Phosphorylation of the cohesin subunit Scc1 by Polo/Cdc5 kinase regulates sister chromatid separation in yeast

At the onset of anaphase, a caspase-related protease (separase) destroys the link between sister chromatids by cleaving the cohesin subunit Scc1. During most of the cell cycle, separase is kept inactive by binding to an inhibitory protein called securin. Separase activation requires proteolysis of securin, which is mediated by an ubiquitin protein ligase called the anaphase-promoting complex. Cells regulate anaphase entry by delaying securin ubiquitination until all chromosomes have attached to the mitotic spindle. Though no longer regulated by this mitotic surveillance mechanism, sister separation remains tightly cell cycle regulated in yeast mutants lacking securin. We show here that the Polo/Cdc5 kinase phosphorylates serine residues adjacent to Scc1 cleavage sites and strongly enhances their cleavage. Phosphorylation of separase recognition sites may be highly conserved and regulates sister chromatid separation independently of securin.     

Phosphorylation by Cdc28 activates the Cdc20-dependent activity of the anaphase-promoting complex

Budding yeast initiates anaphase by activating the Cdc20-dependent anaphase-promoting complex (APC). The mitotic activity of Cdc28 (Cdk1) is required to activate this form of the APC, and mutants that are impaired in mitotic Cdc28 function have difficulty leaving mitosis. This defect can be explained by a defect in APC phosphorylation, which depends on mitotic Cdc28 activity in vivo and can be catalyzed by purified Cdc28 in vitro. Mutating putative Cdc28 phosphorylation sites in three components of the APC, Cdc16, Cdc23, and Cdc27, makes the APC resistant to phosphorylation both in vivo and in vitro. The nonphosphorylatable APC has normal activity in G1, but its mitotic, Cdc20-dependent activity is compromised. These results show that Cdc28 activates the APC in budding yeast to trigger anaphase. Previous reports have shown that the budding yeast Cdc5 homologue, Plk, can also phosphorylate and activate the APC in vitro. We show that, like cdc28 mutants, cdc5 mutants affect APC phosphorylation in vivo. However, although Cdc5 can phosphorylate Cdc16 and Cdc27 in vitro, this in vitro phosphorylation does not occur on in vivo sites of phosphorylation.     

Conservation of function and regulation within the Cdc28/cdc2 protein kinase family: characterization of the human Cdc2Hs protein kinase in Saccharomyces cerevisiae.

Whereas the Cdc28 protein kinase of the budding yeast Saccharomyces cerevisiae plays an essential role in cell cycle progression during the G1 interval, a function in the progression from the G2 interval into M phase has been inferred for its homologs, including the Cdc2Hs protein kinase of humans. To better understand these apparently disparate roles, we constructed a yeast strain in which the resident CDC28 gene was replaced by its human homolog, CDC2Hs. This transgenic yeast strain was able to perform the G1 functions attributed to the Cdc28 protein kinase, including the ability to grow and divide normally, to respond to environmental signals that induce G1 arrest, and to regulate the Cdc2Hs protein kinase appropriately in response to these signals.     

The Yeast Polo Kinase Cdc5 Regulates the Shape of the Mitotic Nucleus

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Invariant phosphorylation of the Saccharomyces cerevisiae Cdc28 protein kinase.

The phosphorylation level of the Saccharomyces cerevisiae Cdc28 protein remained invariant under conditions that resulted in cell cycle arrest in the G1 phase and loss of Cdc28-specific protein kinase activity when the activity was assayed in vitro. These results are in contrast to the proposed regulation of the homologous Cdc2 protein kinase of Schizosaccharomyces pombe.     

Modulation of septin higher-order structure by the Cdc28 protein kinase

Septins are a family of eukaryotic guanosine phosphate-binding proteins that form linear heterooligomeric complexes, which, in turn, polymerize end-on-end into filaments. These filaments further assemble into higher-order structures at distinct subcellular locations. Dynamic changes in the organization of septin cortex structures appear during cell cycle progression. A variety of regulatory proteins and posttranslational modifications are involved in changes to the structure of septin assemblies during the entire cell cycle. In particular, septin-associated protein kinases mediate changes to septin higher order structures or interconnect cellular morphogenesis with the cell cycle. Yeast cyclin-dependent kinase, a master cell cycle regulator, is required for the initiation of a new septin ring. Here, using epifluoresence and electron microscopy, we show that upon phosphorylation by the Cdc28 kinase, septin filaments disassemble into hetero-octameric building blocks, and that filament depolymerization is specifically G1 cyclin-dependent.     

