SPARK

SPARK is a PDK1-like AGC kinase in Toxoplasma gondii that sits at the top of an AGC-kinase signaling hierarchy controlling the parasite's lytic cycle and life-stage transitions (PMID:35484233, PMID:39136687). Despite homology to mammalian PDK1, SPARK lacks the lipid-binding domain of its mammalian counterpart, indicating a rewired pathway in the parasite (PMID:35484233). Conditional depletion impairs host-cell invasion and egress and perturbs intracellular Ca2+ stores (PMID:35484233). Mechanistically, SPARK complexes with an elongin-like protein to maintain the stability of the downstream kinases PKA and PKG, and the invasion/egress defects of SPARK-depleted parasites develop only after PKG and PKA are down-regulated, placing SPARK upstream of both (PMID:39136687). Loss of SPARK also drives differentiation from the replicative tachyzoite into the chronic bradyzoite form, a phenotype linked to reduced activity of a coccidian-specific PKA ortholog acting downstream of SPARK (PMID:39136687).

1. 2022 High

   Establishing that SPARK is a functional kinase required for the lytic cycle answered whether this uncharacterized PDK1-homolog contributes to parasite biology, revealing a role in invasion, egress, and Ca2+ store homeostasis.

   Evidence High-throughput auxin-inducible degron (HiT) tagging with pooled/arrayed fitness screening and phenotypic invasion/egress and Ca2+ assays in Toxoplasma gondii

   PMID:35484233

   Open questions at the time

   • Direct kinase substrates of SPARK not identified
   • How SPARK regulates Ca2+ stores mechanistically is unresolved
   • Absence of a lipid-binding domain implies an unknown mode of activation
2. 2024 High

   Placing SPARK within the AGC signaling hierarchy answered how its phenotypes arise, showing it complexes with an elongin-like protein to stabilize PKA and PKG and thereby acts upstream of both kinases.

   Evidence Conditional depletion, unbiased quantitative proteomics, and Co-IP/complex identification with epistasis analysis in Toxoplasma gondii

   PMID:39136687

   Open questions at the time

   • Whether SPARK directly phosphorylates PKA/PKG or acts only through stabilization is not distinguished
   • The identity and role of the elongin-like partner in the complex is not fully defined
   • Structural basis of the SPARK-elongin complex unknown
3. 2024 Medium

   Testing SPARK's role in life-stage transition answered whether the kinase governs chronic-form conversion, showing its loss triggers bradyzoite differentiation likely via reduced coccidian-specific PKA activity.

   Evidence Conditional SPARK depletion and differentiation assays in Toxoplasma gondii

   PMID:39136687

   Open questions at the time

   • Mechanistic link to the coccidian-specific PKA ortholog is inferred from proteomics/epistasis and not reconstituted
   • Whether differentiation is a direct consequence of PKA loss or a secondary stress response is unclear

• The direct catalytic substrates of SPARK and the biochemical mechanism by which it activates or stabilizes PKA/PKG in the absence of a lipid-binding domain remain unresolved.
• No direct phosphorylation substrate mapped
• No structural or biochemical reconstitution of SPARK activity
• Mode of SPARK activation without a lipid-binding domain unknown