Table of contents

Function and regulation of Wee1 kinase and Myt1, inhibitory kinases that prevents entry into M - The G2 checkpoint

In a cell cycle, the mouse wee1 kinase (Drosophila homolog: Wee) is phosphorylated at M-phase; an in vitro study using a mitotic extract revealed that phosphorylation occurs in the N-terminal domain, which is absent from the human wee1 kinase, resulting in inactivation of the kinase activity. The N-terminal domain or the entire molecule is extensively phosphorylated by cdc2-cyclin B kinase. The activity of the wee1 kinase is reduced by phosphorylation with the mitotic extract that contains cdc2-cyclin B kinase (Honda, 1995).

The Cdc2 protein kinase is a key regulator of the G1-S and G2-M cell cycle transitions in the fission yeast Schizosaccharomyces pombe. The activation of Cdc2 at the G2-M transition is triggered by dephosphorylation at Y15, a conserved tyrosine residue. The level of Y15 phosphorylation is controlled by the Wee1 and Mik1 protein kinases acting in opposition to the Cdc25 protein phosphatase (see Drosophila String). Wee1 overexpression leads to a high stoichiometry of phosphorylation at T14, a previously undetected site in S. pombe Cdc2. T14 phosphorylation is also detected in certain cell cycle mutants blocked in progression through S phase, indicating that T14 phosphorylation might normally occur at low stoichiometry during DNA replication or early G2. Strains in which the chromosomal copy of cdc2 is replaced with either a T14A or a T14S mutant allele were generated. The phenotypes of these strains are consistent with T14 phosphorylation playing an inhibitory role in the activation of Cdc2, as it does in higher eukaryotes. Wee1 (but not Mik1 or Chk1) is required for phosphorylation at this site. The Mik1 and Chk1 protein kinases are unable to drive T14 phosphorylation in vivo: residue 14 phosphorylation requires previous phosphorylation at Y15, and the T14A mutant, unlike Y15F, is recessive to wild-type Cdc2 activity. The normal duration of G2 delay after irradiation or hydroxyurea treatment in a T14A mutant strain indicates that T14 phosphorylation is not required for the DNA damage or replication checkpoint controls (Den Haese, 1995).

The mechanisms that couple cell cycle progression with the organization of the peripheral cytoskeleton are poorly understood. In Saccharomyces cerevisiae, the Swe1 protein phosphorylates and inactivates the cyclin-dependent kinase, Cdc28, thereby delaying the onset of mitosis. The nim1-related protein kinase, Hsl1, induces entry into mitosis by negatively regulating Swe1. Hsl1 physically associates with the septin cytoskeleton in vivo and Hsl1 kinase activity depends on proper septin function. Genetic analysis indicates that two additional Hsl1-related kinases, Kcc4 and Gin4, act redundantly with Hsl1 to regulate Swe1. Kcc4, like Hsl1 and Gin4, localizes to the bud neck in a septin-dependent fashion. Interestingly, hsl1;kcc4;gin4 triple mutants develop a cellular morphology extremely similar to that of septin mutants. Consistent with the idea that Hsl1, Kcc4, and Gin4 link entry into mitosis to proper septin organization, septin mutants incubated at the restrictive temperature trigger a Swe1-dependent mitotic delay that is necessary to maintain cell viability. These results reveal for the first time how cells monitor the organization of their cytoskeleton and demonstrate the existence of a cell cycle checkpoint that responds to defects in the peripheral cytoskeleton. Moreover, Hsl1, Kcc4, and Gin4 have homologs in higher eukaryotes, suggesting that the regulation of Swe1/Wee1 by this class of kinases is highly conserved (Barral, 1999).

Using a polymerase chain reaction-based strategy, a gene encoding a Wee1-like kinase has been isolated from Xenopus eggs. The recombinant Xenopus Wee1 protein efficiently phosphorylates Cdc2 exclusively on Tyr-15 in a cyclin-dependent manner. The addition of exogenous Wee1 protein to Xenopus cell cycle extracts results in a dose-dependent delay of mitotic initiation that is accompanied by enhanced tyrosine phosphorylation of Cdc2. The activity of the Wee1 protein is highly regulated during the cell cycle: the interphase, underphosphorylated form of Wee1 (68 kDa) phosphorylates Cdc2 very efficiently, whereas the mitotic, hyperphosphorylated version (75 kDa) is weakly active as a Cdc2-specific tyrosine kinase. The down-modulation of Wee1 at mitosis is directly attributable to phosphorylation, since dephosphorylation with protein phosphatase 2A restores its kinase activity. During interphase, the activity of this Wee1 homolog does not vary in response to the presence of unreplicated DNA. The mitosis-specific phosphorylation of Wee1 is due to at least two distinct kinases: the Cdc2 protein and another activity (kinase X) that may correspond to an MPM-2 epitope kinase. These studies indicate that the down-regulation of Wee1-like kinase activity at mitosis is a multistep process that occurs after other biochemical reactions have signaled the successful completion of S phase (Mueller, 1995a).

