A quantitative model for ordered Cdk substrate dephosphorylation during mitotic exit

After sister chromatid splitting at anaphase onset, exit from mitosis comprises an ordered series of events. Dephosphorylation of numerous mitotic substrates, which were phosphorylated by cyclin-dependent kinase (Cdk), is thought to bring about mitotic exit, but how temporal ordering of mitotic exit events is achieved is poorly understood. Here, we show, using budding yeast, that dephosphorylation of Cdk substrates involved in sequential mitotic exit events occurs with ordered timing. We test different models of how ordering might be achieved by modulating Cdk and Cdk-counteracting phosphatase Cdc14 activities in vivo, as well as by kinetic analysis of Cdk substrate phosphorylation and dephosphorylation in vitro. Our results suggest that the gradual change of the phosphatase to kinase ratio over the course of mitotic exit is read out by Cdk substrates that respond by dephosphorylation at distinct thresholds. This provides an example and a mechanistic explanation for a quantitative model of cell-cycle progression.     

Cell Cycle Control by a Minimal Cdk Network

In present-day eukaryotes, the cell division cycle is controlled by a complex network of interacting proteins, including members of the cyclin and cyclin-dependent protein kinase (Cdk) families, and the Anaphase Promoting Complex (APC). Successful progression through the cell cycle depends on precise, temporally ordered regulation of the functions of these proteins. In light of this complexity, it is surprising that in fission yeast, a minimal Cdk network consisting of a single cyclin-Cdk fusion protein can control DNA synthesis and mitosis in a manner that is indistinguishable from wild type. To improve our understanding of the cell cycle regulatory network, we built and analysed a mathematical model of the molecular interactions controlling the G1/S and G2/M transitions in these minimal cells. The model accounts for all observed properties of yeast strains operating with the fusion protein. Importantly, coupling the model’s predictions with experimental analysis of alternative minimal cells, we uncover an explanation for the unexpected fact that elimination of inhibitory phosphorylation of Cdk is benign in these strains while it strongly affects normal cells. Furthermore, in the strain without inhibitory phosphorylation of the fusion protein, the distribution of cell size at division is unusually broad, an observation that is accounted for by stochastic simulations of the model. Our approach provides novel insights into the organization and quantitative regulation of wild type cell cycle progression. In particular, it leads us to propose a new mechanistic model for the phenomenon of mitotic catastrophe, relying on a combination of unregulated, multi-cyclin-dependent Cdk activities.     

Phosphoproteome dynamics during mitotic exit in budding yeast

The cell division cycle culminates in mitosis when two daughter cells are born. As cyclin‐dependent kinase (Cdk) activity reaches its peak, the anaphase‐promoting complex/cyclosome (APC/C) is activated to trigger sister chromatid separation and mitotic spindle elongation, followed by spindle disassembly and cytokinesis. Degradation of mitotic cyclins and activation of Cdk‐counteracting phosphatases are thought to cause protein dephosphorylation to control these sequential events. Here, we use budding yeast to analyze phosphorylation dynamics of 3,456 phosphosites on 1,101 proteins with high temporal resolution as cells progress synchronously through mitosis. This reveals that successive inactivation of S and M phase Cdks and of the mitotic kinase Polo contributes to order these dephosphorylation events. Unexpectedly, we detect as many new phosphorylation events as there are dephosphorylation events. These correlate with late mitotic kinase activation and identify numerous candidate targets of these kinases. These findings revise our view of mitotic exit and portray it as a dynamic process in which a range of mitotic kinases contribute to order both protein dephosphorylation and phosphorylation.     

