scholarly journals Genetic and Biochemical Evaluation of the Importance of Cdc6 in Regulating Mitotic Exit

2003 ◽  
Vol 14 (11) ◽  
pp. 4592-4604 ◽  
Author(s):  
Vincent Archambault ◽  
Caihong X. Li ◽  
Alan J. Tackett ◽  
Ralph Wäsch ◽  
Brian T. Chait ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Mitotic Exit ◽  
Terminal Region ◽  
Cyclin Dependent Kinase ◽  
Replication Control ◽  
Mitotic Cyclin ◽  
Biochemical Evaluation ◽  
Inhibitory Domain ◽  
Late Mitosis ◽  
Control Protein

We evaluated the hypothesis that the N-terminal region of the replication control protein Cdc6 acts as an inhibitor of cyclin-dependent kinase (Cdk) activity, promoting mitotic exit. Cdc6 accumulation is restricted to the period from mid-cell cycle until the succeeding G1, due to proteolytic control that requires the Cdc6 N-terminal region. During late mitosis, Cdc6 is present at levels comparable with Sic1 and binds specifically to the mitotic cyclin Clb2. Moderate overexpression of Cdc6 promotes viability of CLB2Δdb strains, which otherwise arrest at mitotic exit, and rescue is dependent on the N-terminal putative Cdk-inhibitory domain. These observations support the potential for Cdc6 to inhibit Clb2-Cdk, thus promoting mitotic exit. Consistent with this idea, we observed a cytokinesis defect in cdh1Δ sic1Δ cdc6Δ2–49 triple mutants. However, we were able to construct viable strains, in three different backgrounds, containing neither SIC1 nor the Cdc6 Cdk-inhibitory domain, in contradiction to previous work. We conclude, therefore, that although both Cdc6 and Sic1 have the potential to facilitate mitotic exit by inhibiting Clb2-Cdk, mitotic exit nevertheless does not require any identified stoichiometric inhibitor of Cdk activity.

scholarly journals Separase cooperates with Zds1 and Zds2 to activate Cdc14 phosphatase in early anaphase

2008 ◽  
Vol 182 (5) ◽  
pp. 873-883 ◽  
Author(s):  
Ethel Queralt ◽  
Frank Uhlmann
Keyword(s):  
Cell Cycle ◽  
Budding Yeast ◽  
Mitotic Exit ◽  
Interacting Proteins ◽  
Cyclin Dependent Kinase ◽  
Down Regulation ◽  
Anaphase Onset ◽  
Early Anaphase ◽  
Key Enzyme ◽  
Mitotic Cyclin

Completion of mitotic exit and cytokinesis requires the inactivation of mitotic cyclin-dependent kinase (Cdk) activity. A key enzyme that counteracts Cdk during budding yeast mitotic exit is the Cdc14 phosphatase. Cdc14 is inactive for much of the cell cycle, sequestered by its inhibitor Net1 in the nucleolus. At anaphase onset, separase-dependent down-regulation of PP2ACdc55 allows phosphorylation of Net1 and consequent Cdc14 release. How separase causes PP2ACdc55 down-regulation is not known. Here, we show that two Cdc55-interacting proteins, Zds1 and Zds2, contribute to timely Cdc14 activation during mitotic exit. Zds1 and Zds2 are required downstream of separase to facilitate nucleolar Cdc14 release. Ectopic Zds1 expression in turn is sufficient to down-regulate PP2ACdc55 and promote Net1 phosphorylation. These findings identify Zds1 and Zds2 as new components of the mitotic exit machinery, involved in activation of the Cdc14 phosphatase at anaphase onset. Our results suggest that these proteins may act as separase-regulated PP2ACdc55 inhibitors.

scholarly journals Budding yeast relies on G1 cyclin specificity to couple cell cycle progression with morphogenetic development

2021 ◽  
Vol 7 (23) ◽  
pp. eabg0007
Author(s):  
Deniz Pirincci Ercan ◽  
Florine Chrétien ◽  
Probir Chakravarty ◽  
Helen R. Flynn ◽  
Ambrosius P. Snijders ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Budding Yeast ◽  
Quantitative Model ◽  
Cyclin Dependent Kinase ◽  
Cycle Progression ◽  
Model Cell ◽  
Mitotic Cyclin ◽  
Substrate Phosphorylation ◽  
The Cost

