Centrosome-associated Chk1 prevents premature activation of cyclin-B-Cdk1 kinase

Entry into mitosis occurs after activation of Cdk1, resulting in chromosome condensation in the nucleus and centrosome separation, as well as increased microtubule nucleation activity in the cytoplasm. The active cyclin-B1-Cdk1 complex first appears at the centrosome, suggesting that the centrosome may facilitate the activation of mitotic regulators required for the commitment of cells to mitosis. However, the signalling pathways involved in controlling the initial activation of Cdk1 at the centrosome remain largely unknown. Here, we show that human Chk1 kinase localizes to interphase, but not mitotic, centrosomes. Chemical inhibition of Chk1 resulted in premature centrosome separation and activation of centrosome-associated Cdk1. Forced immobilization of kinase-inactive Chk1 to centrosomes also resulted in premature Cdk1 activation. Conversely, under such conditions wild-type Chk1 impaired activation of centrosome-associated Cdk1, thereby resulting in DNA endoreplication and centrosome amplification. Activation of centrosomal Cdk1 in late prophase seemed to be mediated by cytoplasmic Cdc25B, whose activity is controlled by centrosome-associated Chk1. These results suggest that centrosome-associated Chk1 shields centrosomal Cdk1 from unscheduled activation by cytoplasmic Cdc25B, thereby contributing to proper timing of the initial steps of cell division, including mitotic spindle formation.     

Cdc25B cooperates with Cdc25A to induce mitosis but has a unique role in activating cyclin B1-Cdk1 at the centrosome

Cdc25 phosphatases are essential for the activation of mitotic cyclin-Cdks, but the precise roles of the three mammalian isoforms (A, B, and C) are unclear. Using RNA interference to reduce the expression of each Cdc25 isoform in HeLa and HEK293 cells, we observed that Cdc25A and -B are both needed for mitotic entry, whereas Cdc25C alone cannot induce mitosis. We found that the G2 delay caused by small interfering RNA to Cdc25A or -B was accompanied by reduced activities of both cyclin B1-Cdk1 and cyclin A-Cdk2 complexes and a delayed accumulation of cyclin B1 protein. Further, three-dimensional time-lapse microscopy and quantification of Cdk1 phosphorylation versus cyclin B1 levels in individual cells revealed that Cdc25A and -B exert specific functions in the initiation of mitosis: Cdc25A may play a role in chromatin condensation, whereas Cdc25B specifically activates cyclin B1-Cdk1 on centrosomes.     

Centrosomal Chk2 in DNA damage responses and cell cycle progession

Two major control systems regulate early stages of mitosis: activation of Cdk1 and anaphase control through assembly and disassembly of the mitotic spindle. In parallel to cell cycle progression, centrosomal duplication is regulated through proteins including Nek2. Recent studies suggest that centrosome-localized Chk1 forestalls premature activation of centrosomal Cdc25b and Cdk1 for mitotic entry, whereas Chk2 binds centrosomes and arrests mitosis only after activation by ATM and ATR in response to DNA damage. Here, we show that Chk2 centrosomal binding does not require DNA damage, but varies according to cell cycle progression. These and other data suggest a model in which binding of Chk2 to the centrosome at multiple cell cycle junctures controls co-localization of Chk2 with other cell cycle and centrosomal regulators.     

Centrosomal Chk2 in DNA damage responses and cell cycle progession

Two major control systems regulate early stages of mitosis: activation of Cdk1 and anaphase control through assembly and disassembly of the mitotic spindle. In parallel to cell cycle progression, centrosomal duplication is regulated through proteins including Nek2. Recent studies suggest that centrosome-localized Chk1 forestalls premature activation of centrosomal Cdc25b and Cdk1 for mitotic entry, whereas Chk2 binds centrosomes and arrests mitosis only after activation by ATM and ATR in response to DNA damage. Here, we show that Chk2 centrosomal binding does not require DNA damage, but varies according to cell cycle progression. These and other data suggest a model in which binding of Chk2 to the centrosome at multiple cell cycle junctures controls co-localization of Chk2 with other cell cycle and centrosomal regulators.Key words: Chk2, centrosome, checkpoint, DNA damage, wild type, kinase-defective     