The role of the polo kinase Cdc5 in controlling Cdc14 localization

In budding yeast, the protein phosphatase Cdc14 controls exit from mitosis. Its activity is regulated by a competitive inhibitor Cfi1/Net1, which binds to and sequesters Cdc14 in the nucleolus. During anaphase, Cdc14 is released from its inhibitor by the action of two regulatory networks. The Cdc Fourteen Early Anaphase Release (FEAR) network initiates Cdc14 release from Cfi1/Net1 during early anaphase, and the Mitotic Exit Network (MEN) promotes Cdc14 release during late anaphase. Here, we investigate the relationship among FEAR network components and propose an order in which they function to promote Cdc14 release from the nucleolus. Furthermore, we examine the role of the protein kinase Cdc5, which is a component of both the FEAR network and the MEN, in Cdc14 release from the nucleolus. We find that overexpression of CDC5 led to Cdc14 release from the nucleolus in S phase-arrested cells, which correlated with the appearance of phosphorylated forms of Cdc14 and Cfi1/Net1. Cdc5 promotes Cdc14 phosphorylation and, by stimulating the MEN, Cfi1/Net1 phosphorylation. Furthermore, we suggest that Cdc14 release from the nucleolus only occurs when Cdc14 and Cfi1/Net1 are both phosphorylated.     

Genetic analysis of Cln/Cdc28 regulation of cell morphogenesis in budding yeast.

The CLN1, CLN2 and CLN3 gene family of G1-acting cyclin homologs of Saccharomyces cerevisiae is functionally redundant: any one of the three Cln proteins is sufficient for activation of Cdc28p protein kinase activity for cell cycle START. The START event leads to multiple processes (including DNA replication and bud emergence); how Cln/Cdc28 activity activates these processes remains unclear. CLN3 is substantially different in structure and regulation from CLN1 and CLN2, so its functional redundancy with CLN1 and CLN2 is also poorly understood. We have isolated mutations that alter this redundancy, making CLN3 insufficient for cell viability in the absence of CLN1 and CLN2 expression. Mutations causing phenotypes specific for the cell division cycle were analyzed in detail. Mutations in one gene result in complete failure of bud formation, leading to depolarized cell growth. This gene was identified as BUD2, previously described as a non-essential gene required for proper bud site selection but not required for budding and viability. Bud2p is probably the GTPase-activating protein for Rsr1p/Bud1p [Park, H., Chant, I. and Herskowitz, I. (1993) Nature, 365, 269-274]; we find that Rsr1p is required for the bud2 lethal phenotype. Mutations in two other genes (ERC10 and ERC19) result in a different morphogenetic defect: failure of cytokinesis resulting in the formation of long multinucleate tubes. These results suggest direct regulation of diverse aspects of bud morphogenesis by Cln/Cdc28p activity.     

TOR signalling regulates mitotic commitment through the stress MAP kinase pathway and the Polo and Cdc2 kinases

The coupling of growth to cell cycle progression allows eukaryotic cells to divide at particular sizes depending on nutrient availability. In fission yeast, this coupling involves the Spc1/Sty1 mitogen-activated protein kinase (MAPK) pathway working through Polo kinase recruitment to the spindle pole bodies (SPBs). Here we report that changes in nutrients influence TOR signalling, which modulates Spc1/Sty1 activity. Rapamycin-induced inhibition of TOR signalling advanced mitotic onset, mimicking the reduction in cell size at division seen after shifts to poor nitrogen sources. Gcn2, an effector of TOR signalling and modulator of translation, regulates the Pyp2 phosphatase that in turn modulates Spc1/Sty1 activity. Rapamycin- or nutrient-induced stimulation of Spc1/Sty1 activity promotes Polo kinase SPB recruitment and Cdc2 activation to advance mitotic onset. This advanced mitotic onset is abolished in cells depleted of Gcn2, Pyp2, or Spc1/Sty1 or on blockage of Spc1/Sty1-dependent Polo SPB recruitment. Therefore, TOR signalling modulates mitotic onset through the stress MAPK pathway via the Pyp2 phosphatase.     

Inhibitory phosphorylation of the APC regulator Hct1 is controlled by the kinase Cdc28 and the phosphatase Cdc14

