Characterization of cyclin-dependent kinases and Cdc2/Cdc28 kinase subunits in Trichomonas vaginalis

SUMMARYCyclin-dependent kinases (CDKs) have important roles in regulating key checkpoints between stages of the cell cycle. Their activity is tightly regulated through a variety of mechanisms, including through binding with cyclin proteins and the Cdc2/Cdc28 kinase subunit (CKS), and their phosphorylation at specific amino acids. Studies of the components involved in cell cycle control in parasitic protozoa are limited. Trichomonas vaginalis is the causative agent of trichomoniasis in humans and is therefore important in public health; however, some of the basic biological processes used by this organism have not been defined. Here, we characterized proteins potentially involved in cell cycle regulation in T. vaginalis. Three genes encoding protein kinases were identified in the T. vaginalis genome, and the corresponding recombinant proteins (TvCRK1, TvCRK2, TvCRK5) were studied. These proteins displayed similar sequence features to CDKs. Two genes encoding CKSs were also identified, and the corresponding recombinant proteins were found to interact with TvCRK1 and TvCRK2 by a yeast two-hybrid system. One putative cyclin B protein from T. vaginalis was found to bind to and activate the kinase activities of TvCRK1 and TvCRK5, but not TvCRK2. This work is the first characterization of proteins involved in cell cycle control in T. vaginalis.

Counteractive Control of Polarized Morphogenesis during Mating by Mitogen-activated Protein Kinase Fus3 and G1 Cyclin-dependent Kinase

Cell polarization in response to external cues is critical to many eukaryotic cells. During pheromone-induced mating in Saccharomyces cerevisiae, the mitogen-activated protein kinase (MAPK) Fus3 induces polarization of the actin cytoskeleton toward a landmark generated by the pheromone receptor. Here, we analyze the role of Fus3 activation and cell cycle arrest in mating morphogenesis. The MAPK scaffold Ste5 is initially recruited to the plasma membrane in random patches that polarize before shmoo emergence. Polarized localization of Ste5 is important for shmooing. In fus3 mutants, Ste5 is recruited to significantly more of the plasma membrane, whereas recruitment of Bni1 formin, Cdc24 guanine exchange factor, and Ste20 p21-activated protein kinase are inhibited. In contrast, polarized recruitment still occurs in a far1 mutant that is also defective in G1 arrest. Remarkably, loss of Cln2 or Cdc28 cyclin-dependent kinase restores polarized localization of Bni1, Ste5, and Ste20 to a fus3 mutant. These and other findings suggest Fus3 induces polarized growth in G1 phase cells by down-regulating Ste5 recruitment and by inhibiting Cln/Cdc28 kinase, which prevents basal recruitment of Ste5, Cdc42-mediated asymmetry, and mating morphogenesis.

Clb6/Cdc28 and Cdc14 Regulate Phosphorylation Status and Cellular Localization of Swi6

ABSTRACT Nuclear export of the transcription factor Swi6 during the budding yeast Saccharomyces cerevisiae cell cycle is known to require phosphorylation of the Swi6 serine 160 residue. We show that Clb6/Cdc28 kinase is required for this nuclear export. Furthermore, Cdc28 combined with the S-phase cyclin Clb6 specifically phosphorylates serine 160 of Swi6 in vitro. Nuclear import of Swi6 occurs concomitantly with dephosphorylation of serine 160 in late M phase. We show that Cdc14 phosphatase, the principal effector of the mitotic exit network, can trigger nuclear import of Swi6 in vivo and that Cdc14 dephosphorylates Swi6 at serine 160 in vitro. Taken together, these observations show how Swi6 dephosphorylation and phosphorylation are integrated into changes of Cdc28 activity governing entry and exit from the G1 phase of the cell cycle.

