (GCaMPs); genetically encoded microneme secretion reporter; pharmacological manipulation (zaprinast, PKG inhibitor); conditional PKG mutant parasites","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal genetic and pharmacological approaches; genetically encoded reporters provide direct functional readout","pmids":["26933037"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2016,"finding":"PKG controls a Ca2+ pool derived from a neutral intracellular store (distinct from the ER) in T. gondii. Two novel ATP-mimetic compounds were identified as PKG inhibitors that prevent zaprinast-induced cytosolic Ca2+ elevation.","method":"Genetically encoded Ca2+ indicator (GCaMP) cell-based screen; pharmacological inhibition with zaprinast; compound library screening; Ca2+ store characterization","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — single lab, genetically encoded reporters plus pharmacological validation, but Ca2+ store identity based on indirect pharmacology","pmids":["26933036"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2017,"finding":"PKG acts downstream of PKA in controlling egress: inhibition of PKG blocks premature egress induced by PKAc1 inactivation or environmental acidification. PKAc1 inactivation alters phosphorylation of a putative cGMP-phosphodiesterase, and a cGMP-PDE inhibitor circumvents egress repression by PKAc1 or pH neutralization, placing PKA-cGMP-PKG in a crosstalk pathway governing egress.","method":"Conditional knockdown (PKAr, PKAc1); phosphoproteomics; pharmacological inhibition of PKG; PDE inhibitor treatment; egress assays","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 2 / Strong — conditional genetics, phosphoproteomics, and pharmacological epistasis combined to place PKG downstream of PKA/cGMP axis in egress control","pmids":["29030485"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2019,"finding":"PKG (together with CDPK1) controls phosphorylation-dependent regulation of the FRM1-initiated apical F-actin flux essential for T. gondii motility, invasion, and egress. This flux is independent of microneme secretion.","method":"F-actin chromobody live imaging; gene disruption of FRM1, myosins A/H; pharmacological inhibition of PKG and CDPK1; motility/invasion/egress assays","journal":"eLife","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — pharmacological and genetic epistasis used to place PKG in actin flux regulation, single lab, multiple orthogonal readouts","pmids":["30753127"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2019,"finding":"The upstream guanylate cyclase TgATPaseP-GC (a plasma membrane-localized protein with coupled P4-ATPase and guanylate cyclase domains) is required for cGMP synthesis; Cre/loxP-mediated knockdown of TgATPaseP-GC reduces cGMP levels and recapitulates the motility-defective egress/invasion phenotypes of TgPKG knockdown, placing TgATPaseP-GC upstream of PKG in the cGMP signaling pathway.","method":"CRISPR/Cas9-assisted disruption; Cre/loxP conditional knockdown; cGMP measurement; phenotypic comparison of TgATPaseP-GC and TgPKG knockdown parasites; IFA localization","journal":"Life science alliance","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — parallel conditional knockdowns with cGMP measurement establish epistasis; single lab","pmids":["31235476"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2024,"finding":"The PDK1 ortholog SPARK regulates the stability of PKG (and PKA) protein levels in T. gondii; SPARK depletion leads to down-regulation of PKG and PKA before phenotypic defects appear, placing SPARK upstream of PKG in the AGC kinase signaling hierarchy.","method":"Conditional genetics (SPARK depletion); unbiased quantitative proteomics; epistasis analysis of PKG/PKA protein levels after SPARK loss","journal":"eLife","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — conditional depletion with proteomics establishes SPARK-PKG hierarchy; single lab, not independently replicated","pmids":["39136687"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2025,"finding":"PKG operates within a positive feedback loop with CDPKs to control motility, invasion, and egress in T. gondii. PKAc1 counteracts this PKG/CDPK pathway. PKAc1 acts on targets between guanylate cyclase and Ca2+ release (i.e., upstream of Ca2+ release but downstream of cGMP production), as shown by perturbation of cGMP vs. Ca2+ in the context of PKAc1 manipulation. PKAc1 also activates PDE2 to consume cAMP in a negative feedback loop that transiently limits its own activity.","method":"Conditional genetic manipulation of PKAc1, PKG, CDPKs; pharmacological perturbation of cGMP and Ca2+; epistasis analysis; egress/motility assays","journal":"Cell reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — conditional genetics and pharmacological epistasis establish pathway topology; single lab, recent single study","pmids":["39903669"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2006,"finding":"The catalytic site hydrophobic pocket of T. gondii PKG mediates binding of imidazopyridine inhibitors through specific contacts analogous to those in CDPK1; analogous amino acid substitutions in this pocket (e.g., T761Q) abolish compound binding to both PKG and CDPK1, establishing the structural basis of inhibitor selectivity.","method":"Radiolabeled