Far1 and Fus3 link the mating pheromone signal transduction pathway to three G1-phase Cdc28 kinase complexes.

In the yeast Saccharomyces cerevisiae, the Cdc28 protein kinase controls commitment to cell division at Start, but no biologically relevant G1-phase substrates have been identified. We have studied the kinase complexes formed between Cdc28 and each of the G1 cyclins Cln1, Cln2, and Cln3. Each complex has a specific array of coprecipitated in vitro substrates. We identify one of these as Far1, a protein required for pheromone-induced arrest at Start. Treatment with alpha-factor induces a preferential association and/or phosphorylation of Far1 by the Cln1, Cln2, and Cln3 kinase complexes. This induced interaction depends upon the Fus3 protein kinase, a mitogen-activated protein kinase homolog that functions near the bottom of the alpha-factor signal transduction pathway. Thus, we trace a path through which a mitogen-activated protein kinase regulates a Cdc2 kinase.     

Enhanced Cell Polarity in Mutants of the Budding Yeast Cyclin-dependent Kinase Cdc28p

The yeast cyclin-dependent kinase Cdc28p regulates bud morphogenesis and cell cycle progression via the antagonistic activities of Cln and Clb cyclins. Cln G1 cyclins direct polarized growth and bud emergence, whereas Clb G2 cyclins promote isotropic growth of the bud and chromosome segregation. Using colony morphology as a screen to dissect regulation of polarity by Cdc28p, we identified nine point mutations that block the apical-isotropic switch while maintaining other functions. Like a clb2 Delta mutation, each confers tubular bud shape, apically polarized actin distribution, unipolar budding, and delayed anaphase. The mutations are all suppressed by CLB2 overexpression and are synthetically lethal with a CLB2 deletion. However, defects in multiple independent pathways may underlie their common phenotype, because the mutations are scattered throughout the CDC28 sequence, complement each other, and confer diverse biochemical properties. Glu12Gly, a mutation that alters a residue involved in Swe1p inhibition of Cdc28p, was unique in being suppressed by deficiency of SWE1 or CLN1. With wild-type CDC28, filament formation induced by CLN1 overexpression was markedly decreased in a SWE1 deletion. These results suggest that Swe1p, via inhibition of Clb2p/Cdc28p, may mediate much of the effect of Cln1p on filamentous morphogenesis.     

Directed evolution to bypass cyclin requirements for the Cdc28p cyclin-dependent kinase.

To identify cyclin-dependent kinase mutants with relaxed cyclin requirements, CDC28 alleles were selected that could rescue a yeast strain expressing as its only CLN G1 cyclin a mutant Cln2p (K129A,E183A) that is defective for Cdc28p binding. Rescue of this strain by mutant CDC28 was dependent upon the mutant cln2-KAEA, but additional mutagenesis and DNA shuffling yielded multiply mutant CDC28-BYC alleles (bypass of CLNs) that could support highly efficient cell cycle initiation in the complete absence of CLN genes. By gel filtration chromatography, one of the mutant Cdc28 proteins exhibited kinase activity associated with cyclin-free monomer. Thus, the mutants' CLN bypass activity might result from constitutive, cyclin-independent activity, suggesting that Cdk targeting by cyclins is not required for cell cycle initiation.     

The cyclin-dependent kinase Cdc28p regulates distinct modes of Cdc6p proteolysis during the budding yeast cell cycle

BACKGROUND: Cdc28p, the major cyclin-dependent kinase in budding yeast, prevents re-replication within each cell cycle by preventing the reassembly of Cdc6p-dependent pre-replicative complexes (pre-RCs) once origins have fired. Cdc6p is a rapidly degraded protein that must be synthesised in each cell cycle and is present only during the G1 phase. RESULTS: We found that, at different times in the cell cycle, there are distinct modes of Cdc6p proteolysis. Before Start, Cdc6p proteolysis did not require either the anaphase-promoting complex (APC/C) or the SCF complex, which mediate the major cell cycle regulated ubiquitination pathways, nor did it require Cdc28p activity or any of the potential Cdc28p phosphorylation sites in Cdc6p. In fact, the activation of B cyclin (Clb)-Cdc28p kinase inactivated this pathway of Cdc6p degradation later in the cell cycle. Activation of the G1 cyclins (Clns) caused Cdc6p degradation to become extremely rapid. This degradation required the SCF(CDC4) and Cdc28p consensus sites in Cdc6p, but did not require Clb5 and Clb6. Later in the cell cycle, SCF(CDC4)-dependent Cdc6p proteolysis remained active but became less rapid. CONCLUSIONS: Levels of Cdc6p are regulated in several ways by the Cdc28p cyclin-dependent kinase. The Cln-dependent elimination of Cdc6p, which does not require the S-phase-promoting cyclins Clb5 and Clb6, suggests that the ability to assemble pre-RCs is lost before, not concomitant with, origin firing.     

