CRK4

TgCrk4 is a CDK-related kinase that governs the G2/M transition of Toxoplasma gondii tachyzoites, where it enforces the coordination of the chromosome and centrosome cycles (PMID:28671988, PMID:38600241). It is essential for tachyzoite growth and specifically required for centrosome duplication during mitosis (PMID:28671988). Functioning as a complex with its cyclin partner TgCyc4, TgCrk4 represses chromosome re-replication and centrosome reduplication and acts upstream of the spindle assembly checkpoint (PMID:38600241). Its kinase activity targets substrates linking it to centrosome and chromosome cycle control, including TgORC4, TgCdc20, TgGCP2, and TgPP2ACA (PMID:38600241), and the complex recruits a dominant interactor, TgiRD1 (related to the DNA replication licensing factor CDT1), which likewise restricts chromosome and centrosome reduplication without acting in G1-phase replication licensing (PMID:38600241). Phylogenetic and structural analyses indicate the Crk4-Cyc4 complex is restricted to a minor group of binary-dividing apicomplexans, suggesting it evolved specifically in this lineage (PMID:38600241).

1. 2017 Medium

   Establishing whether TgCrk4 has a non-redundant cell-cycle function, conditional depletion showed it is essential for growth and specifically required for centrosome duplication, placing it within mitotic control.

   Evidence Conditional tet-OFF knockdown with fluorescence microscopy of centrosome duplication in T. gondii tachyzoites

   PMID:28671988

   Open questions at the time

   • Cyclin partner and direct substrates not identified
   • Molecular mechanism of centrosome control unresolved
2. 2017 Medium

   To test whether TgCrk4 depends on a cyclin-timing mechanism, co-IP against a panel of cyclins gave a negative result, initially indicating no required cyclin partner.

   Evidence Co-immunoprecipitation (negative for cyclin interaction) plus expression profiling across mitosis

   PMID:28671988

   Open questions at the time

   • The tested cyclin panel may have excluded the true partner (later shown to be TgCyc4)
   • Kinase activity requirements not defined
3. 2024 High

   Resolving the earlier negative cyclin result, TgCrk4 was shown to partner with TgCyc4, and the complex was placed mechanistically as a repressor of chromosome re-replication and centrosome reduplication acting upstream of the spindle assembly checkpoint.

   Evidence Conditional knockdown, quantitative fluorescence microscopy, co-IP and global phosphoproteomics in T. gondii

   PMID:38600241

   Open questions at the time

   • Direct enzymatic assay of TgCrk4-TgCyc4 kinase activity not shown
   • How the complex is activated/timed at G2/M not fully defined
4. 2024 Medium

   To connect kinase activity to phenotype, phosphoproteomics identified candidate substrates (TgORC4, TgCdc20, TgGCP2, TgPP2ACA) tying TgCrk4 to both chromosome and centrosome cycle machinery.

   Evidence Global phosphoproteomics in a TgCrk4 conditional knockdown background

   PMID:38600241

   Open questions at the time

   • Direct in vitro phosphorylation of each substrate not demonstrated
   • Phosphosite-level functional consequences not tested
5. 2024 Medium

   To identify complex partners, proteomics revealed the CDT1-related protein TgiRD1 as a dominant interactor that also controls chromosome and centrosome reduplication but, unexpectedly, not G1-phase replication licensing.

   Evidence Co-IP/proteomics followed by conditional knockdown with cell-cycle phenotype readout in T. gondii

   PMID:38600241

   Open questions at the time

   • Whether TgiRD1 is a substrate or purely a scaffold not resolved
   • Mechanism linking TgiRD1 to centrosome control unknown
6. 2024 Low

   To assess conservation, phylogenetic and structural analysis indicated the Crk4-Cyc4 complex is restricted to a minor group of binary-dividing apicomplexans.

   Evidence Phylogenetic analysis and structural comparison (computational only)

   PMID:38600241

   Open questions at the time

   • Computational inference not experimentally validated
   • Functional conservation in other apicomplexans not tested