Cdc2 is the cyclin-dependent kinase that controls entry of cells into mitosis. Phosphorylation of Cdc2 on threonine-14 and tyrosine-15 inhibits the activity of the enzyme and prevents premature initiation of mitosis. Although Wee1 has been identified as the kinase that phosphorylates tyrosine-15 in various organisms, the threonine-14-specific kinase has not been isolated. A complementary DNA was cloned from Xenopus that encodes Myt1 (see Drosophila Myt-1), a member of the Wee1 family that was discovered to phosphorylate Cdc2 efficiently on both threonine-14 and tyrosine-15. Myt1 is a membrane-associated protein that contains a putative transmembrane segment. Immunodepletion studies suggest that Myt1 is the predominant threonine-14-specific kinase in Xenopus egg extracts. Myt1 activity is highly regulated during the cell cycle, suggesting that this relative of Wee1 plays a role in mitotic control (Mueller, 1995b).

Entry into mitosis requires the activity of the Cdc2 kinase. Cdc2 associates with the B-type cyclins, and the Cdc2-cyclin B heterodimer is in turn regulated by phosphorylation. Phosphorylation of threonine 161 is required for the Cdc2-cyclin B complex to be catalytically active, whereas phosphorylation of threonine 14 and tyrosine 15 is inhibitory. Human kinases that catalyze the phosphorylation of threonine 161 and tyrosine 15 have been identified. A novel human cDNA encoding a dual-specificity protein kinase (designated Myt1Hu) preferentially phosphorylates Cdc2 on threonine 14 in a cyclin-dependent manner. Myt1Hu is 46% identical to Myt1Xe, a kinase recently characterized from Xenopus laevis. Myt1Hu localizes to the endoplasmic reticulum and Golgi complex in HeLa cells. A stretch of hydrophobic and uncharged amino acids located outside the catalytic domain of Myt1Hu is the likely membrane-targeting domain, since its deletion results in the localization of Myt1Hu primarily to the nucleus (F. Liu, 1997).

Activation of the Cdc2.cyclin B kinase is a pivotal step of mitotic initiation. This step is mediated principally by the dephosphorylation of residues threonine 14 (Thr14) and tyrosine 15 (Tyr15) on the Cdc2 catalytic subunit. In several organisms homologs of the Wee1 kinase have been shown to be the major activity responsible for phosphorylating the Tyr15 inhibitory site. A membrane-bound kinase capable of phosphorylating residue Thr14, the Myt1 kinase, has been identified in the frog Xenopus laevis and more recently in human. This study examined the substrate specificity and cell cycle regulation of the human Myt1 kinase. Human Myt1 phosphorylates and inactivates Cdc2-containing cyclin complexes but not complexes containing Cdk2 or Cdk4. Analysis of endogenous Myt1 demonstrates that it remains membrane-bound throughout the cell cycle, but its kinase activity decreases during M phase arrest, when Myt1 became hyperphosphorylated. Further, Cdc2. cyclin B1 is capable of phosphorylating Myt1 in vitro, but this phosphorylation does not affect Myt1 kinase activity. These findings suggest that human Myt1 is negatively regulated by an M phase-activated kinase and that Myt1 inhibits mitosis due to its specificity for Cdc2.cyclin complexes (Booher, 1997).

M-phase entry in eukaryotic cells is driven by activation of MPF, a regulatory factor composed of cyclin B and the protein kinase p34(cdc2). In G2-arrested Xenopus oocytes, there is a stock of p34(cdc2)/cyclin B complexes (pre-MPF), which is maintained in an inactive state by p34(cdc2) phosphorylation on Thr14 and Tyr15. This suggests an important role for the p34(cdc2) inhibitory kinase(s) such as Wee1 and Myt1 in regulating the G2-->M transition during oocyte maturation. MAP kinase (MAPK) activation is required for M-phase entry in Xenopus oocytes, but its precise contribution to the activation of pre-MPF is unknown. The C-terminal regulatory domain of Myt1 specifically binds to p90(rsk), a protein kinase that can be phosphorylated and activated by MAPK. In turn, p90(rsk) phosphorylates the C-terminus of Myt1 and down-regulates its inhibitory activity on p34(cdc2)/cyclin B in vitro. Consistent with these results, Myt1 becomes phosphorylated during oocyte maturation, and activation of the MAPK-p90(rsk) cascade can trigger some Myt1 phosphorylation prior to pre-MPF activation. Myt1 preferentially associates with hyperphosphorylated p90(rsk), and complexes can be detected in immunoprecipitates from mature oocytes. These results suggest that during oocyte maturation MAPK activates p90(rsk) and that p90(rsk) in turn down-regulates Myt1, leading to the activation of p34(cdc2)/cyclin B (Palmer, 1998).