Dynamic Changes in Nuclear Architecture during Mitosis: On the Role of Protein Phosphorylation in Spindle Assembly and Chromosome Segregation

During mitosis, the vertebrate cell nucleus undergoes profound changes in architecture. At the onset of mitosis, the nuclear envelope breaks down, the nuclear lamina is depolymerized, and interphase chromatin is condensed to chromosomes. Concomitantly, cytoplasmic microtubules are reorganized into a mitotic spindle apparatus, a highly dynamic structure required for the segregation of sister chromatids. Many of the above events are controlled by reversible phosphorylation. Hence, our laboratory is interested in characterizing the kinases involved in promoting progression through mitosis and in identifying their relevant substrates. Prominent among the kinases responsible for regulating entry into mitosis is the Cdc2 kinase, the first member of the cyclin dependent kinase (Cdk) family. Recently, we found that Cdc2 phosphorylates HsEg5, a human kinesin-related motor protein associated with centrosomes and the spindle apparatus. Our results indicate that phosphorylation regulates the association of HsEg5 with the mitotic spindle and that the function of this plus-end directed motor is essential for centrosome separation and bipolar spindle formation. Another kinase implicated in regulating progression through mitosis is Plk1 (polo-like kinase 1), the human homologue of theDrosophilagene product “polo.” By antibody microinjection we have found that Plk1 is required for the functional maturation of centrosomes and hence for entry into mitosis. Furthermore, we found that microinjected anti-Plk1 antibodies caused a more severe block to cell cycle progression in diploid fibroblasts than in immortalized tumor cells. This observation hints at the existence of a checkpoint linking Cdc2 activation to the presence of functional centrosomes.     

MCPH1 regulates the neuroprogenitor division mode by coupling the centrosomal cycle with mitotic entry through the Chk1-Cdc25 pathway

Primary microcephaly 1 is a neurodevelopmental disorder caused by mutations in the MCPH1 gene, whose product MCPH1 (also known as microcephalin and BRIT1) regulates DNA-damage response. Here we show that Mcph1 disruption in mice results in primary microcephaly, mimicking human MCPH1 symptoms, owing to a premature switching of neuroprogenitors from symmetric to asymmetric division. MCPH1-deficiency abrogates the localization of Chk1 to centrosomes, causing premature Cdk1 activation and early mitotic entry, which uncouples mitosis and the centrosome cycle. This misorients the mitotic spindle alignment and shifts the division plane of neuroprogenitors, to bias neurogenic cell fate. Silencing Cdc25b, a centrosome substrate of Chk1, corrects MCPH1-deficiency-induced spindle misalignment and rescues the premature neurogenic production in Mcph1-knockout neocortex. Thus, MCPH1, through its function in the Chk1-Cdc25-Cdk1 pathway to couple the centrosome cycle with mitosis, is required for precise mitotic spindle orientation and thereby regulates the progenitor division mode to maintain brain size.     

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.     

Human Cdc14B promotes progression through mitosis by dephosphorylating Cdc25 and regulating Cdk1/cyclin B activity

Entry into and progression through mitosis depends on phosphorylation and dephosphorylation of key substrates. In yeast, the nucleolar phosphatase Cdc14 is pivotal for exit from mitosis counteracting Cdk1-dependent phosphorylations. Whether hCdc14B, the human homolog of yeast Cdc14, plays a similar function in mitosis is not yet known. Here we show that hCdc14B serves a critical role in regulating progression through mitosis, which is distinct from hCdc14A. Unscheduled overexpression of hCdc14B delays activation of two master regulators of mitosis, Cdc25 and Cdk1, and slows down entry into mitosis. Depletion of hCdc14B by RNAi prevents timely inactivation of Cdk1/cyclin B and dephosphorylation of Cdc25, leading to severe mitotic defects, such as delay of metaphase/anaphase transition, lagging chromosomes, multipolar spindles and binucleation. The results demonstrate that hCdc14B-dependent modulation of Cdc25 phosphatase and Cdk1/cyclin B activity is tightly linked to correct chromosome segregation and bipolar spindle formation, processes that are required for proper progression through mitosis and maintenance of genomic stability.     