Early Cell Cycle Box-Mediated Transcription ofCLN3 and SWI4 Contributes to the Proper Timing of the G1-to-S Transition in Budding Yeast

ABSTRACT The Cln3-Cdc28 kinase is required to activate the Swi4-Swi6 transcription complex which induces CLN1 andCLN2 transcription in late G1 and drives the transition to S. Cln3 and Swi4 are both rate limiting for G1 progression, and they are coordinately transcribed to peak at the M/G1 boundary. Early cell cycle box (ECB) elements, which confer M/G1-specific transcription, have been found in both promoters, and elimination of all ECB elements from the CLN3 promoter causes both a loss of periodicity and Cln3-deficient phenotypes, which include an extended G1interval and increased cell volume. Mutants lacking the ECB elements in both the CLN3 and SWI4 promoters have low and deregulated levels of CLN transcripts, and the G1-to-S transition for these mutants is delayed and highly variable. These observations support the view that the coordinated rise of Cln3 and Swi4 levels mediated by ECB-dependent transcription controls the timing of the G1-to-S phase transition.

Genetic and Biochemical Characterization of the Yeast Spo12 Protein

We have performed a genetic and biochemical analysis of theSPO12 gene, which regulates meiotic nuclear divisions in budding yeast. When sporulated, spo12 mutants undergo a single meiotic nuclear division most closely resembling meiosis II. We observed that Spo12 protein is localized to the nucleus during both meiotic divisions and that Clb1-Cdc28, Clb3-Cdc28, Clb4-Cdc28, and Clb5-Cdc28 kinase activities during meiosis were not affected by a spo12 mutation. Using two-hybrid analysis, we identified several genes, three of which are meiotically induced, that may code for proteins that interact with Spo12p. We also observed that two genes, BNS1 (Bypasses Need for Spo12p), which has homology to SPO12, andSPO13, whose mutant phenotype is like that ofspo12, can partially suppress the meiotic defect ofspo12 mutants when overexpressed. We found that Spo12p is also localized to the nucleus in vegetative cells and that its level peaks during G2/M. We observed that a spo12 mutation is synthetically lethal in vegetative cells with a mutation inHCT1, a gene necessary for cells to exit mitosis, suggesting that Spo12p may have a role in exit from mitosis.

Phosphorylation of Sic1, a Cyclin-dependent Kinase (Cdk) Inhibitor, by Cdk Including Pho85 Kinase Is Required for Its Prompt Degradation

In the yeast Saccharomyces cerevisiae, Sic1, an inhibitor of Clb-Cdc28 kinases, must be phosphorylated and degraded in G1for cells to initiate DNA replication, and Cln-Cdc28 kinase appears to be primarily responsible for phosphorylation of Sic1. The Pho85 kinase is a yeast cyclin-dependent kinase (Cdk), which is not essential for cell growth unless both CLN1 andCLN2 are absent. We demonstrate that Pho85, when complexed with Pcl1, a G1cyclin homologue, can phosphorylate Sic1 in vitro, and that Sic1 appears to be more stable inpho85Δ cells. Three consensus Cdk phosphorylation sites present in Sic1 are phosphorylated in vivo, and two of them are required for prompt degradation of the inhibitor. Pho85 and other G1Cdks appear to phosphorylate Sic1 at different sites in vivo. Thus at least two distinct Cdks can participate in phosphorylation of Sic1 and may therefore regulate progression through G1.

The Cdk inhibitors p25rum1 and p40SIC1 are functional homologues that play similar roles in the regulation of the cell cycle in fission and budding yeast

p25rum1 and p40SIC1 are specific inhibitors of p34(cdc2/CDC28) kinase complexes with B-type cyclins that play a central role in the regulation of the G1 phase of the cell cycle. We show here that low levels of expression of SIC1 in Schizosaccharomyces pombe rescues all the phenotypes of cells lacking the rum1+ gene. In addition, high level expression of SIC1 in S. pombe induces extra rounds of DNA replication without mitosis, a phenotype very similar to the overexpression of rum1+. Transient expression of rum1+ in S. cerevisiae restores the G1 arrest phenotype of cdc4 sic1Delta double mutants. Overproduction of rum1+ in Saccharomyces cerevisiae causes a cell cycle block in G1 with a phenotype similar to inactivation of all the Clb cyclins. Finally, we have mapped the cyclin interacting domain and Cdk inhibitory domain to a region of about 80 amino acids in p25rum1 that has significant homology to the C-terminal domain of p40SIC1. All these observations suggest that fission yeast p25rum1 and budding yeast p40SIC1 define a family of Cdk inhibitors that specifically down regulate cyclin B/Cdk1 during the G1 phase of the cell cycle.