A common cellular response to DNA damage is cell cycle arrest. This checkpoint control has been the subject of intensive genetic investigation, but the biochemical mechanism that prevents mitosis following DNA damage is unknown. In Schizosaccharomyces pombe, as well as in vertebrates, the timing of mitosis under normal circumstances is determined by the balance of kinases and phosphatases that regulate inhibitory phosphorylation of Cdc2. In S. pombe, the phosphorylation occurs on tyrosine-15. This method of mitotic control is also used in S. pombe to couple mitosis with completion of DNA replication, but the role of Cdc2 tyrosine phosphorylation in the Chk1 kinase-mediated DNA damage checkpoint has remained uncertain. In contrast to recent speculation, the G2 DNA damage checkpoint arrest in S. pombe depends on the inhibitory tyrosine phosphorylation of Cdc2 carried out by the Wee1 and Mik1 kinases. The rate of Cdc2 tyrosine dephosphorylation is reduced by irradiation. This result implicates regulation of Cdc2 tyrosine dephosphorylation, mainly carried out by the Cdc25 tyrosine phosphatase, as an important part of the mechanism by which the DNA damage checkpoint induces Cdc2 inhibition and G2 arrest (Rhind, 1996).

The DNA replication checkpoint inhibits mitosis in cells that are unable to replicate their DNA, as when nucleotide biosynthesis is inhibited by hydroxyurea. In the fission yeast Schizosaccharomyces pombe, genetic evidence suggests that this checkpoint involves the inhibition of Cdc2 activity through the phosphorylation of tyrosine-15. On the contrary, a recent biochemical study indicated that Cdc2 is in an activated state during a replication checkpoint, suggesting that phosphorylation of Cdc2 on tyrosine-15 is not part of the replication checkpoint mechanism. Biochemical and genetic studies have been undertaken to resolve this controversy. The DNA replication checkpoint in S. pombe is abrogated in cells that carry the allele cdc2-Y15F, expressing an unphosphorylatable form of Cdc2. Furthermore, Cdc2 isolated from replication checkpoint-arrested cells can be activated in vitro by Cdc25 (the tyrosine phosphatase responsible for dephosphorylating Cdc2 in vivo) to the same extent as Cdc2 isolated from cdc25ts-blocked cells; this indicates that hydroxyurea treatment causes Cdc2 activity to be maintained at a low level that is insufficient to induce mitosis. These studies show that inhibitory tyrosine-15 phosphorylation of Cdc2 is essential for the DNA replication checkpoint and suggest that Cdc25, and/or one or both of Wee1 and Mik1, the tyrosine kinases that phosphorylate Cdc2, are regulated by the replication checkpoint (Rhind, 1998).

Fission yeast p56(chk1) kinase is known to be involved in the DNA damage checkpoint but not to be required for cell cycle arrest following exposure to the DNA replication inhibitor hydroxyurea (HU). For this reason, p56(chk1) is not considered necessary for the DNA replication checkpoint, which acts through the inhibitory phosphorylation of p34(cdc2) kinase activity. In a search for Schizosaccharomyces pombe mutants that abolish the S phase cell cycle arrest of a thermosensitive DNA polymerase delta strain at 37 degrees C, two chk1 alleles have been isolated. These alleles are proficient for the DNA damage checkpoint, but induce mitotic catastrophe in several S phase thermosensitive mutants. The mitotic catastrophe correlates with a decreased level of tyrosine phosphorylation of p34(cdc2). In addition, the deletion of chk1 and the chk1 alleles abolish the cell cycle arrest and induce mitotic catastrophe in cells exposed to HU, if the cells are grown at 37 degrees C. These findings suggest that chk1 is important for the maintenance of the DNA replication checkpoint in S phase thermosensitive mutants and that the p56(chk1) kinase must possess a novel function that prevents premature activation of p34(cdc2) kinase under conditions of impaired DNA replication at 37 degrees C (Francesconi, 1997).

The G2 DNA damage checkpoint ensures maintenance of cell viability by delaying progression into mitosis in cells that have suffered genomic damage. It is controlled by a number of proteins that are hypothesized to transduce signals through cell cycle regulators to delay activation of p34cdc2. Studies in mammalian cells have correlated induction of inhibitory tyrosine 15 (Y15) phosphorylation on p34cdc2 with the response to DNA damage. However, genetic studies in fission yeast have suggested that the major Y15 kinase, p107wee1, is not required for the cell cycle delay in response to DNA damage, although it is required for survival after irradiation. Thus, the target of the checkpoint, and hence the mechanism of cell cycle delay, remains unknown. Y15 phosphorylation is maintained in checkpoint-arrested fission yeast cells. wee1 is required for cell cycle arrest induced by up-regulation of an essential component of this checkpoint, chk1. p107wee1 is hyperphosphorylated in cells delayed by chk1 overexpression or UV irradiation, and p56chk1 can phosphorylate p107wee1 directly in vitro. These observations suggest that in response to DNA damage p107wee1 is phosphorylated by p56chk1 in vivo, and this results in maintenance of Y15 phosphorylation and hence G2 delay. In the absence of wee1, other Y15 kinases, such as p66mik1, may partially substitute for p107wee1 to induce cell cycle delay, but this wee1-independent delay is insufficient to maintain full viability. This study establishes a link between a G2 DNA damage checkpoint function and a core cell cycle regulator (O'Connell, 1997).