Checking Out the Centrosome

Centrosomes consist of a pair of barrel-shaped microtubule assemblies called centrioles, surrounded by a pericentriolar matrix. The only well-characterized function of centrosomes is to organize both interphase microtubule arrays responsible for cell polarity and the mitotic spindle, which mediates the strictly bipolar separation of chromosomes. In addition to these established functions it has been speculated that centrosomes might be involved in several different cell cycle regulatory events like entry into mitosis, cytokinesis, G1/S transition and monitoring of DNA damage. These assumptions are mainly based on a rapidly growing list of centrosome-associated regulatory proteins such as p53, Brca1, Chk1, Chk2, TopBP1, Aurora-A, Plk1, cyclin B1, and Cdk1. However, only very few direct links between their localization to the centrosome and specific cellular functions have been unraveled until recently. This review will focus on recent advances in the understanding of the role of centrosomes as integrators of positive and negative pathways for mitotic entry.     

Human Cdc14A phosphatase modulates the G2/M transition through Cdc25A and Cdc25B

The Cdc14 family of serine-threonine phosphatases antagonizes CDK activity by reversing CDK-dependent phosphorylation events. It is well established that the yeast members of this family bring about the M/G1 transition. Budding yeast Cdc14 is essential for CDK inactivation at the end of mitosis and fission yeast Cdc14 homologue Flp1/Clp1 down-regulates Cdc25 to ensure the inactivation of mitotic CDK complexes to trigger cell division. However, the functions of human Cdc14 homologues remain poorly understood. Here we have tested the hypothesis that Cdc14A might regulate Cdc25 mitotic inducers in human cells. We found that increasing levels of Cdc14A delay entry into mitosis by inhibiting Cdk1-cyclin B1 activity. By contrast, lowering the levels of Cdc14A accelerates mitotic entry. Biochemical analyses revealed that Cdc14A acts through key Cdk1-cyclin B1 regulators. We observed that Cdc14A directly bound to and dephosphorylated Cdc25B, inhibiting its catalytic activity. Cdc14A also regulated the activity of Cdc25A at the G2/M transition. Our results indicate that Cdc14A phosphatase prevents premature activation of Cdk1 regulating Cdc25A and Cdc25B at the entry into mitosis.     

Chk1 kinase negatively regulates mitotic function of Cdc25A phosphatase through 14-3-3 binding

The order and fidelity of cell cycle events in mammals is intimately linked to the integrity of the Chk1 kinase-Cdc25A phosphatase pathway. Chk1 phosphorylation targets Cdc25A for destruction and, as shown here, inhibits interactions between Cdc25A and its mitotic substrate cyclin B1-Cdk1. Phosphorylation of Cdc25A on serine 178 and threonine 507 facilitates 14-3-3 binding, and Chk1 phosphorylates both residues in vitro. Mutation of T507 to alanine (T507A) enhanced the biological activity of Cdc25A. Cdc25A(T507A) was more efficient in binding to cyclin B1, activating cyclin B1-Cdk1, and promoting premature entry into mitosis. We propose that the Chk1/Cdc25A/14-3-3 pathway functions to prevent cells from entering into mitosis prior to replicating their genomes to ensure the fidelity of the cell division process.     

Drosophila grapes/CHK1 mutants are defective in cyclin proteolysis and coordination of mitotic events.

The Drosophila grapes (grp) gene, which encodes a homolog of the Schizosaccharomyces pombe Chk1 kinase, provides a cell-cycle checkpoint that delays mitosis in response to inhibition of DNA replication [1]. Grp is also required in the undisturbed early embryonic cycles: in its absence, mitotic abnormalities appear in cycle 12 and chromosomes fail to fully separate in subsequent cycles [2] [3]. In other systems, Chk1 kinase phosphorylates and suppresses the activity of Cdc25 phosphatase: the resulting failure to remove inhibitory phosphate from cyclin-dependent kinase 1 (Cdk1) prevents entry into mitosis [4] [5]. Because in Drosophila embryos Cdk1 lacks inhibitory phosphate during cycles 11-13 [6], it is not clear that known actions of Grp/Chk1 suffice in these cycles. We found that the loss of grp compromised cyclin A proteolysis and delayed mitotic disjunction of sister chromosomes. These defects occurred before previously reported grp phenotypes. We conclude that Grp activates cyclin A degradation, and functions to time the disjunction of chromosomes in the early embryo. As cyclin A destruction is required for sister chromosome separation [7], a failure in Grp-promoted cyclin destruction can also explain the mitotic phenotype. The mitotic failure described previously for cycle 12 grp embryos might be a more severe form of the phenotypes that we describe in earlier embryos and we suggest that the underlying defect is reduced degradation of cyclin A.     

