Genetic analysis of the relationship between activation loop phosphorylation and cyclin binding in the activation of the Saccharomyces cerevisiae Cdc28p cyclin-dependent kinase.
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We showed recently that a screen for mutant CDC28 with improved binding to a defective Cln2p G1 cyclin yielded a spectrum of mutations similar to those yielded by a screen for intragenic suppressors of the requirement for activation loop phosphorylation (T169E suppressors). Recombination among these mutations yielded CDC28 mutants that bypassed the G1 cyclin requirement. Here we analyze further the interrelationship between T169E suppression, interaction with defective cyclin, and G1 cyclin bypass. DNA shuffling of mutations from the various screens and recombination onto a T169E-encoding 3' end yielded CDC28 mutants with strong T169E suppression. Some of the strongest T169E suppressors could suppress the defective Cln2p G1 cyclin even while retaining T169E. The strong T169E suppressors did not exhibit bypass of the G1 cyclin requirement but did so when T169E was reverted to T. These results suggested that for these mutants, activation loop phosphorylation and cyclin binding might be alternative means of activation rather than independent requirements for activation (as with wild type). These results suggest mechanistic overlap between the conformational shift induced by cyclin binding and that induced by activation loop phosphorylation. This conclusion was supported by analysis of suppressors of a mutation in the Cdk phosphothreonine-binding pocket created by cyclin binding. (+info)
Activating phosphorylation of the Saccharomyces cerevisiae cyclin-dependent kinase, cdc28p, precedes cyclin binding.
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Eukaryotic cell cycle progression is controlled by a family of protein kinases known as cyclin-dependent kinases (Cdks). Two steps are essential for Cdk activation: binding of a cyclin and phosphorylation on a conserved threonine residue by the Cdk-activating kinase (CAK). We have studied the interplay between these regulatory mechanisms during the activation of the major Saccharomyces cerevisiae Cdk, Cdc28p. We found that the majority of Cdc28p was phosphorylated on its activating threonine (Thr-169) throughout the cell cycle. The extent of Thr-169 phosphorylation was similar for monomeric Cdc28p and Cdc28p bound to cyclin. By varying the order of the addition of cyclin and Cak1p, we determined that Cdc28p was activated most efficiently when it was phosphorylated before cyclin binding. Furthermore, we found that a Cdc28p(T169A) mutant, which cannot be phosphorylated, bound cyclin less well than wild-type Cdc28p in vivo. These results suggest that unphosphorylated Cdc28p may be unable to bind tightly to cyclin. We propose that Cdc28p is normally phosphorylated by Cak1p before it binds cyclin. This activation pathway contrasts with that in higher eukaryotes, in which cyclin binding appears to precede activating phosphorylation. (+info)
Exit from mitosis in budding yeast: biphasic inactivation of the Cdc28-Clb2 mitotic kinase and the role of Cdc20.
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Cdc20, an activator of the anaphase-promoting complex (APC), is also required for the exit from mitosis in Saccharomyces cerevisiae. Here we show that during mitosis, both the inactivation of Cdc28-Clb2 kinase and the degradation of mitotic cyclin Clb2 occur in two steps. The first phase of Clb2 proteolysis, which commences at the metaphase-to-anaphase transition when Clb2 abundance is high, is dependent on Cdc20. The second wave of Clb2 destruction in telophase requires activation of the Cdc20 homolog, Hct1/Cdh1. The first phase of Clb2 destruction, which lowers the Cdc28-Clb2 kinase activity, is a prerequisite for the second. Thus, Clb2 proteolysis is not solely mediated by Hct1 as generally believed; instead, it requires a sequential action of both Cdc20 and Hct1. (+info)
Regulation of the yeast transcriptional factor PHO2 activity by phosphorylation.
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The induction of yeast Saccharomyces cerevisiae gene PHO5 expression is mediated by transcriptional factors PHO2 and PHO4. PHO4 protein has been reported to be phosphorylated and inactivated by a cyclin-CDK (cyclin-dependent kinase) complex, PHO80-PHO85. We report here that PHO2 can also be phosphorylated. A Ser-230 to Ala mutation in the consensus sequence (SPIK) recognized by cdc2/CDC28-related kinase in PHO2 protein led to complete loss of its ability to activate the transcription of PHO5 gene. Further investigation showed that the Pro-231 to Ser mutation inactivated PHO2 protein as well, whereas the Ser-230 to Asp mutation did not affect PHO2 activity. Since the PHO2 Asp-230 mutant mimics Ser-230-phosphorylated PHO2, we postulate that only phosphorylated PHO2 protein could activate the transcription of PHO5 gene. Two hybrid assays showed that yeast CDC28 could interact with PHO2. CDC28 immunoprecipitate derived from the YPH499 strain grown under low phosphate conditions phosphorylated GST-PHO2 in vitro. A phosphate switch regulates the transcriptional activation activity of PHO2, and mutations of the (SPIK) site affect the transcriptional activation activity of PHO2 and the interaction between PHO2 and PHO4. BIAcore(R) analysis indicated that the negative charge in residue 230 of PHO2 was sufficient to help PHO2 interact with PHO4 in vitro. (+info)
Phosphorylation of Cdc28 and regulation of cell size by the protein kinase CKII in Saccharomyces cerevisiae.
