Affinage · Toxoplasma gondii

PKG

protein kinase G AGC kinase family member PKG · UniProt A0A125YJK5

Annotated
2026-09-08
21 papers in source corpus 12 papers cited in narrative 12 extracted findings

Mechanistic narrative

Synthesis pass · prose summary of the discoveries below

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).

Mechanistic history

Synthesis pass · year-by-year structured walk · 11 steps
  1. 2002 High

    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

    PMID:12455981

    Open questions at the time
    • Downstream substrates and cellular processes not yet defined
    • Isoform architecture not yet resolved
  2. 2002 High

    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

    PMID:11897122

    Open questions at the time
    • Functional distinction between isoforms not yet established
    • Physiological role of the apicomplexa-specific third cGMP-binding site incompletely defined
  3. 2006 Medium

    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

    PMID:16765465

    Open questions at the time
    • No crystal structure of the inhibitor-bound kinase in this work
    • Selectivity across the full apicomplexan kinome not addressed
  4. 2016 High

    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

    PMID:26933037 PMID:26933036

    Open questions at the time
    • Molecular identity of the neutral Ca2+ store not established
    • Direct PKG substrates linking cGMP to secretion not identified
  5. 2017 High

    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

    PMID:28465425

    Open questions at the time
    • Why cytosolic PKGII is retained is unexplained
    • Local membrane substrates not defined
  6. 2017 High

    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

    PMID:29030485

    Open questions at the time
    • Identity/regulation of the implicated cGMP-PDE not fully defined
    • Direct PKA-PKG connecting substrates not mapped
  7. 2019 Medium

    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

    PMID:30753127

    Open questions at the time
    • Direct PKG substrates within the actin-flux pathway not identified
    • Mechanism relating flux control to secretion-independent motility unresolved
  8. 2019 Medium

    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

    PMID:31235476

    Open questions at the time
    • How the P4-ATPase domain contributes to cyclase regulation not resolved
    • Direct biochemical coupling to PKG not shown
  9. 2022 Medium

    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

    PMID:35976251

    Open questions at the time
    • Individual substrates not independently validated as direct PKG targets
    • Direct versus indirect phosphorylation events not distinguished
  10. 2024 Medium

    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

    PMID:39136687

    Open questions at the time
    • Whether SPARK phosphorylates PKG directly not established
    • Mechanism of PKG destabilization upon SPARK loss unknown
  11. 2025 Medium

    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

    PMID:39903669

    Open questions at the time
    • Molecular targets of PKAc1 between cGMP and Ca2+ not identified
    • Direct components of the PKG-CDPK feedback loop not defined

Open questions

Synthesis pass · forward-looking unresolved questions
  • 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.
  • 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

Mechanism profile

Synthesis pass · controlled-vocabulary classification
Partners
CDPKS / CDPK1 (POSITIVE-FEEDBACK PARTNERS IN MOTILITY AND F-ACTIN FLUX)FRM1 (FORMIN INITIATING APICAL F-ACTIN FLUX)PKAC1 (ANTAGONISTIC KINASE ACTING BETWEEN CGMP AND CA2+ RELEASE)SPARK (PDK1 ORTHOLOG, PKG PROTEIN-STABILITY REGULATOR)TGATPASEP-GC (UPSTREAM GUANYLATE CYCLASE / CGMP SOURCE)