ligand binding assay with parasite extracts; amino acid sequence comparison; site-directed mutagenesis; transgenic parasite lines expressing compound-refractory PKG (T761Q-KO)","journal":"Molecular and biochemical parasitology","confidence":"Medium","confidence_rationale":"Tier 1–2 / Moderate — mutagenesis and binding assay define catalytic site contacts; single lab","pmids":["16765465"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2022,"finding":"Time-resolved phosphoproteomics following PKG activation mapped the downstream waves of phosphoregulation in T. gondii, identifying specific proteins phosphorylated upon PKG-dependent signaling. This approach corroborated known physiological roles of PKG and identified novel substrates operating in these pathways.","method":"Time-resolved phosphoproteomics after PKG activation; thermal proteome profiling","journal":"eLife","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — phosphoproteomics with PKG activation provides substrate landscape; single lab, no independent validation of individual substrates in this abstract","pmids":["35976251"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]}],"current_model":"In Toxoplasma gondii, PKG is an essential cGMP-dependent serine/threonine kinase whose activity is restricted to the plasma membrane via N-terminal myristoylation (Gly2) and palmitoylation (Cys4) of its PKGI isoform; this membrane localization—regulated upstream by the guanylate cyclase TgATPaseP-GC and the AGC kinase SPARK—is absolutely required for PKG to drive microneme secretion, F-actin flux, motility, invasion, and egress through a positive feedback loop with CDPKs, while PKAc1 antagonizes this pathway by acting between cGMP production and Ca2+ release, and a transient PKAc1-dependent negative feedback loop on cAMP ensures that parasites remain responsive to environmental signals while sustaining stable intracellular replication."},"narrative":{"mechanistic_narrative":"PKG is an essential cGMP-dependent serine/threonine protein kinase that serves as the master regulator of the lytic cycle in Toxoplasma gondii, driving microneme secretion, motility, host-cell invasion, and egress [PMID:12455981, PMID:26933037]. The gene is expressed as two isoforms generated by alternative translational initiation: a membrane-associated isoform-I (~110 kDa) and a slightly smaller cytosolic isoform-II; membrane targeting of isoform-I depends on N-terminal myristoylation at Gly2 and palmitoylation at Cys4 [PMID:11897122]. Plasma-membrane-associated PKGI is necessary and fully sufficient for all PKG-dependent processes, whereas the cytosolic isoform is functionally insufficient, establishing that spatial restriction to the plasma membrane defines PKG's signaling output [PMID:28465425]. Upstream, the plasma-membrane guanylate cyclase TgATPaseP-GC generates the cGMP that activates PKG, and its loss phenocopies PKG depletion [PMID:31235476]; the PDK1 ortholog SPARK sustains PKG protein stability [PMID:39136687]. Activated PKG triggers Ca2+ release from a neutral intracellular store distinct from the ER and can drive microneme secretion independently of a rise in cytosolic Ca2+, with a host serum-albumin cue elevating cGMP to initiate this cascade [PMID:26933037, PMID:26933036]. PKG additionally governs FRM1-initiated apical F-actin flux together with CDPK1, a function independent of microneme secretion [PMID:30753127], and operates within a positive-feedback loop with CDPKs that is antagonized by PKAc1 acting between cGMP production and Ca2+ release [PMID:29030485, PMID:39903669]. Time-resolved phosphoproteomics has mapped the downstream waves of PKG-dependent phosphoregulation and identified its substrate landscape [PMID:35976251]. A catalytic-site threonine (Thr761) forms the hydrophobic-pocket contact that makes PKG the selective molecular target of trisubstituted pyrrole and imidazopyridine antiparasitic inhibitors [PMID:12455981, PMID:16765465].","teleology":[{"year":2002,"claim":"Establishing that PKG is essential and is the direct target of an antiparasitic compound converted a candidate kinase into a validated drug target and a genetically tractable pathway node.","evidence":"Genetic disruption with trans-complementation, active-site mutagenesis (T761Q/T761M), recombinant kinase assay, and mouse infection in T. gondii","pmids":["12455981"],"confidence":"High","gaps":["Downstream substrates and cellular processes not yet defined","Isoform architecture not yet resolved"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2002,"claim":"Resolving that PKG exists as membrane-associated and cytosolic isoforms via alternative translational initiation and defining lipid-based membrane targeting explained how PKG activity is spatially organized.","evidence":"Translational initiation-site mutagenesis, metabolic labeling, IFA, and kinase assays in T. gondii and Eimeria tenella","pmids":["11897122"],"confidence":"High","gaps":["Functional distinction between isoforms not yet established","Physiological role of the apicomplexa-specific third cGMP-binding site incompletely defined"],"attribution":[{"species":"Toxoplasma gondii","strain":""},{"species":"Eimeria tenella","strain":""}]},{"year":2006,"claim":"Mapping the catalytic-site hydrophobic pocket contacts shared with CDPK1 explained the