Successful recovery from DNA damage requires coordination of several biological processes. Eukaryotic cell cycle progression is delayed when the cells encounter DNA-damaging agents. This cell cycle delay allows the cells to cope with DNA damage by utilizing DNA repair enzymes. Thus, at least two processes, induction of the cell cycle delay and repair of damaged DNA, are coordinately required for recovery. In this study, a fission yeast rad mutant (slp1-362) was genetically investigated. In response to radiation, slp1 stops cell division; however, it does not restart it. This defect is suppressed when slp1-362 is combined with wee1-50 or cdc2-3w: in these mutants, the onset of mitosis is advanced due to the premature activation of p34cdc2. In contrast, slp1 is synthetically lethal with cdc25, nim1/cdr1, or cdr2, all of which are unable to activate the p34cdc2 kinase correctly. These genetic interactions of slp1 with cdc2 and its modulators imply that slp1 is not defective in either "induction of cell cycle delay" or "DNA repair." slp1+ may be involved in a critical process that restarts cell cycle progression after the completion of DNA repair. Molecular cloning of slp1+ reveals that slp1+ encodes a putative 488-amino-acid polypeptide exhibiting significant homology to WD-domain proteins, namely, CDC20 (budding yeast), p55CDC (human), and Fizzy (a Drosophila protein that promotes degradation of cyclins). A possible role of slp1+ is proposed (Matsumoto, 1997).

It has been suggested that the survival response of p53 defective tumor cells to agents that inhibit DNA replication or damage DNA may be largely dependent on cell cycle checkpoints that regulate the onset of mitosis. In human cells, the mitosis-inducing kinase CDC2/cyclin B is inhibited by phosphorylation of threonine-14 and tyrosine-15, but the roles of these phosphorylations in enforcing checkpoints is not known. In a human cervical carcinoma cell line (HeLa cells) low level expression of a mutant nonphosphorylatable form of CDC2 abrogates regulation of the endogenous CDC2/cyclin B. Disruption of this pathway is toxic and renders cells highly sensitive to killing by DNA damage or by inhibition of DNA replication. These findings establish the importance of inhibitory phosphorylation of CDC2 in the survival mechanism used by human cells when exposed to some of the most common forms of anticancer therapy (Blasina, 1997).

During the meiotic cell cycle in Xenopus oocytes, p90rsk, the downstream kinase of the Mos-MAPK pathway, interacts with and inhibits the Cdc2 inhibitory kinase Myt1/Wee1. However, p90rsk is inactivated after fertilization due to the degradation of Mos. The Polo-like kinase Plx1, instead of p90rsk, interacts with and inhibits Myt1 after fertilization of Xenopus eggs. At the M phase of the embryonic cell cycle, Cdc2 phosphorylates Myt1 on Thr478 and thereby creates a docking site for Plx1. Plx1 can phosphorylate Myt1 and inhibit its kinase activity both in vitro and in vivo. The interaction between Myt1 and Plx1 is required, at least in part, for normal embryonic cell divisions. Finally, and interestingly, Myt1 is phosphorylated on Thr478 even during the meiotic cell cycle, but its interaction with Plx1 is largely inhibited by p90rsk-mediated phosphorylation. These results indicate a switchover in the Myt1 inhibition mechanism at fertilization of Xenopus eggs, and strongly suggest that Plx1 acts as a direct inhibitory kinase of Myt1 in the mitotic cell cycles in Xenopus (Inoue, 2005).

Although the interruption of the Plx1-Myt1 interaction by T478A mutation largely impaired the Plx1 phosphorylation and inhibition of Myt1, it seemed to have a relatively small (although significant) effect on early embryonic cell divisions. This is presumably due, however, to the dramatic increase in levels of the Myt1/Wee1-antagonizing Cdc25A phosphatase during this period. In somatic cells, however, the level of Cdc25A is considerably lower; therefore, the interruption of the Plx1-Myt1 interaction in somatic cells might have a significantly larger effect on cell divisions. In human somatic cells, Myt1 localizes to the endoplasmic reticulum and Golgi complex. Therefore, the binding of Plk1/Plx1 or the Myt1 substrate Cdc2-cyclin B to Myt1 could also be responsible, at least in part, for their known localization and function at the Golgi complex. Indeed, in Drosophila embryos, Myt1 has been implicated in Golgi fragmentation (Cornwell, 2002), a mitotic event involving both Cdc2 and Plk1 (Lin, 2000; Sutterlin, 2001; Inoue, 2005 and references therein).

