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Cyclin-dependent kinase TPK2 is a critical cell cycle regulator in Toxoplasma gondii
Farzana Khan1, Jianzhong Tang, Chang-Le Qin
1Department of Medicine, Division of Infectious Diseases, Albert Einstein College of Medicine, Bronx, NY 10461, USA.
Abstract:
The Apicomplexan parasite Toxoplasma gondii replicates by endodyogeny, an unusual form of binary fission. We tested the role of TPK2, a homologue of the CDC2 cyclin-dependent kinases, in cell cycle regulation. TPK2 tagged with HA epitope (TPK2-HA-wt) was expressed in mammalian cells as confirmed by Western blot analysis using HA tag and PSTAIRE antibodies. TPK2-HA-wt phosphorylated a peptide from Histone H1, proving that TPK2 is a functional kinase. TPK2-HA-wt coimmunoprecipitated with mammalian cyclins A, B1, D3 and E. Despite being a functional kinase, TPK2 did not rescue Schizosaccharomyces pombe cdc2 and Saccharomyces cerevisiae cdc28 mutant strains. Overexpression of a dominant-negative mutant of TPK2 (TPK2-HA-dn) in T. gondii tachyzoites arrested replication. FACS analysis of tachyzoites expressing TPK2-HA-dn revealed an increase in the fraction of cells in S-phase when compared with TPK2-HA-wt transfected parasites. Expression of TPK2-HA-wt did not arrest tachyzoite replication. No discernable G2 cell cycle block was evident suggesting that cell cycle checkpoints differ in T. gondii from most other eukaryotic cells. These data suggest that TPK2 executes an essential function in T. gondii cell cycle and is likely to be the T. gondii CDC2 orthologue.
Insights
The study identifies TPK2 as a crucial protein kinase for Toxoplasma gondii replication. Overexpressing a dominant-negative TPK2 mutant arrests parasite growth, suggesting TPK2 is essential for the T. gondii cell cycle.
Area of Science:
- Parasitology
- Cell Biology
- Molecular Biology
Background:
- Apicomplexan parasites like Toxoplasma gondii replicate via endodyogeny.
- Cell cycle regulation is critical for parasite proliferation.
- Cyclin-dependent kinases (CDKs) are key regulators of eukaryotic cell cycles.
Purpose of the Study:
- To investigate the role of TPK2, a homologue of CDC2 cyclin-dependent kinases, in Toxoplasma gondii cell cycle regulation.
- To determine if TPK2 functions as the CDC2 orthologue in T. gondii.
Main Methods:
- TPK2 tagged with an HA epitope (TPK2-HA-wt) was expressed in mammalian cells and T. gondii tachyzoites.
- Western blot analysis and co-immunoprecipitation were used to confirm TPK2 expression and interactions.
- Kinase activity was assessed using a Histone H1 peptide phosphorylation assay.
- Functional complementation assays were performed in yeast mutant strains.
- A dominant-negative TPK2 mutant (TPK2-HA-dn) was overexpressed in T. gondii tachyzoites.
- Flow cytometry (FACS) analysis was used to assess cell cycle progression.
Main Results:
- TPK2-HA-wt demonstrated functional kinase activity by phosphorylating Histone H1 and co-immunoprecipitated with mammalian cyclins.
- TPK2 did not rescue yeast cdc2 or cdc28 mutants.
- Overexpression of TPK2-HA-dn in T. gondii tachyzoites arrested parasite replication.
- FACS analysis showed an increased fraction of T. gondii cells in S-phase upon TPK2-HA-dn expression.
- No G2 cell cycle block was observed, suggesting unique cell cycle checkpoints in T. gondii.
Conclusions:
- TPK2 is a functional kinase with essential roles in Toxoplasma gondii cell cycle regulation.
- TPK2 is likely the functional CDC2 orthologue in T. gondii.
- T. gondii possesses distinct cell cycle control mechanisms compared to other eukaryotes.