structural basis of inhibitor selectivity and cross-sensitivity.","evidence":"Radiolabeled ligand binding, sequence comparison, and T761Q mutant transgenic lines in T. gondii","pmids":["16765465"],"confidence":"Medium","gaps":["No crystal structure of the inhibitor-bound kinase in this work","Selectivity across the full apicomplexan kinome not addressed"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2016,"claim":"Placing PKG at the center of microneme secretion and identifying a Ca2+-independent secretory branch redefined the signaling hierarchy, showing cGMP/PKG can act upstream and partly independently of Ca2+.","evidence":"Genetically encoded Ca2+ and secretion reporters, pharmacology (zaprinast, PKG inhibitor), and conditional PKG mutants in T. gondii","pmids":["26933037","26933036"],"confidence":"High","gaps":["Molecular identity of the neutral Ca2+ store not established","Direct PKG substrates linking cGMP to secretion not identified"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2017,"claim":"Isoform-specific rescue proved that plasma-membrane PKGI alone drives all PKG functions, establishing that membrane localization is the functional determinant.","evidence":"Auxin-inducible degron depletion, CRISPR editing, isoform-specific and N-terminal domain-swap rescue with invasion/egress assays in T. gondii","pmids":["28465425"],"confidence":"High","gaps":["Why cytosolic PKGII is retained is unexplained","Local membrane substrates not defined"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2017,"claim":"Epistasis positioning PKG downstream of a PKA-cGMP-PDE axis in egress control revealed inter-kinase crosstalk governing when parasites exit the host cell.","evidence":"Conditional PKAr/PKAc1 knockdown, phosphoproteomics, PKG inhibition, and PDE inhibitor treatment with egress assays in T. gondii","pmids":["29030485"],"confidence":"High","gaps":["Identity/regulation of the implicated cGMP-PDE not fully defined","Direct PKA-PKG connecting substrates not mapped"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2019,"claim":"Linking PKG (with CDPK1) to FRM1-initiated apical F-actin flux showed PKG controls the cytoskeletal machinery of motility independently of microneme secretion.","evidence":"F-actin chromobody live imaging, gene disruption of FRM1/myosins, and pharmacological inhibition of PKG/CDPK1 in T. gondii","pmids":["30753127"],"confidence":"Medium","gaps":["Direct PKG substrates within the actin-flux pathway not identified","Mechanism relating flux control to secretion-independent motility unresolved"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2019,"claim":"Identifying the plasma-membrane guanylate cyclase TgATPaseP-GC as the cGMP source placed a defined enzyme immediately upstream of PKG in the signaling cascade.","evidence":"CRISPR disruption, Cre/loxP conditional knockdown, cGMP measurement, and phenotypic comparison to PKG knockdown in T. gondii","pmids":["31235476"],"confidence":"Medium","gaps":["How the P4-ATPase domain contributes to cyclase regulation not resolved","Direct biochemical coupling to PKG not shown"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2022,"claim":"Time-resolved phosphoproteomics after PKG activation provided the first systematic substrate landscape, corroborating known roles and nominating new effectors.","evidence":"Time-resolved phosphoproteomics and thermal proteome profiling following PKG activation in T. gondii","pmids":["35976251"],"confidence":"Medium","gaps":["Individual substrates not independently validated as direct PKG targets","Direct versus indirect phosphorylation events not distinguished"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2024,"claim":"Showing that the PDK1 ortholog SPARK maintains PKG (and PKA) protein levels added an upstream stability-control layer to the AGC kinase hierarchy.","evidence":"Conditional SPARK depletion with unbiased quantitative proteomics and epistasis analysis in T. gondii","pmids":["39136687"],"confidence":"Medium","gaps":["Whether SPARK phosphorylates PKG directly not established","Mechanism of PKG destabilization upon SPARK loss unknown"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2025,"claim":"Defining a PKG/CDPK positive-feedback loop antagonized by PKAc1 acting between cGMP production and Ca2+ release integrated the pathway topology governing egress and motility.","evidence":"Conditional genetics of PKAc1/PKG/CDPKs and pharmacological perturbation of cGMP and Ca2+ with epistasis in T. gondii","pmids":["39903669"],"confidence":"Medium","gaps":["Molecular targets of PKAc1 between cGMP and Ca2+ not identified","Direct components of the PKG-CDPK feedback loop not defined"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":null,"claim":"The direct PKG substrates that mechanistically couple cGMP to Ca2+ release, the neutral Ca2+ store, and F-actin flux remain to be defined at biochemical resolution.","evidence":"","pmids":[],"confidence":"Medium","gaps":["Identity of the neutral, non-ER Ca2+ store unresolved","No validated direct substrate list linking PKG to secretion/motility","Structural model of the full-length activated kinase absent"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]}],"mechanism_profile":{"molecular_activity":[],"localization":[],"pathway":[],"complexes":[],"partners":["TGATPASEP-GC (UPSTREAM GUANYLATE CYCLASE / CGMP SOURCE)","SPARK (PDK1 ORTHOLOG, PKG PROTEIN-STABILITY REGULATOR)","CDPKS / CDPK1 (POSITIVE-FEEDBACK PARTNERS IN MOTILITY AND F-ACTIN FLUX)","PKAC1 (ANTAGONISTIC KINASE ACTING BETWEEN CGMP AND CA2+ RELEASE)","FRM1 (FORMIN INITIATING APICAL F-ACTIN FLUX)"],"other_free_text":["Two isoforms arise by alternative translational initiation; PKGI is membrane-targeted by N-terminal myristoylation (Gly2) and palmitoylation (Cys4) and is necessary and sufficient for function, while cytosolic PKGII is dispensable.","Catalytic-site residue Thr761 defines sensitivity to trisubstituted pyrrole (compound 1) and imidazopyridine inhibitors; T761Q/T761M substitutions confer resistance.","PKG activation triggers Ca2+ release from a neutral intracellular store distinct from the ER and can drive microneme secretion independently of cytosolic Ca2+ elevation, with host serum albumin acting as an upstream cGMP-elevating cue.","An apicomplexa-specific third allosteric cGMP-binding site contributes to cGMP-dependent kinase activity."]},"strain_conflicts":[]},"prefetch_data":{},"corpus_meta":[{"pmid":"28465425","id":"PMC_28465425","title":"Plasma Membrane Association by N-Acylation Governs PKG Function in Toxoplasma gondii.","date":"2017","source":"mBio","url":"https://pubmed.ncbi.nlm.nih.gov/28465425","citation_count":154,"is_preprint":false},{"pmid":"15023358","id":"PMC_15023358","title":"Protein kinases as targets for anti-parasitic chemotherapy.","date":"2004","source":"Biochimica et biophysica acta","url":"https://pubmed.ncbi.nlm.nih.gov/15023358","citation_count":113,"is_preprint":false},{"pmid":"12455981","id":"PMC_12455981","title":"Toxoplasma gondii cyclic GMP-dependent kinase: chemotherapeutic targeting of an essential parasite protein 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parasitology","url":"https://pubmed.ncbi.nlm.nih.gov/11897122","citation_count":54,"is_preprint":false},{"pmid":"26933036","id":"PMC_26933036","title":"Using a Genetically Encoded Sensor to Identify Inhibitors of Toxoplasma gondii Ca2+ Signaling.","date":"2016","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/26933036","citation_count":54,"is_preprint":false},{"pmid":"33581139","id":"PMC_33581139","title":"Protein kinases in Toxoplasma gondii.","date":"2021","source":"International journal for parasitology","url":"https://pubmed.ncbi.nlm.nih.gov/33581139","citation_count":35,"is_preprint":false},{"pmid":"35976251","id":"PMC_35976251","title":"Temporal and thermal profiling of the Toxoplasma proteome implicates parasite Protein Phosphatase 1 in the regulation of Ca2+-responsive pathways.","date":"2022","source":"eLife","url":"https://pubmed.ncbi.nlm.nih.gov/35976251","citation_count":24,"is_preprint":false},{"pmid":"31235476","id":"PMC_31235476","title":"An unusual and vital protein with guanylate cyclase and P4-ATPase domains in a pathogenic protist.","date":"2019","source":"Life science alliance","url":"https://pubmed.ncbi.nlm.nih.gov/31235476","citation_count":22,"is_preprint":false},{"pmid":"29946311","id":"PMC_29946311","title":"Effects of Estradiol and Progesterone-Induced Intracellular Calcium Fluxes on Toxoplasma gondii Gliding, Microneme Secretion, and Egress.","date":"2018","source":"Frontiers in microbiology","url":"https://pubmed.ncbi.nlm.nih.gov/29946311","citation_count":17,"is_preprint":false},{"pmid":"31508380","id":"PMC_31508380","title":"iTRAQ-Based Global Phosphoproteomics Reveals Novel Molecular Differences Between Toxoplasma gondii Strains of Different Genotypes.","date":"2019","source":"Frontiers in cellular and infection 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gondii.","date":"2017","source":"mBio","url":"https://pubmed.ncbi.nlm.nih.gov/28743818","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":11933,"output_tokens":3556,"usd":0.044569,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":9691,"output_tokens":4895,"usd":0.085415,"stage2_stop_reason":"end_turn"},"total_usd":0.129984,"stage1_batch_id":"msgbatch_01S6GjoYQm8LTPUsdhASdqag","stage2_batch_id":"msgbatch_01VX63RTMdBJqsG6gQMqoCjz","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2002,\n      \"finding\": \"T. gondii PKG is an essential protein; genetic disruption of the PKG locus is only achievable when a complementing copy is provided in trans. A catalytic site substitution (T761Q or T761M) renders PKG refractory to compound 1 (trisubstituted pyrrole inhibitor) without affecting other kinase properties, confirming PKG as the primary molecular target of this antiparasitic compound.\",\n      \"method\": \"Genetic disruption with trans-complementation; active-site mutagenesis; recombinant expression; in vitro kinase assay; mouse infection model\",\n      \"journal\": \"Eukaryotic cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — active-site mutagenesis combined with genetic complementation, recombinant enzyme characterization, and in vivo validation in mouse model\",\n      \"pmids\": [\"12455981\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"T. gondii PKG exists as two isoforms: isoform-I (~110 kDa, membrane-associated) and isoform-II (slightly smaller, cytosolic). Isoform-II arises from alternative translational initiation at an internal methionine codon. Membrane association of isoform-I is mediated by N-terminal myristoylation at G2 and palmitoylation at C4. A putative third allosteric cGMP-binding site unique to apicomplexan PKGs is functionally significant; mutation of this site markedly reduces cGMP-dependent kinase activity in transiently transfected parasites.