In fully grown oocytes, meiosis is arrested at first prophase until species-specific initiation signals trigger maturation. Meiotic resumption universally involves early activation of M phase-promoting factor (Cdc2 kinase-Cyclin B complex, MPF) by dephosphorylation of the inhibitory Thr14/Tyr15 sites of Cdc2. However, underlying mechanisms vary. In Xenopus oocytes, deciphering the intervening chain of events has been hampered by a sensitive amplification loop involving Cdc2-Cyclin B, the inhibitory kinase Myt1 and the activating phosphatase Cdc25. This study provides evidence that the critical event in meiotic resumption is a change in the balance between inhibitory Myt1 activity and Cyclin B neosynthesis. First, this study shows that in fully grown oocytes Myt1 is essential for maintaining prophase I arrest. Second, it was demonstrated that, upon upregulation of Cyclin B synthesis in response to progesterone, rapid inactivating phosphorylation of Myt1 occurs, mediated by Cdc2 and without any significant contribution of Mos/MAPK or Plx1. A model in which the appearance of active MPF complexes following increased Cyclin B synthesis causes Myt1 inhibition, upstream of the MPF/Cdc25 amplification loop (Gaffré, 2011).

This study focused on the role and regulation of Myt1, the member of the Wee1 family of inhibitory kinases that is expressed in Xenopus prophase I oocytes. The Wee1 family of kinases comprises Wee1, which is present in all eukaryotes, and Myt1, which is restricted to metazoans. Wee1 is a nuclear kinase, phosphorylating Cdc2 on Tyr15, whereas Myt1 possesses a dual Thr14/Tyr15 Cdc2-phosphorylating activity and associates with cell membranes. In Xenopus oocytes only Myt1 is detectable at the protein level during the last stages of growth and the early steps of meiosis reinitiation. The two forms of Xenopus Wee1 (Wee1A and Wee1B) are not expressed in the prophase I-arrested oocytes. Wee1A becomes detectable only after completion of the first meiotic division. Myt1 kinase has been implicated in both male and female gametogenesis in various animals, either alone, as is the case in C. elegans, Drosophila, starfish and Xenopus, or in concert with Wee1, as in mouse oocytes. In starfish, Myt1 has been clearly demonstrated to be involved both in the G2 arrest of oocytes and in meiosis re-entry, which involves downregulation of Myt1 activity by Akt phosphorylation. Whether Myt1 also plays an essential role in Xenopus oocyte meiotic resumption remains unclear (Gaffré, 2011).

The main aim of this study was to determine the contribution of Myt1 to the prophase arrest of fully grown Xenopus oocytes and to meiosis re-entry. A crucial issue was to determine which kinases are responsible for the initial phosphorylation and downregulation of Myt1 activity following progesterone treatment. As well as Cdc2, candidate kinases activated at around the same time include Plx1 (the Xenopus homolog of Drosophila Polo kinase) and members of the MAPK cascade, including Mos and p90Rsk kinase (Gaffré, 2011).

This study found that Myt1 function is required to maintain prophase I arrest in fully grown oocytes, such that experimental Myt1 inhibition promoted meiosis re-entry. At the onset of maturation, the inactivating phosphorylation of Myt1 was found to precede the activating phosphorylation of Cdc25 and to be mediated principally by Cdc2 itself in association with newly synthesized Cyclins, rather than by the Mos/MAPK cascade or Plx1. A model is proposed in which the significant upregulation of Cyclin B synthesis following progesterone stimulation produces a small population of active Cdc2-Cyclin B, which is responsible for early Myt1 phosphorylation and inhibition, ahead of full Cdc25 activation and thus entry into the auto-amplification loop. In Xenopus, the change in the balance between Cyclin B synthesis and Myt1 activity following hormone stimulation is therefore a key feature of meiotic re-entry (Gaffré, 2011).

Cdc2, the mitotic spindle, and mitotic spindle checkpoint

The spindle checkpoint prevents anaphase onset until completion of mitotic spindle assembly by restraining activation of the ubiquitin ligase anaphase-promoting complex/cyclosome-Cdc20 (APC/CCdc20: Fizzy is the Drosophila homolog of Cdc20). The spindle checkpoint requires mitotic cyclin-dependent kinase (cdk) activity. Inhibiting cdk activity overrides checkpoint-dependent arrest in Xenopus egg extracts and human cells. Following inhibition, the interaction between APC/C and Cdc20 transiently increases while the inhibitory checkpoint protein Mad2 dissociates from Cdc20. Cdk inhibition also overcomes Mad2-induced mitotic arrest. In addition, in vitro cdk1-phosphorylated Cdc20 interacts with Mad2 rather than APC/C. Thus, cdk activity is required to restrain APC/CCdc20 activation until completion of spindle assembly (D'Angiolella, 2003).