Effect of positive feedback loops on the robustness of oscillations in the network of cyclin-dependent kinases driving the mammalian cell cycle

The transitions between the G(1) , S, G(2) and M phases of the mammalian cell cycle are driven by a network of cyclin-dependent kinases (Cdks), whose sequential activation is regulated by intertwined negative and positive feedback loops. We previously proposed a detailed computational model for the Cdk network, and showed that this network is capable of temporal self-organization in the form of sustained oscillations, which govern ordered progression through the successive phases of the cell cycle [Gérard and Goldbeter (2009) Proc Natl Acad Sci USA106, 21643-21648]. We subsequently proposed a skeleton model for the cell cycle that retains the core regulatory mechanisms of the detailed model [Gérard and Goldbeter (2011) Interface Focus1, 24-35]. Here we extend this skeleton model by incorporating Cdk regulation through phosphorylation/dephosphorylation and by including the positive feedback loops that underlie the dynamics of the G(1) /S and G(2) /M transitions via phosphatase Cdc25 and via phosphatase Cdc25 and kinase Wee1, respectively. We determine the effects of these positive feedback loops and ultrasensitivity in phosphorylation/dephosphorylation on the dynamics of the Cdk network. The multiplicity of positive feedback loops as well as the existence of ultrasensitivity promote the occurrence of bistability and increase the amplitude of the oscillations in the various cyclin/Cdk complexes. By resorting to stochastic simulations, we further show that the presence of multiple, redundant positive feedback loops in the G(2) /M transition of the cell cycle markedly enhances the robustness of the Cdk oscillations with respect to molecular noise.     

Cdk-counteracting phosphatases unlock mitotic exit

Entry into mitosis of the eukaryotic cell cycle is driven by rising cyclin-dependent kinase (Cdk) activity. During exit from mitosis, Cdk activity must again decline. Cdk downregulation by itself, however, is not able to guide mitotic exit, if not a phosphatase reverses mitotic Cdk phosphorylation events. In budding yeast, this role is played by the Cdc14 phosphatase. We are gaining an increasingly detailed picture of its regulation during anaphase, and of the way it orchestrates ordered progression through mitosis. Much less is known about protein dephosphorylation during mitotic exit in organisms other than budding yeast, but evidence is now mounting for crucial contributions of regulated phosphatases also in metazoan cells.     

The Coordination of Centrosome Reproduction with Nuclear Events of the Cell Cycle in the Sea Urchin Zygote

Centrosomes repeatedly reproduce in sea urchin zygotes arrested in S phase, whether cyclin-dependent kinase 1–cyclin B (Cdk1-B) activity remains at prefertilization levels or rises to mitotic values. In contrast, when zygotes are arrested in mitosis using cyclin B Δ-90, anaphase occurs at the normal time, yet centrosomes do not reproduce. Together, these results reveal the cell cycle stage specificity for centrosome reproduction and demonstrate that neither the level nor the cycling of Cdk1-B activity coordinate centrosome reproduction with nuclear events. In addition, the proteolytic events of the metaphase–anaphase transition do not control when centrosomes duplicate. When we block protein synthesis at first prophase, the zygotes divide and arrest before second S phase. Both blastomeres contain just two complete centrosomes, which indicates that the cytoplasmic conditions between mitosis and S phase support centrosome reproduction. However, the fact that these daughter centrosomes do not reproduce again under such supportive conditions suggests that they are lacking a component required for reproduction. The repeated reproduction of centrosomes during S phase arrest points to the existence of a necessary “licensing” event that restores this component to daughter centrosomes during S phase, preparing them to reproduce in the next cell cycle.     

Drosophila Wee1 kinase regulates Cdk1 and mitotic entry during embryogenesis

Cyclin-dependent kinases (Cdks) are the central regulators of the cell division cycle. Inhibitors of Cdks ensure proper coordination of cell cycle events and help regulate cell proliferation in the context of tissues and organs. Wee1 homologs phosphorylate a conserved tyrosine to inhibit the mitotic cyclin-dependent kinase Cdk1. Loss of Wee1 function in fission or budding yeast causes premature entry into mitosis. The importance of metazoan Wee1 homologs for timing mitosis, however, has been demonstrated only in Xenopus egg extracts and via ectopic Cdk1 activation . Here, we report that Drosophila Wee1 (dWee1) regulates Cdk1 via phosphorylation of tyrosine 15 and times mitotic entry during the cortical nuclear cycles of syncytial blastoderm embryos, which lack gap phases. Loss of maternal dwee1 leads to premature entry into mitosis, mitotic spindle defects, chromosome condensation problems, and a Chk2-dependent block of subsequent development, and then embryonic lethality. These findings modify previous models about cell cycle regulation in syncytial embryos and demonstrate that Wee1 kinases can regulate mitotic entry in vivo during metazoan development even in cycles that lack a G2 phase.     