Mechanisms involved in regulating DNA replication origins during the cell cycle and in response to DNA damage

Replication origins in eukaryotic cells never fire more than once in a given S phase. Here, we summarize the role of cyclin-dependent kinases in limiting DNA replication origin usage to once per cell cycle in the budding yeast Saccharomyces cerevisiae. We have examined the role of different cyclins in the phosphorylation and regulation of several replication/regulatory factors including Cdc6, Sic1, ORC and DNA polymerase alpha-primase. In addition to being regulated by the cell cycle machinery, replication origins are also regulated by the genome integrity checkpoint kinases, Mec1 and Rad53. In response to DNA damage or drugs which interfere with the progression of replication forks, the activation of late-firing replication origins is inhibited. There is evidence indicating that the temporal programme of origin firing depends upon the local histone acetylation state. We have attempted to test the possibility that checkpoint regulation of late-origin firing operates through the regulation of the acetylation state. We found that overexpression of the essential histone acetylase, Esal, cannot override checkpoint regulation of origin firing. We have also constructed a temperature-sensitive esa1 mutant. This mutant is unable to resume cell cycle progression after alpha-factor arrest. This can be overcome by overexpression of the G1 cyclin, Cln2, revealing a novel role for Esal in regulating Start.     

Cks1 is required for G(1) cyclin-cyclin-dependent kinase activity in budding yeast

p13(suc1) (Cks) proteins have been implicated in the regulation of cyclin-dependent kinase (CDK) activity. However, the mechanism by which Cks influences the function of cyclin-CDK complexes has remained elusive. We show here that Cks1 is required for the protein kinase activity of budding yeast G(1) cyclin-CDK complexes. Cln2 and Cdc28 subunits coexpressed in baculovirus-infected insect cells fail to exhibit protein kinase activity towards multiple substrates in the absence of Cks1. Cks1 can both stabilize Cln2-Cdc28 complexes and activate intact complexes in vitro, suggesting that it plays multiple roles in the biogenesis of active G(1) cyclin-CDK complexes. In contrast, Cdc28 forms stable, active complexes with the B-type cyclins Clb4 and Clb5 regardless of whether Cks1 is present. The levels of Cln2-Cdc28 and Cln3-Cdc28 protein kinase activity are severely reduced in cks1-38 cell extracts. Moreover, phosphorylation of G(1) cyclins, which depends on Cdc28 activity, is reduced in cks1-38 cells. The role of Cks1 in promoting G(1) cyclin-CDK protein kinase activity both in vitro and in vivo provides a simple molecular rationale for the essential role of CKS1 in progression through G(1) phase in budding yeast.     

Cdc37 promotes the stability of protein kinases Cdc28 and Cak1

In the budding yeast Saccharomyces cerevisiae, Cdc37 is required for the productive formation of Cdc28-cyclin complexes. The cdc37-1 mutant arrests at Start with low levels of Cdc28 protein, which is predominantly unphosphorylated at Thr169, fails to bind cyclin, and has little protein kinase activity. We show here that Cdc28 and not cyclin is specifically defective in the cdc37-1 mutant and that Cdc37 likely does not act as an assembly factor for Cdc28-cyclin complex formation. We have also found that the levels and activity of the protein kinase Cak1 are significantly reduced in the cdc37-1 mutant. Pulse-chase analysis indicates that Cdc28 and Cak1 proteins are both destabilized when Cdc37 function is absent during but not after translation. In addition, Cdc37 promotes the production of Cak1, but not that of Cdc28, when coexpressed in insect cells. We conclude that budding yeast Cdc37, like its higher eukaryotic homologs, promotes the physical integrity of multiple protein kinases, perhaps by virtue of a cotranslational role in protein folding.     

First the CDKs, now the DDKs.

In budding yeast, Dbf4p and Cdc7p control initiation of DNA synthesis. They form a protein kinase - Cdc7p being the catalytic subunit and Dbf4p a cyclin-like molecule that activates the kinase in late G1 phase. Dbf4p also targets Cdc7p to origins of replication, where probable substrates include certain Mcm proteins. Recent studies have identified Dbf4p- and Cdc7p-related proteins in fission yeast and metazoans. These homologues also phosphorylate Mcm proteins and could have a similar function to that of Dbf4p-Cdc7p in budding yeast. Thus, it seems likely that, like the cyclin-dependent kinases (CDKs), the Dbf4p-Cdc7p activity is conserved in all eukaryotes.     

A mathematical model of mitotic exit in budding yeast: the role of Polo kinase

Cell cycle progression in eukaryotes is regulated by periodic activation and inactivation of a family of cyclin-dependent kinases (Cdk's). Entry into mitosis requires phosphorylation of many proteins targeted by mitotic Cdk, and exit from mitosis requires proteolysis of mitotic cyclins and dephosphorylation of their targeted proteins. Mitotic exit in budding yeast is known to involve the interplay of mitotic kinases (Cdk and Polo kinases) and phosphatases (Cdc55/PP2A and Cdc14), as well as the action of the anaphase promoting complex (APC) in degrading specific proteins in anaphase and telophase. To understand the intricacies of this mechanism, we propose a mathematical model for the molecular events during mitotic exit in budding yeast. The model captures the dynamics of this network in wild-type yeast cells and 110 mutant strains. The model clarifies the roles of Polo-like kinase (Cdc5) in the Cdc14 early anaphase release pathway and in the G-protein regulated mitotic exit network.