BACKGROUND: Exit from mitosis requires inactivation of mitotic cyclin-dependent kinases (CDKs). A key mechanism of CDK inactivation is ubiquitin-mediated cyclin proteolysis, which is triggered by the late mitotic activation of a ubiquitin ligase known as the anaphase-promoting complex (APC). Activation of the APC requires its association with substoichiometric activating subunits termed Cdc20 and Hct1 (also known as Cdh1). Here, we explore the molecular function and regulation of the APC regulatory subunit Hct1 in Saccharomyces cerevisiae. RESULTS: Recombinant Hct1 activated the cyclin-ubiquitin ligase activity of APC isolated from multiple cell cycle stages. APC isolated from cells arrested in G1, or in late mitosis due to the cdc14-1 mutation, was more responsive to Hct1 than APC isolated from other stages. We found that Hct1 was phosphorylated in vivo at multiple CDK consensus sites during cell cycle stages when activity of the cyclin-dependent kinase Cdc28 is high and APC activity is low. Purified Hct1 was phosphorylated in vitro at these sites by purified Cdc28-cyclin complexes, and phosphorylation abolished the ability of Hct1 to activate the APC in vitro. The phosphatase Cdc14, which is known to be required for APC activation in vivo, was able to reverse the effects of Cdc28 by catalyzing Hct1 dephosphorylation and activation. CONCLUSIONS: We conclude that Hct1 phosphorylation is a key regulatory mechanism in the control of cyclin destruction. Phosphorylation of Hct1 provides a mechanism by which Cdc28 blocks its own inactivation during S phase and early mitosis. Following anaphase, dephosphorylation of Hct1 by Cdc14 may help initiate cyclin destruction.     

Rac and Cdc42-dependent regulation of c-Jun N-terminal kinases by the delta-opioid receptor

Heptahelical opioid receptors utilize Gi proteins to regulate a multitude of effectors including the classical adenylyl cyclases and the more recently discovered mitogen-activated protein kinases (MAPKs). The c-Jun NH2-terminal kinases (JNKs) belong to one of three subgroups of MAPKs. In NG108-15 neuroblastoma x glioma hybrid cells that endogenously express delta-opioid receptors, delta-agonist dose-dependently stimulated JNK activity in a pertussis toxin-sensitive manner. By using COS-7 cells transiently transfected with the cDNAs of delta-opioid receptor and hemagglutinin (HA)-tagged JNK, we delineated the signaling components involved in this pathway. Sequestration of Gbetagamma subunits by transducin suppressed the opioid-induced JNK activity. The possible involvement of the small GTPases was also examined. Expression of dominant negative mutants of Rac and Cdc42 blocked the opioid-induced JNK activation, and a partial inhibition was observed in the presence of the dominant negative mutant of Ras. In contrast, the dominant negative mutant of Rho did not affect the opioid-induced JNK activation. In addition, the receptor-mediated JNK activation was dependent on Src family tyrosine kinases, but independent of phosphatidylinositol-3 kinase and EGF receptor tyrosine kinases. Collectively, these results demonstrate functional regulation of JNK by the delta-opioid receptor, and this pathway requires Gbetagamma, Src kinases and the small GTPases Rac and Cdc42.     

Control of the yeast cell cycle is associated with assembly/disassembly of the Cdc28 protein kinase complex

The Saccharomyces cerevisiae gene CDC28 encodes a protein kinase required for progression from G1 to S phase in the cell cycle. We present evidence that the active form of the Cdc28 protein kinase is a complex of approximately 160 kd containing an endogenous substrate, p40, and possibly other polypeptides. This complex phosphorylates p40 and exogenous histone H1 in vitro. Cell cycle arrest during G1 results in inactivation of the protein kinase accompanied by the disassembly of the complex. Furthermore, assembly of the complex is regulated during the cell cycle, reaching a maximum during G1. Partial complexes thought to be intermediates in the assembly process phosphorylate histone H1 but not p40. Addition of soluble factors to these partial complexes in vitro restores p40 phosphorylation and causes the complex to increase to the mature size. A model is presented in which p40 phosphorylation is required during G1 for cells to initiate a new cell cycle.     

An essential function of yeast cyclin-dependent kinase Cdc28 maintains chromosome stability

Multiple surveillance pathways maintain genomic integrity in yeast during mitosis. Although the cyclin-dependent kinase Cdc28 is a well established regulator of mitotic progression, evidence for a direct role in mitotic surveillance has been lacking. We have now implicated a conserved sequence in the Cdc28 carboxyl terminus in maintaining chromosome stability through mitosis. Six temperature-sensitive mutants were isolated via random mutagenesis of 13 carboxyl-terminal residues. These mutants identify a Cdc28 domain necessary for proper mitotic arrest in the face of kinetochore defects or microtubule inhibitors. These chromosome stability-defective cdc28(CST) mutants inappropriately continue mitosis when the mitotic spindle is disrupted at 23 degrees C, display high rates of spontaneous chromosome loss at 30 degrees C, and suffer catastrophic aneuploidy at 35 degrees C. A dosage suppression screen identified Cak1, a kinase known to phosphorylate and activate Cdc28, as a specific high copy suppressor of cdc28(CST) temperature sensitivity and chromosome instability. Suppression is independent of the kinase activity of Cak1, suggesting that Cak1 may bind to the carboxyl terminus to serve a non-catalytic role in assembly and/or stabilization of active Cdc28 complexes. Significantly, these studies implicate Cdc28 and Cak1 in an essential surveillance function required to maintain genetic stability through mitosis.     