\",\n      \"method\": \"Targeted mutagenesis of translational initiation sites; metabolic labeling; immunofluorescence; in vitro kinase activity assays with transiently transfected parasites\",\n      \"journal\": \"Molecular and biochemical parasitology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — multiple orthogonal methods (mutagenesis, metabolic labeling, IFA, kinase assay) in a single rigorous study\",\n      \"pmids\": [\"11897122\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}, {\"species\": \"Eimeria tenella\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"PKGI (plasma membrane-associated isoform) is necessary and fully sufficient for all PKG-dependent cellular processes (microneme secretion, motility, invasion, egress), whereas PKGII (cytosolic isoform) is functionally insufficient and dispensable when PKGI is present. The first 15 residues of PKGI, including a myristoylated Gly2, are critical for membrane association and PKG function; swapping this N-terminal region abolishes function.\",\n      \"method\": \"Auxin-inducible degron (AID) conditional depletion; CRISPR-Cas9 genome editing; isoform-specific rescue constructs; N-terminal mutagenesis; invasion/egress assays\",\n      \"journal\": \"mBio\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — conditional genetic depletion with domain-swap rescue and mutagenesis, multiple cellular phenotype readouts, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"28465425\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Microneme secretion in T. gondii is centrally controlled by PKG in a cGMP-dependent manner. Serum albumin, a host protein, triggers microneme secretion by elevating cGMP and activating PKG independently of elevated intracellular Ca2+; Ca2+ elevation augments but is not required for PKG-dependent secretion. PKG also controls a downstream Ca2+ release from a neutral intracellular store distinct from the ER.\",\n      \"method\": \"Genetically encoded Ca2+ indicators (GCaMPs); genetically encoded microneme secretion reporter; pharmacological manipulation (zaprinast, PKG inhibitor); conditional PKG mutant parasites\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal genetic and pharmacological approaches; genetically encoded reporters provide direct functional readout\",\n      \"pmids\": [\"26933037\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"PKG controls a Ca2+ pool derived from a neutral intracellular store (distinct from the ER) in T. gondii. Two novel ATP-mimetic compounds were identified as PKG inhibitors that prevent zaprinast-induced cytosolic Ca2+ elevation.\",\n      \"method\": \"Genetically encoded Ca2+ indicator (GCaMP) cell-based screen; pharmacological inhibition with zaprinast; compound library screening; Ca2+ store characterization\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — single lab, genetically encoded reporters plus pharmacological validation, but Ca2+ store identity based on indirect pharmacology\",\n      \"pmids\": [\"26933036\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"PKG acts downstream of PKA in controlling egress: inhibition of PKG blocks premature egress induced by PKAc1 inactivation or environmental acidification. PKAc1 inactivation alters phosphorylation of a putative cGMP-phosphodiesterase, and a cGMP-PDE inhibitor circumvents egress repression by PKAc1 or pH neutralization, placing PKA-cGMP-PKG in a crosstalk pathway governing egress.\",\n      \"method\": \"Conditional knockdown (PKAr, PKAc1); phosphoproteomics; pharmacological inhibition of PKG; PDE inhibitor treatment; egress assays\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — conditional genetics, phosphoproteomics, and pharmacological epistasis combined to place PKG downstream of PKA/cGMP axis in egress control\",\n      \"pmids\": [\"29030485\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"PKG (together with CDPK1) controls phosphorylation-dependent regulation of the FRM1-initiated apical F-actin flux essential for T. gondii motility, invasion, and egress. This flux is independent of microneme secretion.\",\n      \"method\": \"F-actin chromobody live imaging; gene disruption of FRM1, myosins A/H; pharmacological inhibition of PKG and CDPK1; motility/invasion/egress assays\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — pharmacological and genetic epistasis used to place PKG in actin flux regulation, single lab, multiple orthogonal readouts\",\n      \"pmids\": [\"30753127\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"The upstream guanylate cyclase TgATPaseP-GC (a plasma membrane-localized protein with coupled P4-ATPase and guanylate cyclase domains) is required for cGMP synthesis; Cre/loxP-mediated knockdown of TgATPaseP-GC reduces cGMP levels and recapitulates the motility-defective egress/invasion phenotypes of TgPKG knockdown, placing TgATPaseP-GC upstream of PKG in the cGMP signaling pathway.