Two models have been put forward for cyclin-dependent kinase (Cdk) control of the cell cycle. In the qualitative model, cell cycle events are ordered by distinct substrate specificities of successive cyclin waves. Alternatively, in the quantitative model, the gradual rise of Cdk activity from G1 phase to mitosis leads to ordered substrate phosphorylation at sequential thresholds. Here, we study the relative contributions of qualitative and quantitative Cdk control in Saccharomyces cerevisiae. All S phase and mitotic cyclins can be replaced by a single mitotic cyclin, albeit at the cost of reduced fitness. A single cyclin can also replace all G1 cyclins to support ordered cell cycle progression, fulfilling key predictions of the quantitative model. However, single-cyclin cells fail to polarize or grow buds and thus cannot survive. Our results suggest that budding yeast has become dependent on G1 cyclin specificity to couple cell cycle progression to essential morphogenetic events.

scholarly journals Disruption of Centrosome Structure, Chromosome Segregation, and Cytokinesis by Misexpression of Human Cdc14A Phosphatase

2002 ◽  
Vol 13 (7) ◽  
pp. 2289-2300 ◽  
Author(s):  
Brett K. Kaiser ◽  
Zachary A. Zimmerman ◽  
Harry Charbonneau ◽  
Peter K. Jackson
Keyword(s):  
Cell Cycle ◽  
Phosphatase Activity ◽  
Chromosome Segregation ◽  
Mitotic Spindle ◽  
Genomic Stability ◽  
Mitotic Exit ◽  
Protein Abundance ◽  
Cyclin Dependent Kinase ◽  
Chromosome Partitioning

In budding yeast, the Cdc14p phosphatase activates mitotic exit by dephosphorylation of specific cyclin-dependent kinase (Cdk) substrates and seems to be regulated by sequestration in the nucleolus until its release in mitosis. Herein, we have analyzed the two human homologs of Cdc14p, hCdc14A and hCdc14B. We demonstrate that the human Cdc14A phosphatase is selective for Cdk substrates in vitro and that although the protein abundance and intrinsic phosphatase activity of hCdc14A and B vary modestly during the cell cycle, their localization is cell cycle regulated. hCdc14A dynamically localizes to interphase but not mitotic centrosomes, and hCdc14B localizes to the interphase nucleolus. These distinct patterns of localization suggest that each isoform of human Cdc14 likely regulates separate cell cycle events. In addition, hCdc14A overexpression induces the loss of the pericentriolar markers pericentrin and γ-tubulin from centrosomes. Overproduction of hCdc14A also causes mitotic spindle and chromosome segregation defects, defective karyokinesis, and a failure to complete cytokinesis. Thus, the hCdc14A phosphatase appears to play a role in the regulation of the centrosome cycle, mitosis, and cytokinesis, thereby influencing chromosome partitioning and genomic stability in human cells.

scholarly journals Intrinsic and Cyclin-dependent Kinase-dependent Control of Spindle Pole Body Duplication in Budding Yeast

2008 ◽  
Vol 19 (8) ◽  
pp. 3243-3253 ◽  
Author(s):  
Laura A. Simmons Kovacs ◽  
Christine L. Nelson ◽  
Steven B. Haase
Keyword(s):  
Cell Cycle ◽  
Budding Yeast ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Cyclin Dependent Kinase ◽  
Multipolar Spindle ◽  
Cyclin Dependent Kinases ◽  
Pole Body ◽  
Intrinsic Mechanism ◽  
Mitotic Cyclin

Centrosome duplication must be tightly controlled so that duplication occurs only once each cell cycle. Accumulation of multiple centrosomes can result in the assembly of a multipolar spindle and lead to chromosome mis-segregation and genomic instability. In metazoans, a centrosome-intrinsic mechanism prevents reduplication until centriole disengagement. Mitotic cyclin/cyclin-dependent kinases (CDKs) prevent reduplication of the budding yeast centrosome, called a spindle pole body (SPB), in late S-phase and G2/M, but the mechanism remains unclear. How SPB reduplication is prevented early in the cell cycle is also not understood. Here we show that, similar to metazoans, an SPB-intrinsic mechanism prevents reduplication early in the cell cycle. We also show that mitotic cyclins can inhibit SPB duplication when expressed before satellite assembly in early G1, but not later in G1, after the satellite had assembled. Moreover, electron microscopy revealed that SPBs do not assemble a satellite in cells expressing Clb2 in early G1. Finally, we demonstrate that Clb2 must localize to the cytoplasm in order to inhibit SPB duplication, suggesting the possibility for direct CDK inhibition of satellite components. These two mechanisms, intrinsic and extrinsic control by CDK, evoke two-step system that prevents SPB reduplication throughout the cell cycle.