A possible target for cdk action in regulating Cdc20 interaction with APC/C and Mad2 could be Cdc20 itself. Cdc20 contains multiple cdk phosphorylation sites and is phosphorylated during mitosis and under checkpoint conditions. Mutation of several of these sites substantially reduces Cdc20 phosphorylation by mitotic egg extracts and by cdk1. In addition, it has been shown by in vitro studies that phosphorylated Cdc20 is a poorer stimulator of APC/C activity than non- or hypo-phosphorylated Cdc20 and that cdk-dependent phosphorylation of Cdc20 inhibits binding to APC componenet Cdc27 (see Drosophila Cdc27/Makos). Thus, under checkpoint conditions, cdk1-dependent phosphorylation of Cdc20 may affect the ability of Cdc20 to bind APC/C and Mad2. Phosphorylated Cdc20 has retarded mobility on SDS-PAGE. To analyze the phosphorylation status of Cdc20 in checkpoint-arrested cells after cdk inhibition, HeLa proteins were separated in longer SDS-PAGE runs to better visualize changes in Cdc20 mobility. Cdc20 mobility increases after addition of cdk inhibitor Roscovitine to checkpoint-arrested cells, suggesting that Cdc20 becomes dephosphorylated. Cdc20 isolated from checkpoint-arrested cells also shows increased mobility after treatment with lambda protein phosphatase, indicating that the increased mobility can be ascribed to dephosphorylation. The timing of Cdc20 dephosphorylation is compatible with the hypothesis that dephosphorylation of Cdc20 increases binding to APC/C while it decreases it to Mad2 (D'Angiolella, 2003).

Egg extracts were used to study in more detail the relevance of Cdc20 phosphorylation for the interaction with APC/C and Mad2. Does Cdc20 phosphorylation depend on cdk1 or cdk2 activity in egg extracts? The results indicate that cdk1 rather than cdk2 is responsible for Cdc20 phosphorylation (D'Angiolella, 2003).

To know whether cdk1-dependent phosphorylation affects the ability of Cdc20 to interact with Cdc27 and Mad2 in egg extracts, [35S]-labeled Cdc20 wild type and a mutant Cdc20 version nonphosphorylatable at seven cdk phosphorylation sites (7A) were used. Using this mutant, it has been shown that cdk1-phosphorylated Cdc20 binds poorly to Cdc27 in vitro. Cdc20 wild type and 7A were independently pretreated with active cdk1 and then incubated in CSF-arrested extracts with recombinant Mad2. Cdc27 or Mad2 were isolated 30 min later and the amount of bound Cdc20 wild type or Cdc20 7A was determined. Cdc27 binds more highly to Cdc20 7A than to Cdc20 wild type. Conversely, Mad2 preferentially binds to Cdc20 wild type than to Cdc20 7A. Although cdk-dependent phosphorylation of Cdc20 in vivo may not be so extensive as in vitro, these data show that cdk-phosphorylated Cdc20 interacts more efficiently with Mad2 than Cdc27. Mad2-dependent arrest is maintained in the presence of the cdk1-phosphorylated wild-type Cdc20 but not in the presence of the phosphorylation-resistant mutant Cdc20 version. These findings are consistent with the in vivo findings that cdk inhibition induces dissociation of Cdc20 from Mad2 and a transient increase of Cdc20 binding to Cdc27 (D'Angiolella, 2003).

These observations, from Xenopus egg extracts and human somatic cells, indicate a crucial role for cdk1 activity in the regulation of the spindle checkpoint. Cdk1 activity appears to be required to inhibit APC/CCdc20 activation by stabilizing Cdc20-Mad2 interaction and reducing Cdc20 binding to APC/C via direct phosphorylation of Cdc20 (D'Angiolella, 2003).

In Xenopus egg extracts and embryos, cyclin E-cdk2 activity is relevant for many aspects of the M phase; it is also involved in checkpoint control. It is possible that, under checkpoint conditions, cyclin E-cdk2 is required to sustain cdk1 activity, and that this is ultimately responsible to inhibit APC/CCdc20 activation. It is presently unclear why cyclin E-cdk2 plays a role in mitotic events in this system, while it does not appear to be so relevant for mitosis in somatic cells. Many reports also indicate that MAPK participates in checkpoint regulation in egg extracts. MAPK may participate directly in checkpoint regulation, and also indirectly, like cdk2, by sustaining cdk1 activity (D'Angiolella, 2003).

In mammalian cells, cdk2 may not have a crucial role in spindle checkpoint-dependent arrest because the abundance of its partners, cyclin A and E, is very low under checkpoint conditions, and the relevance of the MAPK pathway in mitosis and spindle checkpoint is not yet completely understood . Some human cancers, however, show deregulated cyclin E expression and cyclin E-cdk2 activity is present at high levels also in mitosis. Observations in the embryonic system may provide a framework to explain some aspects of the oncogenic potential of deregulated cyclin E expression and why this causes chromosomal instability in somatic mammalian cells (D'Angiolella, 2003).