Cell Cycle Sibling Rivalry: Cdc2 Versus Cdk2

It has been long believed that the cyclin-dependent kinase 2 [Cdk2] binds to cyclin E or cyclin Aand exclusively promotes the G1/S phase transition and that Cdc2/cyclin B complexes play a majorrole in mitosis. We now provide evidence that Cdc2 binds to cyclin E [in addition to cyclin A & B]and is able to promote the G1/S transition. This new concept indicates that both Cdk2 and/or Cdc2can drive cells through G1/S phase in parallel. In this review we discuss the classic cell cycle modeland how results from knockout mice provide new evidence that refute this model. We focus on newroles of Cdc2 and p27 in regulating the mammalian cell cycle and propose a new model for cellcycle regulation that accommodates these novel findings.     

Minimal Models for Cell-Cycle Control Based on Competitive Inhibition and?Multisite Phosphorylations of Cdk Substrates

The eukaryotic cell cycle is characterized by alternating oscillations in the activities of cyclin-dependent kinase (Cdk) and the anaphase-promoting complex (APC). Successful completion of the cell cycle is dependent on the precise, temporally ordered appearance of these activities. A modest level of Cdk activity is sufficient to initiate DNA replication, but mitosis and APC activation require an elevated Cdk activity. In present-day eukaryotes, this temporal order is provided by a complex network of regulatory proteins that control both Cdk and APC activities via sharp thresholds, bistability, and time delays. Using simple computational models, we show here that these dynamical features of cell-cycle organization could emerge in a control system driven by a single Cdk/cyclin complex and APC wired in a negative-feedback loop. We show that ordered phosphorylation of cellular proteins could be explained by multisite phosphorylation/dephosphorylation and competition of substrates for interconverting kinase (Cdk) and phosphatase. In addition, the competition of APC substrates for ubiquitylation can create and maintain sustained oscillations in cyclin levels. We propose a sequence of models that gets closer and closer to a realistic model of cell-cycle control in yeast. Since these models lack the elaborate control mechanisms characteristic of modern eukaryotes, they suggest that bistability and time delay may have characterized eukaryotic cell divisions before the current cell-cycle control network evolved in all its complexity.     

The DASH complex component Ask1 is a cell cycle-regulated Cdk substrate in Saccharomyces cerevisiae

The proper timing and fidelity of cell cycle transitions is critical for the survival of organisms. Cyclin-dependent kinases orchestrate many cell cycle transitions in eukaryotes including S phase entry and mitosis. Accurate chromosome segregation during mitosis is one of the key events regulated by the cell cycle and many proteins function together to ensure the fidelity of this process. In S. cerevisiae, the DASH complex is essential for chromosome segregation. The DASH complex binds to microtubules and kinetochores and regulates their association. Here we report that Askl, one component of DASH, is phosphorylated during the cell cycle. This phosphorylation is dependent on Cdks in vivo, and in vitro Cdc28 can phosphorylate Askl. We identify two Cdk phosphorylation sites in Askl and find that the phosphorylation of Askl is important for its full activity in vivo. Thus, the DASH complex is directly regulated by cyclin-dependent kinases to facilitate chromosome segregation.     