Structure and regulation of the myotonic dystrophy kinase-related Cdc42-binding kinase

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Morphogenesis in the yeast cell cycle: regulation by Cdc28 and cyclins

Analysis of cell cycle regulation in the budding yeast Saccharomyces cerevisiae has shown that a central regulatory protein kinase, Cdc28, undergoes changes in activity through the cell cycle by associating with distinct groups of cyclins that accumulate at different times. The various cyclin/Cdc28 complexes control different aspects of cell cycle progression, including the commitment step known as START and mitosis. We found that altering the activity of Cdc28 had profound effects on morphogenesis during the yeast cell cycle. Our results suggest that activation of Cdc28 by G1 cyclins (Cln1, Cln2, or Cln3) in unbudded G1 cells triggers polarization of the cortical actin cytoskeleton to a specialized pre-bud site at one end of the cell, while activation of Cdc28 by mitotic cyclins (Clb1 or Clb2) in budded G2 cells causes depolarization of the cortical actin cytoskeleton and secretory apparatus. Inactivation of Cdc28 following cyclin destruction in mitosis triggers redistribution of cortical actin structures to the neck region for cytokinesis. In the case of pre-bud site assembly following START, we found that the actin rearrangement could be triggered by Cln/Cdc28 activation in the absence of de novo protein synthesis, suggesting that the kinase may directly phosphorylate substrates (such as actin-binding proteins) that regulate actin distribution in cells.     

Requirement of Hsp90 for centrosomal function reflects its regulation of Polo kinase stability

We have previously shown that the molecular chaperone heat shock protein 90 (Hsp90) is required to ensure proper centrosome function in Drosophila and vertebrate cells. This observation led to the hypothesis that this chaperone could be required for the stability of one or more centrosomal proteins. We have found that one of these is Polo, a protein kinase known to regulate several aspects of cell division including centrosome maturation and function. Inhibition of Hsp90 results in the inactivation of Polo kinase activity. It also leads to a loss in the ability of cytoplasmic extracts to complement the failure of salt-stripped preparations of centrosomes to nucleate microtubules. This effect can be rescued upon addition of active recombinant POLO: We also show that Polo and Hsp90 are part of a complex and conclude that stabilization of Polo is one of the mechanisms by which Hsp90 contributes to the maintenance of functional centrosomes.     

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.     

Analysis of Polo-like kinase Cdc5 in the meiosis recombination checkpoint

In meiosis, accumulation of recombination intermediates or defects in chromosome synapsis trigger checkpoint-mediated arrest in prophase I. Such 'checkpoints' are important surveillance mechanisms that ensure temporal dependence of cell cycle events. The budding yeast Polo-like kinase, Cdc5, has been identified as a key regulator of the meiosis I chromosome segregation pattern. Here we have analysed the role of Cdc5 in the recombination checkpoint and observed that Polo-like kinase is not required for checkpoint activation in yeast meiosis. Surprisingly, depletion of CDC5 in the Drad17 checkpoint-defective background resulted in nuclear fragmentation to levels even higher than that observed inDdmc1 Drad17 cells that bypass the checkpoint arrest despite accumulating DNA double-strand breaks. The spindle morphology of Cdc5-depleted cells included short, thick metaphase I spindles in mononucleate cells and disassembled spindles in binucleate and tetranucleate cells, although this phenotype does not appear to be the cause of the nuclear fragmentation. An exaggeration of chromosome synapsis defects occurred in Cdc5-depleted Drad17 cells and may contribute to the nuclear fragmentation phenotype. The analysis also uncovered a role for Cdc5 in maintaining spindle integrity in Ddmc1 Drad17 cells. Further analysis confirmed that adaptation to DNA damage does occur in meiosis and that CDC5 is required for this process. The cdc5-ad mutation that renders cells unable to adapt to DNA damage in mitosis did not affect checkpoint adaptation in meiosis, indicating that the mechanisms of checkpoint adaptation in mitosis and meiosis are not fully conserved.     

Mitotic exit regulation through distinct domains within the protein kinase Cdc15

The mitotic exit network (MEN), a Ras-like signaling cascade, promotes the release of the protein phosphatase Cdc14 from the nucleolus and is essential for cells to exit from mitosis in Saccharomyces cerevisiae. We have characterized the functional domains of one of the MEN components, the protein kinase Cdc15, and investigated the role of these domains in mitotic exit. We show that a region adjacent to Cdc15's kinase domain is required for self-association and for binding to spindle pole bodies and that this domain is essential for CDC15 function. Furthermore, we find that overexpression of CDC15 lacking the C-terminal 224 amino acids results in hyperactivation of MEN and premature release of Cdc14 from the nucleolus, suggesting that this domain within Cdc15 functions to inhibit MEN signaling. Our findings indicate that multiple modes of MEN regulation occur through the protein kinase Cdc15.