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The CDK (cyclin-dependent kinase) family of enzymes is required for the G(1)-to-S-phase and G(2)-to-M-phase transitions during the cell-division cycle of eukaryotes. We have shown previously that the protein kinase CKII catalyses the phosphorylation of Ser-39 in Cdc2 during the G(1) phase of the HeLa cell-division cycle [Russo, Vandenberg, Yu, Bae, Franza and Marshak (1992) J. Biol. Chem. 267, 20317-20325]. To identify a functional role for this phosphorylation, we have studied the homologous enzymes in the budding yeast Saccharomyces cerevisiae. The S. cerevisiae homologue of Cdc2, Cdc28, contains a consensus CKII site (Ser-46), which is homologous with that of human Cdc2. Using in vitro kinase assays, metabolic labelling, peptide mapping and phosphoamino acid analysis, we demonstrate that this site is phosphorylated in Cdc28 in vivo as well in vitro. In addition, S. cerevisiae cells in which Ser-46 has been mutated to alanine show a decrease in both cell volume and protein content of 33%, and this effect is most pronounced in the stationary phase. Because cell size in S. cerevisiae is regulated primarily at the G(1) stage, we suggest that CKII contributes to the regulation of the cell cycle in budding yeast by phosphorylation of Cdc28 as a checkpoint for G(1) progression. (+info)
Bud6 directs sequential microtubule interactions with the bud tip and bud neck during spindle morphogenesis in Saccharomyces cerevisiae.
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In budding yeast, spindle polarity relies on a precise temporal program of cytoplasmic microtubule-cortex interactions throughout spindle assembly. Loss of Clb5-dependent kinase activity under conditions of attenuated Cdc28 function disrupts this program, resulting in diploid-specific lethality. Here we show that polarity loss is tolerated by haploids due to a more prominent contribution of microtubule-neck interactions to spindle orientation inherent to haploids. These differences are mediated by the relative partition of Bud6 between the bud tip and bud neck, distinguishing haploids from diploids. Bud6 localizes initially to the bud tip and accumulates at the neck concomitant with spindle assembly. bud6Delta mutant phenotypes are consistent with Bud6's role as a cortical cue for cytoplasmic microtubule capture. Moreover, mutations that affect Bud6 localization and partitioning disrupt the sequential program of microtubule-cortex interactions accordingly. These data support a model whereby Bud6 sequentially cues microtubule capture events at the bud tip followed by capture events at the bud neck, necessary for correct spindle morphogenesis and polarity. (+info)
Nuclear-specific degradation of Far1 is controlled by the localization of the F-box protein Cdc4.
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Far1 is a bifunctional protein that is required to arrest the cell cycle and establish cell polarity during yeast mating. Here we show that SCF(Cdc4) ubiquitylates Far1 in the nucleus, which in turn targets the multi-ubiquitylated protein to 26S proteasomes most likely located at the nuclear envelope. In response to mating pheromones, a fraction of Far1 was stabilized after its export into the cytoplasm by Ste21/Msn5. Preventing nuclear export destabilized Far1, while conversely cytoplasmic Far1 was stabilized, although the protein was efficiently phosphorylated in a Cdc28-Cln-dependent manner. The core SCF subunits Cdc53, Hrt1 and Skp1 were distributed in the nucleus and the cytoplasm, whereas the F-box protein Cdc4 was exclusively nuclear. A cytoplasmic form of Cdc4 was unable to complement the growth defect of cdc4-1 cells, but it was sufficient to degrade Far1 in the cytoplasm. Our results illustrate the importance of subcellular localization of F-box proteins, and provide an example of how an extracellular signal regulates protein stability at the level of substrate localization. (+info)
Phosphorylation of the Cdc42 exchange factor Cdc24 by the PAK-like kinase Cla4 may regulate polarized growth in yeast.
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Rho-type GTPases control many cytoskeletal rearrangements, but their regulation remains poorly understood. Here, we show that in S. cerevisiae, activation of the CDK Cdc28-Cln2 at bud emergence triggers relocalization of Cdc24, the GEF for Cdc42, from the nucleus to the polarization site, where it is stably maintained by binding to the adaptor Bem1. Locally activated Cdc42 then polarizes the cytoskeleton in a manner dependent on its effectors Bni1 and the PAK-like kinase Cla4. In addition, Cla4 induces phosphorylation of Cdc24, leading to its dissociation from Bem1 at bud tips, thereby ending polarized bud growth in vivo. Our results thus suggest a dynamic temporal and spatial regulation of the Cdc42 module: Cdc28-Cln triggers actin polarization by activating Cdc42, which in turn restricts its own activation via a negative feedback loop acting on its GEF Cdc24. (+info)