\",\n      \"method\": \"CRISPR/Cas9-assisted disruption; Cre/loxP conditional knockdown; cGMP measurement; phenotypic comparison of TgATPaseP-GC and TgPKG knockdown parasites; IFA localization\",\n      \"journal\": \"Life science alliance\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — parallel conditional knockdowns with cGMP measurement establish epistasis; single lab\",\n      \"pmids\": [\"31235476\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"The PDK1 ortholog SPARK regulates the stability of PKG (and PKA) protein levels in T. gondii; SPARK depletion leads to down-regulation of PKG and PKA before phenotypic defects appear, placing SPARK upstream of PKG in the AGC kinase signaling hierarchy.\",\n      \"method\": \"Conditional genetics (SPARK depletion); unbiased quantitative proteomics; epistasis analysis of PKG/PKA protein levels after SPARK loss\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — conditional depletion with proteomics establishes SPARK-PKG hierarchy; single lab, not independently replicated\",\n      \"pmids\": [\"39136687\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"PKG operates within a positive feedback loop with CDPKs to control motility, invasion, and egress in T. gondii. PKAc1 counteracts this PKG/CDPK pathway. PKAc1 acts on targets between guanylate cyclase and Ca2+ release (i.e., upstream of Ca2+ release but downstream of cGMP production), as shown by perturbation of cGMP vs. Ca2+ in the context of PKAc1 manipulation. PKAc1 also activates PDE2 to consume cAMP in a negative feedback loop that transiently limits its own activity.\",\n      \"method\": \"Conditional genetic manipulation of PKAc1, PKG, CDPKs; pharmacological perturbation of cGMP and Ca2+; epistasis analysis; egress/motility assays\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — conditional genetics and pharmacological epistasis establish pathway topology; single lab, recent single study\",\n      \"pmids\": [\"39903669\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"The catalytic site hydrophobic pocket of T. gondii PKG mediates binding of imidazopyridine inhibitors through specific contacts analogous to those in CDPK1; analogous amino acid substitutions in this pocket (e.g., T761Q) abolish compound binding to both PKG and CDPK1, establishing the structural basis of inhibitor selectivity.\",\n      \"method\": \"Radiolabeled ligand binding assay with parasite extracts; amino acid sequence comparison; site-directed mutagenesis; transgenic parasite lines expressing compound-refractory PKG (T761Q-KO)\",\n      \"journal\": \"Molecular and biochemical parasitology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — mutagenesis and binding assay define catalytic site contacts; single lab\",\n      \"pmids\": [\"16765465\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Time-resolved phosphoproteomics following PKG activation mapped the downstream waves of phosphoregulation in T. gondii, identifying specific proteins phosphorylated upon PKG-dependent signaling. This approach corroborated known physiological roles of PKG and identified novel substrates operating in these pathways.\",\n      \"method\": \"Time-resolved phosphoproteomics after PKG activation; thermal proteome profiling\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — phosphoproteomics with PKG activation provides substrate landscape; single lab, no independent validation of individual substrates in this abstract\",\n      \"pmids\": [\"35976251\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    }\n  ],\n  \"current_model\": \"In Toxoplasma gondii, PKG is an essential cGMP-dependent serine/threonine kinase whose activity is restricted to the plasma membrane via N-terminal myristoylation (Gly2) and palmitoylation (Cys4) of its PKGI isoform; this membrane localization—regulated upstream by the guanylate cyclase TgATPaseP-GC and the AGC kinase SPARK—is absolutely required for PKG to drive microneme secretion, F-actin flux, motility, invasion, and egress through a positive feedback loop with CDPKs, while PKAc1 antagonizes this pathway by acting between cGMP production and Ca2+ release, and a transient PKAc1-dependent negative feedback loop on cAMP ensures that parasites remain responsive to environmental signals while sustaining stable intracellular replication.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"PKG is an essential cGMP-dependent serine/threonine protein kinase that serves as the master regulator of the lytic cycle in Toxoplasma gondii, driving microneme secretion, motility, host-cell invasion, and egress [#0, #3]. The gene is expressed as two isoforms generated by alternative translational initiation: a membrane-associated isoform-I (~110 kDa) and a slightly smaller cytosolic isoform-II; membrane targeting of isoform-I depends on N-terminal myristoylation at Gly2 and palmitoylation at Cys4 [#1]. Plasma-membrane-associated PKGI is necessary and fully sufficient for all PKG-dependent processes, whereas the cytosolic isoform is functionally insufficient, establishing that spatial restriction to the plasma membrane defines PKG's signaling output [#2]. Upstream, the plasma-membrane guanylate cyclase TgATPaseP-GC generates the cGMP that activates PKG, and its loss phenocopies PKG depletion [#7]; the PDK1 ortholog SPARK sustains PKG protein stability [#8]. Activated PKG triggers Ca2+ release from a neutral intracellular store distinct from the ER and can drive microneme secretion independently of a rise in cytosolic Ca2+, with a host serum-albumin cue elevating cGMP to initiate this cascade [#3, #4]. PKG additionally governs FRM1-initiated apical F-actin flux together with CDPK1, a function independent of microneme secretion [#6], and operates within a positive-feedback loop with CDPKs that is antagonized by PKAc1 acting between cGMP production and Ca2+ release [#5, #9]. Time-resolved phosphoproteomics has mapped the downstream waves of PKG-dependent phosphoregulation and identified its substrate landscape [#11]. A catalytic-site threonine (Thr761) forms the hydrophobic-pocket contact that makes PKG the selective molecular target of trisubstituted pyrrole and imidazopyridine antiparasitic inhibitors [#0, #10].