Inactivating Cdc25, Mitotic Style

Mitotic entry and exit require activation and inactivation of the Cdk1-cyclin B kinase complex, respectively. The Cdc25 protein phosphatase family activates Cdk1-cyclin B at the G2/M transition by removing inhibitory phosphate groups. Cdc25 family members, held inactive during interphase, are activated during mitotic progression in an amplification loop involving Cdk1-cyclin B. While Cdc25 activation at the G2/M transition is required for the timely initiation of mitosis, recent evidence suggests that the inactivation of Cdc25 in late mitosis may play a role in supporting Cdk1-cyclin B inactivation. Here, we discuss the mechanisms of Cdc25 regulation and how they pertain to both mitotic entry and exit.     

Inactivating Cdc25, mitotic style

Mitotic entry and exit require activation and inactivation of the Cdk1-cyclin B kinase complex, respectively. The Cdc25 protein phosphatase family activates Cdk1-cyclin B at the G2/M transition by removing inhibitory phosphate groups. Cdc25 family members, held inactive during interphase, are activated during mitotic progression in an amplification loop involving Cdk1-cyclin B. While Cdc25 activation at the G2/M transition is required for the timely initiation of mitosis, recent evidence suggests that the inactivation of Cdc25 in late mitosis may play a role in supporting Cdk1-cyclin B inactivation. Here, we discuss the mechanisms of Cdc25 regulation and how they pertain to both mitotic entry and exit.     

Mitotic progression becomes irreversible in prometaphase and collapses when Wee1 and Cdc25 are inhibited

Mitosis requires precise coordination of multiple global reorganizations of the nucleus and cytoplasm. Cyclin-dependent kinase 1 (Cdk1) is the primary upstream kinase that directs mitotic progression by phosphorylation of a large number of substrate proteins. Cdk1 activation reaches the peak level due to positive feedback mechanisms. By inhibiting Cdk chemically, we showed that, in prometaphase, when Cdk1 substrates approach the peak of their phosphorylation, cells become capable of proper M-to-G1 transition. We interfered with the molecular components of the Cdk1-activating feedback system through use of chemical inhibitors of Wee1 and Myt1 kinases and Cdc25 phosphatases. Inhibition of Wee1 and Myt1 at the end of the S phase led to rapid Cdk1 activation and morphologically normal mitotic entry, even in the absence of G2. Dampening Cdc25 phosphatases simultaneously with Wee1 and Myt1 inhibition prevented Cdk1/cyclin B kinase activation and full substrate phosphorylation and induced a mitotic "collapse," a terminal state characterized by the dephosphorylation of mitotic substrates without cyclin B proteolysis. This was blocked by the PP1/PP2A phosphatase inhibitor, okadaic acid. These findings suggest that the positive feedback in Cdk activation serves to overcome the activity of Cdk-opposing phosphatases and thus sustains forward progression in mitosis.     

Novel Positive Feedback Loop between Cdk1 and Chk1 in the Nucleus during G2/M Transition

Chk1, one of the critical transducers in DNA damage/replication checkpoints, prevents entry into mitosis through inhibition of Cdk1 activity. However, it has remained unclear how this inhibition is cancelled at the G₂/M transition. We reported recently that Chk1 is phosphorylated at Ser²⁸⁶ and Ser³⁰¹ by Cdk1 during mitosis. Here, we show that mitotic Chk1 phosphorylation is accompanied by Chk1 translocation from the nucleus to the cytoplasm in prophase. This translocation advanced in accordance with prophase progression and was regulated by Crm-1-dependent nuclear export. Exogenous Chk1 mutated at Ser²⁸⁶ and Ser³⁰¹ to Ala (S286A/S301A) was observed mainly in the nuclei of prophase cells, although such nuclear accumulation was hardly observed in wild-type Chk1. Induction of S286A/S301A resulted in the delay of mitotic entry. Biochemical analyses using immunoprecipitated cyclin B₁-Cdk1 complexes revealed S286A/S301A expression to block the adequate activation of Cdk1. In support of this, S286A/S301A expression retained Wee1 at higher levels and Cdk1-induced phosphorylation of cyclin B₁ and vimentin at lower levels. A kinase-dead version of S286A/S301A also localized predominantly in the nucleus but lost the ability to delay mitotic entry. These results indicate that Chk1 phosphorylation by Cdk1 participates in cytoplasmic sequestration of Chk1 activity, which releases Cdk1 inhibition in the nucleus and promotes mitotic entry.     