Human Cdc14A becomes a cell cycle gene in controlling Cdk1 activity at the G2/M transition

Cdc14 belongs to a dual-specificity phosphatase family highly conserved through evolution that preferentially reverses CDK (Cyclin dependent kinases) –dependent phosphorylation events. In the yeast Saccharomyces cerevisiae, Cdc14 is an essential regulator of late mitotic events and exit from mitosis by counteracting CDK activity at the end of mitosis. However, many studies have shown that Cdc14 is dispensable for exiting mitosis in all other model systems analyzed. In fission yeast, the Cdc14 homologue Flp1/Clp1 regulates the stability of the mitotic inducer Cdc25 at the end of mitosis to ensure Cdk1 inactivation before cytokinesis. We have recently reported that human Cdc14A, the Cdc14 isoform located at the centrosomes during interphase, down-regulates Cdc25 activity at the G2/M transition to prevent premature activation of Cdk1-Cyclin B1 complexes and untimely entry into mitosis. Here we speculate about new molecular mechanisms for Cdc14A and discuss the current evidence suggesting that Cdc14 phosphatase plays a role in cell cycle control in higher eukaryotes.     

The DASH Complex Component Ask1 Is a Cell Cycle-Regulated Cdk Substrate in Saccharomyces cerevisiae

The proper timing and fidelity of cell cycle transitions is critical for the survival of organisms. Cyclin-dependent kinases orchestrate many cell cycle transitions in eukaryotes including S phase entry and mitosis. Accurate chromosome segregation during mitosis is one of the key events regulated by the cell cycle and many proteins function together to ensure the fidelity of this process. In S. cerevisiae, the DASH complex is essential for chromosome segregation. The DASH complex binds to microtubules and kinetochores and regulates their association. Here we report that Ask1, one component of DASH, is phosphorylated during the cell cycle. This phosphorylation is dependent on Cdks in vivo, and in vitro Cdc28 can phosphorylate Ask1. We identify two Cdk phosphorylation sites in Ask1 and find that the phosphorylation of Ask1 is important for its full activity in vivo. Thus, the DASH complex is directly regulated by cyclin-dependent kinases to facilitate chromosome segregation.     

Cyclin D1 serves as a cell cycle regulatory switch in actively proliferating cells

Much of our current understanding of the cell cycle involves analyses of its induction in quiescent cells. To better understand the control of cell cycle propagation and termination, studies have been performed in actively cycling cultures using time-lapse photography and quantitative image analysis. These studies reveal a highly ordered sequence of events required for promotion of continued proliferation. The decision to continue cell cycle progression takes place in G2 phase, when cellular Ras induces the elevation of cyclin D1 levels. These levels are maintained through G1 phase and are required for the initiation of S phase, at which time cyclin D1 levels are automatically reduced to low levels. The reduction of cyclin D1 to low levels during S phase is required for DNA synthesis, and forces the cell to induce high cyclin D1 levels once again when it enters G2 phase. In this way, cyclin D1 is proposed to serve as an active switch in the regulation of continued cell cycle progression.     

S and G2 phase roles for Cdk2 revealed by inducible expression of a dominant-negative mutant in human cells

Cyclin-dependent kinase 2 (Cdk2) is essential for initiation of DNA synthesis in higher eukaryotes. Biochemical studies in Xenopus egg extracts and microinjection studies in human cells have suggested an additional function for Cdk2 in activation of Cdk1 and entry into mitosis. To further examine the role of Cdk2 in human cells, we generated stable clones with inducible expression of wild-type and dominant-negative forms of the enzyme (Cdk2-wt and Cdk2-dn, respectively). Both exogenous proteins associated efficiently with endogenous cyclins. Cdk2-wt had no apparent effect on the cell division cycle, whereas Cdk2-dn inhibited progression through several distinct stages. Cdk2-dn induction could arrest cells at the G1/S transition, as previously observed in transient expression studies. However, under normal culture conditions, Cdk2-dn induction primarily arrested cells with S and G2/M DNA contents. Several observations suggested that the latter cells were in G2 phase, prior to the onset of mitosis: these cells contained uncondensed chromosomes, low levels of cyclin B-associated kinase activity, and high levels of tyrosine-phosphorylated Cdk1. Furthermore, Cdk2-dn did not delay progression through mitosis upon release of cells from a nocodazole block. Although the G2 arrest imposed by Cdk2-dn was similar to that imposed by the DNA damage checkpoint, the former was distinguished by its resistance to caffeine. These findings provide evidence for essential functions of Cdk2 during S and G2 phases of the mammalian cell cycle.