CRK1

TgCrk1 is a CDK-related kinase that drives daughter bud assembly during Toxoplasma gondii tachyzoite endodyogeny (PMID:28671988). It functions in complex with the atypical cyclin TgCycL, and conditional depletion of TgCrk1 blocks daughter bud formation, establishing it as a mitotic kinase essential for cytokinesis (PMID:28671988). TgCrk1 is embedded in a phosphatase-dependent regulatory pathway: its phosphorylation state is altered upon depletion of the plant-like phosphatase PPKL, placing TgCrk1 downstream of PPKL in signaling that controls daughter parasite development (PMID:37877735). Beyond this cyclin partnership and PPKL-dependent regulation, the substrates, specific phosphosites, and structural basis of TgCrk1 activity have not been characterized in the available corpus.

1. 2017 High

   Whether a CDK-related kinase governs the assembly of daughter parasites was unknown; identifying TgCrk1 as essential for bud formation established it as a mitotic kinase controlling tachyzoite cytokinesis.

   Evidence Conditional tet-OFF knockdown with quantitative fluorescence microscopy in T. gondii tachyzoites

   PMID:28671988

   Open questions at the time

   • Direct kinase substrates driving bud assembly not identified
   • No structural or catalytic characterization
   • Activation mechanism of the kinase unresolved
2. 2017 Medium

   The cyclin partner conferring specificity was unknown; Co-IP identified the atypical cyclin TgCycL as the binding partner, defining the functional TgCrk1-TgCycL complex.

   Evidence Co-immunoprecipitation with supporting genetic loss-of-function phenotype in T. gondii

   PMID:28671988

   Open questions at the time

   • Single Co-IP without reciprocal biochemical reconstitution
   • Whether TgCycL activates TgCrk1 kinase activity not shown directly
   • Stoichiometry and additional complex members unknown
3. 2023 Low

   Whether TgCrk1 activity is regulated by upstream signaling was unknown; phosphoproteomics upon PPKL depletion showed altered TgCrk1 phosphorylation, placing the kinase in a PPKL-dependent pathway controlling daughter cell assembly.

   Evidence Conditional knockdown of PPKL with global phosphoproteomics, Co-IP and proximity labeling in T. gondii (initially bioRxiv preprint, corroborated in mBio)

   PMID:37398039 PMID:37877735

   Open questions at the time

   • No direct biochemical reconstitution of PPKL acting on TgCrk1
   • Specific phosphosites and writer/eraser identity not established
   • Functional rescue of TgCrk1 phosphostate not tested

• The direct substrates of TgCrk1, the phosphosites governing its activity, and the biochemical mechanism linking PPKL dephosphorylation to TgCrk1 function during endodyogeny remain unresolved.
• No TgCrk1 substrate identified
• No direct enzyme-substrate reconstitution of the PPKL-TgCrk1 relationship
• No structural model of the TgCrk1-TgCycL complex