\"\n,\n  \"teleology\": [\n    {\n      \"year\": 2002,\n      \"claim\": \"Establishing that PKG is essential and is the direct target of an antiparasitic compound converted a candidate kinase into a validated drug target and a genetically tractable pathway node.\",\n      \"evidence\": \"Genetic disruption with trans-complementation, active-site mutagenesis (T761Q/T761M), recombinant kinase assay, and mouse infection in T. gondii\",\n      \"pmids\": [\"12455981\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Downstream substrates and cellular processes not yet defined\", \"Isoform architecture not yet resolved\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Resolving that PKG exists as membrane-associated and cytosolic isoforms via alternative translational initiation and defining lipid-based membrane targeting explained how PKG activity is spatially organized.\",\n      \"evidence\": \"Translational initiation-site mutagenesis, metabolic labeling, IFA, and kinase assays in T. gondii and Eimeria tenella\",\n      \"pmids\": [\"11897122\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional distinction between isoforms not yet established\", \"Physiological role of the apicomplexa-specific third cGMP-binding site incompletely defined\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}, {\"species\": \"Eimeria tenella\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Mapping the catalytic-site hydrophobic pocket contacts shared with CDPK1 explained the structural basis of inhibitor selectivity and cross-sensitivity.\",\n      \"evidence\": \"Radiolabeled ligand binding, sequence comparison, and T761Q mutant transgenic lines in T. gondii\",\n      \"pmids\": [\"16765465\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No crystal structure of the inhibitor-bound kinase in this work\", \"Selectivity across the full apicomplexan kinome not addressed\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Placing PKG at the center of microneme secretion and identifying a Ca2+-independent secretory branch redefined the signaling hierarchy, showing cGMP/PKG can act upstream and partly independently of Ca2+.\",\n      \"evidence\": \"Genetically encoded Ca2+ and secretion reporters, pharmacology (zaprinast, PKG inhibitor), and conditional PKG mutants in T. gondii\",\n      \"pmids\": [\"26933037\", \"26933036\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular identity of the neutral Ca2+ store not established\", \"Direct PKG substrates linking cGMP to secretion not identified\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Isoform-specific rescue proved that plasma-membrane PKGI alone drives all PKG functions, establishing that membrane localization is the functional determinant.\",\n      \"evidence\": \"Auxin-inducible degron depletion, CRISPR editing, isoform-specific and N-terminal domain-swap rescue with invasion/egress assays in T. gondii\",\n      \"pmids\": [\"28465425\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Why cytosolic PKGII is retained is unexplained\", \"Local membrane substrates not defined\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Epistasis positioning PKG downstream of a PKA-cGMP-PDE axis in egress control revealed inter-kinase crosstalk governing when parasites exit the host cell.\",\n      \"evidence\": \"Conditional PKAr/PKAc1 knockdown, phosphoproteomics, PKG inhibition, and PDE inhibitor treatment with egress assays in T. gondii\",\n      \"pmids\": [\"29030485\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Identity/regulation of the implicated cGMP-PDE not fully defined\", \"Direct PKA-PKG connecting substrates not mapped\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Linking PKG (with CDPK1) to FRM1-initiated apical F-actin flux showed PKG controls the cytoskeletal machinery of motility independently of microneme secretion.\",\n      \"evidence\": \"F-actin chromobody live imaging, gene disruption of FRM1/myosins, and pharmacological inhibition of PKG/CDPK1 in T. gondii\",\n      \"pmids\": [\"30753127\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct PKG substrates within the actin-flux pathway not identified\", \"Mechanism relating flux control to secretion-independent motility unresolved\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Identifying the plasma-membrane guanylate cyclase TgATPaseP-GC as the cGMP source placed a defined enzyme immediately upstream of PKG in the signaling cascade.