scholarly journals Mitotic Exit in the Absence of Separase Activity

2009 ◽  
Vol 20 (5) ◽  
pp. 1576-1591 ◽  
Author(s):  
Ying Lu ◽  
Frederick Cross
Keyword(s):  
Nuclear Export ◽  
Spindle Pole Body ◽  
Quantitative Measure ◽  
Spindle Pole ◽  
Mitotic Exit ◽  
Cyclin Dependent Kinase ◽  
Sister Chromatids ◽  
Early Anaphase ◽  
Mitotic Cyclin ◽  
A Minor

In budding yeast, three interdigitated pathways regulate mitotic exit (ME): mitotic cyclin–cyclin-dependent kinase (Cdk) inactivation; the Cdc14 early anaphase release (FEAR) network, including a nonproteolytic function of separase (Esp1); and the mitotic exit network (MEN) driven by interaction between the spindle pole body and the bud cortex. Here, we evaluate the contributions of these pathways to ME kinetics. Reducing Cdk activity is critical for ME, and the MEN contributes strongly to ME efficiency. Esp1 contributes to ME kinetics mainly through cohesin cleavage: the Esp1 requirement can be largely bypassed if cells are provided Esp1-independent means of separating sister chromatids. In the absence of Esp1 activity, we observed only a minor ME delay consistent with a FEAR defect. Esp1 overexpression drives ME in Cdc20-depleted cells arrested in metaphase. We have found that this activity of overexpressed Esp1 depended on spindle integrity and the MEN. We defined the first quantitative measure for Cdc14 release based on colocalization with the Net1 nucleolar anchor. This measure indicates efficient Cdc14 release upon MEN activation; release driven by Esp1 in the absence of microtubules was inefficient and incapable of driving ME. We also found a novel role for the MEN: activating Cdc14 nuclear export, even in the absence of Net1.

scholarly journals A Late Mitotic Regulatory Network Controlling Cyclin Destruction in Saccharomyces cerevisiae

1998 ◽  
Vol 9 (10) ◽  
pp. 2803-2817 ◽  
Author(s):  
Sue L. Jaspersen ◽  
Julia F. Charles ◽  
Rachel L. Tinker-Kulberg ◽  
David O. Morgan
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Regulatory Network ◽  
Growth Defect ◽  
Cyclin Dependent Kinase ◽  
Anaphase Promoting Complex ◽  
Yeast Saccharomyces Cerevisiae ◽  
Mitotic Cyclin ◽  
Low Levels ◽  
Late Mitosis ◽  
Mutant Cells

Exit from mitosis requires the inactivation of mitotic cyclin-dependent kinase–cyclin complexes, primarily by ubiquitin-dependent cyclin proteolysis. Cyclin destruction is regulated by a ubiquitin ligase known as the anaphase-promoting complex (APC). In the budding yeast Saccharomyces cerevisiae, members of a large class of late mitotic mutants, including cdc15,cdc5, cdc14, dbf2, andtem1, arrest in anaphase with a phenotype similar to that of cells expressing nondegradable forms of mitotic cyclins. We addressed the possibility that the products of these genes are components of a regulatory network that governs cyclin proteolysis. We identified a complex array of genetic interactions among these mutants and found that the growth defect in most of the mutants is suppressed by overexpression of SPO12, YAK1, andSIC1 and is exacerbated by overproduction of the mitotic cyclin Clb2. When arrested in late mitosis, the mutants exhibit a defect in cyclin-specific APC activity that is accompanied by high Clb2 levels and low levels of the anaphase inhibitor Pds1. Mutant cells arrested in G1 contain normal APC activity. We conclude that Cdc15, Cdc5, Cdc14, Dbf2, and Tem1 cooperate in the activation of the APC in late mitosis but are not required for maintenance of that activity in G1.

scholarly journals Oscillations in Cdc14 release and sequestration reveal a circuit underlying mitotic exit

2010 ◽  
Vol 190 (2) ◽  
pp. 209-222 ◽  
Author(s):  
Romilde Manzoni ◽  
Francesca Montani ◽  
Clara Visintin ◽  
Fabrice Caudron ◽  
Andrea Ciliberto ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Feedback Loop ◽  
Mitotic Exit ◽  
Centrosome Duplication ◽  
Negative Feedback Loop ◽  
Cyclin Dependent Kinase ◽  
Bud Formation ◽  
Mitotic Exit Network ◽  
Early Anaphase ◽  
Periodic Cycles