During mitosis, cdk1 may perform positive phosphorylations of APC/C, a prerequisite for its activity, but at the same time, by phosphorylating Cdc20, it lessens interaction between APC/C and Cdc20 and provides a precondition for inhibitory checkpoint proteins, activated at unattached kinetochores like Mad2, to interact with Cdc20. Thus, upon completion of mitotic spindle assembly, activated checkpoint proteins are no longer produced and active APC/Cdc20 can begin to form. An alternative hypothesis could be that the checkpoint mechanism acts also by sustaining the pathways that control and maintain cdk activation. Thus, upon completion of mitotic spindle assembly, an early and transient proteolysis-independent drop in cdk1 activity may help rapid APC/Cdc20 reactivation by increasing the Cdc20-APC/C interaction and inducing the dissociation of activated checkpoint proteins from Cdc20 (D'Angiolella, 2003).

The bipolar mitotic spindle is responsible for segregating sister chromatids at anaphase. Microtubule motor proteins generate spindle bipolarity and enable the spindle to perform mechanical work. A major change in spindle architecture occurs at anaphase onset when central spindle assembly begins. This structure regulates the initiation of cytokinesis and is essential for its completion. Central spindle assembly requires the centralspindlin complex composed of the Caenorhabditis elegans ZEN-4 (mammalian orthologue MKLP1) kinesin-like protein and the Rho family GAP CYK-4 (MgcRacGAP). This study describes a regulatory mechanism that controls the timing of central spindle assembly. The mitotic kinase Cdk1/cyclin B phosphorylates the motor domain of ZEN-4 on a conserved site within a basic amino-terminal extension characteristic of the MKLP1 subfamily. Phosphorylation by Cdk1 diminishes the motor activity of ZEN-4 by reducing its affinity for microtubules. Preventing Cdk1 phosphorylation of ZEN-4/MKLP1 causes enhanced metaphase spindle localization and defects in chromosome segregation. Thus, phosphoregulation of the motor domain of MKLP1 kinesin ensures that central spindle assembly occurs at the appropriate time in the cell cycle and maintains genomic stability (Mishima, 2004).

GRASP65, a structural protein of the Golgi apparatus, has been linked to the sensing of Golgi structure and the integration of this information with the control of mitotic entry in the form of a Golgi checkpoint. Cdk1-cyclin B is the major kinase phosphorylating GRASP65 in mitosis, and phosphorylated GRASP65 interacts with the polo box domain of the polo-like kinase Plk1. GRASP65 is phosphorylated in its C-terminal domain at four consensus sites by Cdk1-cyclin B, and mutation of these residues to alanine essentially abolishes both mitotic phosphorylation and Plk1 binding. Expression of the wild-type GRASP65 C-terminus but not the phosphorylation defective mutant in normal rat kidney cells causes a delay but not the block in mitotic entry expected if this were a true cell cycle checkpoint. These findings identify a Plk1-dependent signalling mechanism potentially linking Golgi structure and cell cycle control, but suggest that this may not be a cell cycle checkpoint in the classical sense (Preisinger, 2005).

Activation of anaphase-promoting complex/cyclosome (APC/CCdc20) by Cdc20 is delayed by the spindle assembly checkpoint (SAC). When all kinetochores come under tension, the SAC is turned off and APC/CCdc20 degrades cyclin B and securin, which activates separase. The latter then cleaves cohesin holding sister chromatids together. Because cohesin cleavage also destroys the tension responsible for turning off the SAC, cells must possess a mechanism to prevent SAC reactivation during anaphase, which could be conferred by a dependence of the SAC on Cdk1. To test this, mouse oocytes and embryos expressing nondegradable cyclin B were analyzed together with a Cdk1-resistant form of separase. After biorientation and SAC inactivation, APC/CCdc20 activates separase but the resulting loss of (some) cohesion is accompanied by SAC reactivation and APC/CCdc20 inhibition, which aborts the process of further securin degradation. Cyclin B is therefore the only APC/CCdc20 substrate whose degradation at the onset of anaphase is necessary to prevent SAC reactivation. The mutual activation of tension sensitive SAC and Cdk1 creates a bistable system that ensures complete activation of separase and total downregulation of Cdk1 when all chromosomes have bioriented (Rattani, 2014).

Cdk1 inactivation terminates mitotic checkpoint surveillance and stabilizes kinetochore attachments in anaphase

Two mechanisms safeguard the bipolar attachment of chromosomes in mitosis. A correction mechanism destabilizes erroneous attachments that do not generate tension across sister kinetochores. In response to unattached kinetochores, the mitotic checkpoint delays anaphase onset by inhibiting the anaphase-promoting complex/cyclosome (APC/C(Cdc20)). Upon satisfaction of both pathways, the APC/C(Cdc20) elicits the degradation of securin (see Drosophila Pimples) and cyclin B. This liberates separase triggering sister chromatid disjunction and inactivates cyclin-dependent kinase 1 (Cdk1) causing mitotic exit. How eukaryotic cells avoid the engagement of attachment monitoring mechanisms when sister chromatids split and tension is lost at anaphase is poorly understood. This study shows that Cdk1 inactivation disables mitotic checkpoint surveillance at anaphase onset in human cells. Preventing cyclin B1 proteolysis at the time of sister chromatid disjunction destabilizes kinetochore-microtubule attachments and triggers the engagement of the mitotic checkpoint. As a consequence, mitotic checkpoint proteins accumulate at anaphase kinetochores, the APC/C(Cdc20) is inhibited, and securin reaccumulates. Conversely, acute pharmacological inhibition of Cdk1 abrogates the engagement and maintenance of the mitotic checkpoint upon microtubule depolymerization. It is proposed that the simultaneous destruction of securin and cyclin B elicited by the APC/C(Cdc20) couples chromosome segregation to the dissolution of attachment monitoring mechanisms during mitotic exit (Vazquez-Novelle, 2014).