Budding Yeast Swe1 Is Involved in the Control of Mitotic Spindle Elongation and Is Regulated by Cdc14 Phosphatase during Mitosis

Cyclin-dependent kinase (Cdk1) activity is required for mitotic entry, and this event is restrained by an inhibitory phosphorylation of the catalytic subunit Cdc28 on a conserved tyrosine (Tyr¹⁹). This modification is brought about by the protein kinase Swe1 that inhibits Cdk1 activation thus blocking mitotic entry. Swe1 levels are regulated during the cell cycle, and they decrease during G₂/M concomitantly to Cdk1 activation, which drives entry into mitosis. However, after mitotic entry, a pool of Swe1 persists, and we collected evidence that it is involved in controlling mitotic spindle elongation. We also describe that the protein phosphatase Cdc14 is implicated in Swe1 regulation; in fact, we observed that Swe1 dephosphorylation in vivo depends on Cdc14 that, in turn, is able to control its subcellular localization. In addition we show that the lack of Swe1 causes premature mitotic spindle elongation and that high levels of Swe1 block mitotic spindle elongation, indicating that Swe1 inhibits this process. Importantly, these effects are not dependent upon the role of in Cdk1 inhibition. These data fit into a model in which Cdc14 binds and inhibits Swe1 to allow timely mitotic spindle elongation.     

The role of Cdc25 phosphatases in cell cycle checkpoints

Summary The major driving forces in the eukaryotic cell cycle are the cyclin-dependent kinases (Cdk). Cdks can be activated through dephosphorylation of inhibitory phosphorylations catalyzed by the Cdc25 phosphatase family. In higher-eukaryotic cells, there exist three Cdc25 family members, Cdc25A, Cdc25B, and Cdc25C. While Cdc25A plays a major role at the G₁-to-S phase transition, Cdc25B and C are required for entry into mitosis. The regulation of Cdc25C is crucial for the operation of the DNA-damage checkpoint. Two protein kinases, Chk1 and Cds1, can be activated in response to DNA damage or in the presence of unreplicated DNA. Chk1 and Cds1 may phosphorylate Cdc25C to prevent entry into mitosis through inhibition of Cdc2 (Cdk1) dephosphorylation.     

Mitotic regulation of the human anaphase-promoting complex by phosphorylation

The anaphase-promoting complex (APC) or cyclosome is a ubiquitin ligase that initiates anaphase and mitotic exit. APC activation is thought to depend on APC phosphorylation and Cdc20 binding. We have identified 43 phospho-sites on APC of which at least 34 are mitosis specific. Of these, 32 sites are clustered in parts of Apc1 and the tetratricopeptide repeat (TPR) subunits Cdc27, Cdc16, Cdc23 and Apc7. In vitro, at least 15 of the mitotic phospho-sites can be generated by cyclin-dependent kinase 1 (Cdk1), and 3 by Polo-like kinase 1 (Plk1). APC phosphorylation by Cdk1, but not by Plk1, is sufficient for increased Cdc20 binding and APC activation. Immunofluorescence microscopy using phospho-antibodies indicates that APC phosphorylation is initiated in prophase during nuclear uptake of cyclin B1. In prometaphase phospho-APC accumulates on centrosomes where cyclin B ubiquitination is initiated, appears throughout the cytosol and disappears during mitotic exit. Plk1 depletion neither prevents APC phosphorylation nor cyclin A destruction in vivo. These observations imply that APC activation is initiated by Cdk1 already in the nuclei of late prophase cells.