In budding yeast, the phosphatase Cdc14 orchestrates progress through anaphase and mitotic exit, thereby resetting the cell cycle for a new round of cell division. Two consecutive pathways, Cdc fourteen early anaphase release (FEAR) and mitotic exit network (MEN), contribute to the progressive activation of Cdc14 by regulating its release from the nucleolus, where it is kept inactive by Cfi1. In this study, we show that Cdc14 activation requires the polo-like kinase Cdc5 together with either Clb–cyclin-dependent kinase (Cdk) or the MEN kinase Dbf2. Once active, Cdc14 triggers a negative feedback loop that, in the presence of stable levels of mitotic cyclins, generates periodic cycles of Cdc14 release and sequestration. Similar phenotypes have been described for yeast bud formation and centrosome duplication. A common theme emerges where events that must happen only once per cycle, although intrinsically capable of oscillations, are limited to one occurrence by the cyclin–Cdk cell cycle engine.

scholarly journals The budding yeast Dbf2 protein kinase localises to the centrosome and moves to the bud neck in late mitosis

2000 ◽  
Vol 113 (19) ◽  
pp. 3399-3408 ◽  
Author(s):  
L.M. Frenz ◽  
S.E. Lee ◽  
D. Fesquet ◽  
L.H. Johnston
Keyword(s):  
Cell Cycle ◽  
Protein Kinase ◽  
Preliminary Investigation ◽  
Spindle Pole ◽  
Mitotic Exit ◽  
Temperature Sensitive ◽  
Multifunctional Protein ◽  
Double Ring ◽  
Bud Neck ◽  
Late Mitosis

Dbf2 is a multifunctional protein kinase in Saccharomyces cerevisiae that functions in transcription, the stress response and as part of a network of genes in exit from mitosis. By analogy with fission yeast it seemed likely that these mitotic exit genes would be involved in cytokinesis. As a preliminary investigation of this we have used Dbf2 tagged with GFP to examine intracellular localisation of the protein in living cells. Dbf2 is found on the centrosomes/spindle pole bodies (SPBs) and also at the bud neck where it forms a double ring. The localisation of Dbf2 is cell cycle regulated. It is on the SPBs for much of the cell cycle and migrates from there to the bud neck in late mitosis, consistent with a role in cytokinesis. Dbf2 partly co-localises with septins at the bud neck. A temperature-sensitive mutant of dbf2 also blocks progression of cytokinesis at 37 degrees C. Following cytokinesis some Dbf2 moves into the nascent bud. Localisation to the bud neck depends upon the septins and also the mitotic exit network proteins Mob1, Cdc5, Cdc14 and Cdc15. The above data are consistent with Dbf2 acting downstream in a pathway controlling cytokinesis.

scholarly journals The yeast cyclin-dependent kinase inhibitor Sic1 and mammalian p27Kip1 are functional homologues with a structurally conserved inhibitory domain

2005 ◽  
Vol 387 (3) ◽  
pp. 639-647 ◽  
Author(s):  
Matteo BARBERIS ◽  
Luca DE GIOIA ◽  
Maria RUZZENE ◽  
Stefania SARNO ◽  
Paola COCCETTI ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Dna Replication ◽  
Kinase Inhibitor ◽  
Molecular Model ◽  
Cyclin A ◽  
Cyclin Dependent Kinase ◽  
Protein Fusion ◽  
Inhibitory Domain ◽  
Functional Homologues

In Saccharomyces cerevisiae, Sic1, an inhibitor of Cdk (cyclin-dependent kinase), blocks the activity of S-Cdk1 (Cdk1/Clb5,6) kinase that is required for DNA replication. Deletion of Sic1 causes premature DNA replication from fewer origins, extension of the S phase and inefficient separation of sister chromatids during anaphase. Despite the well-documented relevance of Sic1 inhibition of S-Cdk1 for cell cycle control and genome instability, the molecular mechanism by which Sic1 inhibits S-Cdk1 activity remains obscure. In this paper, we show that Sic1 is functionally and structurally related to the mammalian Cki (Cdk inhibitor) p27Kip1 of the Kip/Cip family. A molecular model of the inhibitory domain of Sic1 bound to the Cdk2–cyclin A complex suggested that the yeast inhibitor might productively interface with the mammalian Cdk2–cyclin A complex. Consistent with this, Sic1 is able to bind to, and strongly inhibit the kinase activity of, the Cdk2–cyclin A complex. In addition, comparison of the different inhibitory patterns obtained using histone H1 or GST (glutathione S-transferase)–pRb (retinoblastoma protein) fusion protein as substrate (the latter of which recognizes both the docking site and the catalytic site of Cdk2–cyclin A) offers interesting suggestions for the inhibitory mechanism of Sic1. Finally, overexpression of the KIP1 gene in vivo in Saccharomyces cerevisiae, like overexpression of the related SIC1 gene, rescues the cell cycle-related phenotype of a sic1Δ strain. Taken together, these findings strongly indicate that budding yeast Sic1 and mammalian p27Kip1 are functional homologues with a structurally conserved inhibitory domain.

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