\",\n      \"evidence\": \"CRISPR disruption, Cre/loxP conditional knockdown, cGMP measurement, and phenotypic comparison to PKG knockdown in T. gondii\",\n      \"pmids\": [\"31235476\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"How the P4-ATPase domain contributes to cyclase regulation not resolved\", \"Direct biochemical coupling to PKG not shown\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Time-resolved phosphoproteomics after PKG activation provided the first systematic substrate landscape, corroborating known roles and nominating new effectors.\",\n      \"evidence\": \"Time-resolved phosphoproteomics and thermal proteome profiling following PKG activation in T. gondii\",\n      \"pmids\": [\"35976251\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Individual substrates not independently validated as direct PKG targets\", \"Direct versus indirect phosphorylation events not distinguished\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Showing that the PDK1 ortholog SPARK maintains PKG (and PKA) protein levels added an upstream stability-control layer to the AGC kinase hierarchy.\",\n      \"evidence\": \"Conditional SPARK depletion with unbiased quantitative proteomics and epistasis analysis in T. gondii\",\n      \"pmids\": [\"39136687\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether SPARK phosphorylates PKG directly not established\", \"Mechanism of PKG destabilization upon SPARK loss unknown\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Defining a PKG/CDPK positive-feedback loop antagonized by PKAc1 acting between cGMP production and Ca2+ release integrated the pathway topology governing egress and motility.\",\n      \"evidence\": \"Conditional genetics of PKAc1/PKG/CDPKs and pharmacological perturbation of cGMP and Ca2+ with epistasis in T. gondii\",\n      \"pmids\": [\"39903669\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular targets of PKAc1 between cGMP and Ca2+ not identified\", \"Direct components of the PKG-CDPK feedback loop not defined\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The direct PKG substrates that mechanistically couple cGMP to Ca2+ release, the neutral Ca2+ store, and F-actin flux remain to be defined at biochemical resolution.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Identity of the neutral, non-ER Ca2+ store unresolved\", \"No validated direct substrate list linking PKG to secretion/motility\", \"Structural model of the full-length activated kinase absent\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0004692\", \"supporting_discovery_ids\": [0, 1, 3], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]},\n      {\"term_id\": \"GO:0004672\", \"supporting_discovery_ids\": [0, 11], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]},\n      {\"term_id\": \"GO:0030553\", \"supporting_discovery_ids\": [1], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [1, 2, 7], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [1, 2], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"GO:0007165\", \"supporting_discovery_ids\": [3, 5, 7, 9], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]},\n      {\"term_id\": \"GO:0006874\", \"supporting_discovery_ids\": [3, 4], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]},\n      {\"term_id\": \"GO:0030050\", \"supporting_discovery_ids\": [6], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\n      \"TgATPaseP-GC (upstream guanylate cyclase / cGMP source)\",\n      \"SPARK (PDK1 ortholog, PKG protein-stability regulator)\",\n      \"CDPKs / CDPK1 (positive-feedback partners in motility and F-actin flux)\",\n      \"PKAc1 (antagonistic kinase acting between cGMP and Ca2+ release)\",\n      \"FRM1 (formin initiating apical F-actin flux)\"\n    ],\n    \"other_free_text\": [\n      \"Two isoforms arise by alternative translational initiation; PKGI is membrane-targeted by N-terminal myristoylation (Gly2) and palmitoylation (Cys4) and is necessary and sufficient for function, while cytosolic PKGII is dispensable.\",\n      \"Catalytic-site residue Thr761 defines sensitivity to trisubstituted pyrrole (compound 1) and imidazopyridine inhibitors; T761Q/T761M substitutions confer resistance.\",\n      \"PKG activation triggers Ca2+ release from a neutral intracellular store distinct from the ER and can drive microneme secretion independently of cytosolic Ca2+ elevation, with host serum albumin acting as an upstream cGMP-elevating cue.\",\n      \"An apicomplexa-specific third allosteric cGMP-binding site contributes to cGMP-dependent kinase activity.\"\n    ]\n  },\n  \"strain_conflicts\": []\n}","audit_flag":null,"strains":[{"strain":"ME49","gene_id":"TGME49_311360","name":"PKG","product":"protein kinase G AGC kinase family member PKG","uniprot_acc":"A0A125YJK5","chromosome":"TGME49_chrXI","start_pos":1992306},{"strain":"GT1","gene_id":"TGGT1_311360","name":null,"product":"protein kinase G AGC kinase family member PKG","uniprot_acc":null,"chromosome":"TGGT1_chrXI","start_pos":1956120},{"strain":"RH88","gene_id":"TGRH88_050830","name":null,"product":"protein kinase G AGC kinase family member PKG","uniprot_acc":null,"chromosome":"CM023092","start_pos":2041767},{"strain":"VEG","gene_id":"TGVEG_311360","name":null,"product":"protein kinase G AGC kinase family member PKG","uniprot_acc":"B6K8K4","chromosome":"TGVEG_chrXI","start_pos":2004899}]}