Evidence

Reading pass · 12 per-paper findings extracted from the source corpus
Year Finding Method Journal Conf PMIDs
2002 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. Genetic disruption with trans-complementation; active-site mutagenesis; recombinant expression; in vitro kinase assay; mouse infection model Eukaryotic cell High 12455981
2002 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. Targeted mutagenesis of translational initiation sites; metabolic labeling; immunofluorescence; in vitro kinase activity assays with transiently transfected parasites Molecular and biochemical parasitology High 11897122
2017 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. Auxin-inducible degron (AID) conditional depletion; CRISPR-Cas9 genome editing; isoform-specific rescue constructs; N-terminal mutagenesis; invasion/egress assays mBio High 28465425
2016 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. Genetically encoded Ca2+ indicators (GCaMPs); genetically encoded microneme secretion reporter; pharmacological manipulation (zaprinast, PKG inhibitor); conditional PKG mutant parasites The Journal of biological chemistry High 26933037
2016 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. Genetically encoded Ca2+ indicator (GCaMP) cell-based screen; pharmacological inhibition with zaprinast; compound library screening; Ca2+ store characterization The Journal of biological chemistry Medium 26933036
2017 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. Conditional knockdown (PKAr, PKAc1); phosphoproteomics; pharmacological inhibition of PKG; PDE inhibitor treatment; egress assays The EMBO journal High 29030485
2019 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. F-actin chromobody live imaging; gene disruption of FRM1, myosins A/H; pharmacological inhibition of PKG and CDPK1; motility/invasion/egress assays eLife Medium 30753127
2019 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. CRISPR/Cas9-assisted disruption; Cre/loxP conditional knockdown; cGMP measurement; phenotypic comparison of TgATPaseP-GC and TgPKG knockdown parasites; IFA localization Life science alliance Medium 31235476
2024 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. Conditional genetics (SPARK depletion); unbiased quantitative proteomics; epistasis analysis of PKG/PKA protein levels after SPARK loss eLife Medium 39136687
2025 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. Conditional genetic manipulation of PKAc1, PKG, CDPKs; pharmacological perturbation of cGMP and Ca2+; epistasis analysis; egress/motility assays Cell reports Medium 39903669
2006 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. Radiolabeled ligand binding assay with parasite extracts; amino acid sequence comparison; site-directed mutagenesis; transgenic parasite lines expressing compound-refractory PKG (T761Q-KO) Molecular and biochemical parasitology Medium 16765465
2022 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. Time-resolved phosphoproteomics after PKG activation; thermal proteome profiling eLife Medium 35976251

Source papers

Stage 0 corpus · 21 papers · ranked by NIH iCite citations
Year Title Journal Citations PMID
2017 Plasma Membrane Association by N-Acylation Governs PKG Function in Toxoplasma gondii. mBio 154 28465425
2004 Protein kinases as targets for anti-parasitic chemotherapy. Biochimica et biophysica acta 113 15023358
2002 Toxoplasma gondii cyclic GMP-dependent kinase: chemotherapeutic targeting of an essential parasite protein kinase. Eukaryotic cell 109 12455981
2006 Anticoccidial kinase inhibitors: identification of protein kinase targets secondary to cGMP-dependent protein kinase. Molecular and biochemical parasitology 87 16765465
2017 Crosstalk between PKA and PKG controls pH-dependent host cell egress of Toxoplasma gondii. The EMBO journal 83 29030485
2015 Identification of potent phosphodiesterase inhibitors that demonstrate cyclic nucleotide-dependent functions in apicomplexan parasites. ACS chemical biology 80 25555060
2019 Three F-actin assembly centers regulate organelle inheritance, cell-cell communication and motility in Toxoplasma gondii. eLife 68 30753127
2016 Serum Albumin Stimulates Protein Kinase G-dependent Microneme Secretion in Toxoplasma gondii. The Journal of biological chemistry 59 26933037
2016 Using a Genetically Encoded Sensor to Identify Inhibitors of Toxoplasma gondii Ca2+ Signaling. The Journal of biological chemistry 54 26933036
2002 Molecular characterization of a coccidian parasite cGMP dependent protein kinase. Molecular and biochemical parasitology 54 11897122
2021 Protein kinases in Toxoplasma gondii. International journal for parasitology 35 33581139
2022 Temporal and thermal profiling of the Toxoplasma proteome implicates parasite Protein Phosphatase 1 in the regulation of Ca2+-responsive pathways. eLife 24 35976251
2019 An unusual and vital protein with guanylate cyclase and P4-ATPase domains in a pathogenic protist. Life science alliance 22 31235476
2019 iTRAQ-Based Global Phosphoproteomics Reveals Novel Molecular Differences Between Toxoplasma gondii Strains of Different Genotypes. Frontiers in cellular and infection microbiology 17 31508380
2018 Effects of Estradiol and Progesterone-Induced Intracellular Calcium Fluxes on Toxoplasma gondii Gliding, Microneme Secretion, and Egress. Frontiers in microbiology 17 29946311
2020 The protozoan parasite Toxoplasma gondii encodes a gamut of phosphodiesterases during its lytic cycle in human cells. Computational and structural biotechnology journal 9 33335684
2016 PKA and Apicomplexan Parasite Diseases. Hormone and metabolic research = Hormon- und Stoffwechselforschung = Hormones et metabolisme 9 27835919
2024 SPARK regulates AGC kinases central to the Toxoplasma gondii asexual cycle. eLife 5 39136687
2025 Phospho-relay feedback loops control egress vs. intracellular development in Toxoplasma gondii. Cell reports 3 39903669
2024 SPARK regulates AGC kinases central to the Toxoplasma gondii asexual cycle. bioRxiv : the preprint server for biology 1 37961644
2017 AID-ing Signaling in Toxoplasma gondii. mBio 0 28743818