Cyclin-dependent kinase TPK2 is a critical cell cycle regulator in Toxoplasma gondii.
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.
Department of Medicine, Division of Infectious Diseases, Albert Einstein College of Medicine, Bronx, NY 10461, USA.
Farzana
F
Khan
Jianzhong
J
Tang
Chang-Le
CL
Qin
Kami
K
Kim
D000595:Amino Acid Sequence; D000818:Animals; D016203:CDC2 Protein Kinase / Q000172:deficiency / Q000235:genetics; D017489:CDC28 Protein Kinase, S cerevisiae / Q000172:deficiency / Q000235:genetics; D019556:COS Cells; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D018844:Cyclin-Dependent Kinases / Q000235:genetics / Q000502:physiology*; D016213:Cyclins / Q000378:metabolism; D005816:Genetic Complementation Test; D008969:Molecular Sequence Data; D015800:Protozoan Proteins / Q000235:genetics / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000502:physiology; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000502:physiology; D029702:Schizosaccharomyces pombe Proteins / Q000235:genetics / Q000502:physiology; D013045:Species Specificity; D014122:Toxoplasma / Q000166:cytology / Q000201:enzymology*
D016213:Cyclins; D015800:Protozoan Proteins; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D029702:Schizosaccharomyces pombe Proteins; D016203:CDC2 Protein Kinase; D017489:CDC28 Protein Kinase, S cerevisiae; D018844:Cyclin-Dependent Kinases; C465638:TPK2 protein, Toxoplasma gondii