Cdc2 and damage checkpoint control

DNA damage and replication checkpoints in eukaryotic cells ensure that progression through the cell cycle is restrained while chromosomes undergo repair or replication. Damage and replication defects are recognized by the putative protein complex containing protein kinases such as human ATM and ATR, fission yeast Rad3, and budding yeast Mec1p. These kinases contain a lipid kinase motif, are highly conserved through evolution, and are thought to convey the checkpoint signal by phosphorylation to other downstream kinases, Chk1 and Cds1, through which regulators of the major cell cycle machinery are regulated. Chk1 kinase becomes phosphorylated in response to DNA damage, and this phosphorylation depends on Rad3 kinase. In fission yeast, Chk1 is targeted to restrain the Cdc25 mitotic inducer. Cds1 is activated upon replication arrest and DNA damage occurring in S phase. It is unknown, however, whether the checkpoint signal is sent directly from the checkpoint Rad complex to Chk1 in vivo. A fission yeast protein, Crb2/Rhp9, resembling budding yeast Rad9p and a human tumor suppressor protein BRCA1, is required for the damage checkpoint. Chk1 kinase is not activated in the crb2 deletion mutant (designated Deltacrb2) after DNA damage. Crb2 was identified by genetic and two-hybrid interactions with Cut5/Rad4 essential for replication and replication checkpoint. They form the complex in vitro, but the stable complex has not been found in vivo. Crb2 also shows genetic and two-hybrid interactions with Chk1. Hyperphosphorylation of Crb2 after DNA damage depends on the presence of Rad3 kinase, but not Chk1. Overproduction of Chk1, however, rescues the phenotype of Deltacrb2 damage. These results suggested that Crb2 might be placed upstream of Chk1 and downstream of Rad3. Alternatively, Crb2 and Rad3 may be independently required for the activation of Chk1 (Esashi, 1999 and references).

The C-terminal half of Crb2 contains two BRCT motifs present in a wide range of proteins implicated in DNA damage and repair. The BRCT domain of Crb2 is highly similar to that of budding yeast Rad9p, and to a lesser degree, to human BRCA1 and 53BP1. Plasmid carrying the RAD9 gene, however, fails to rescue the UV sensitivity of Deltacrb2. Crb2 and Rad9p are hyperphosphorylated after DNA damage, revealing upper band shift in the immunoblot patterns. Crb2 is phosphorylated not only when damaged but also during the normal mitotic cell cycle. A Cdc2 site, T215, was identified in the N terminus of Crb2. This T215 site undergoes stage-specific phosphorylation in the cell cycle and also is phosphorylated when damaged. The nonphosphorylatable T215A mutant abolishes hyperphosphorylation of Crb2 upon damage, and it allows cells to enter the arrested state but fails to reenter cell cycle progression. Thus fission yeast Crb2, which is required for the damage checkpoint, is the target of Cdc2 during normal mitosis and also in response to damage. Cdc2 kinase phosphorylation is necessary for reentering the cell cycle after the checkpoint. It may be noteworthy that finding Cdk phosphorylation sites for which phenotypes can be adduced is fairly unusual. Since only dividing cells undergo checkpoint arrest, active participation of Cdc2 in checkpoint control may have universal significance (Esashi, 1999).

Why is the Cdc2 site implicated in checkpoint control? The requirement of Cdc2 kinase in checkpoint control was unexpected. Cdc2 was considered to be the target of repression during checkpoint arrest because checkpoint was not activated in the presence of hyperactive Cdc2. This study, however, demonstrates that Cdc2 phosphorylation is involved in reestablishing cell cycle progression after checkpoint. The fact that the T215A mutant has problems reentering the cell cycle is clear from the results obtained. Furthermore, the level of thymine dimer generated by UV irradiation is reduced in the T215A mutant at a rate similar to that in wild-type cells. Difficulty in reentering the cell cycle thus may not be due to remaining damages but, rather, it may be caused by the failure of Crb2 to be the target of Cdc2 kinase (Esashi, 1999).

Why does T215A result in prolonged cell cycle delay? One possible explanation is that the T215A-unphosphorylatable Crb2 mutant protein retains the ability to inhibit Cdc2 kinase or loses the ability to activate Cdc2 kinase through downregulating Chk1, so that irradiated cells cannot reestablish cell cycle progression, bringing cells into permanent arrest. The T215A mutant is UV sensitive for the opposite reason than is the crb2 null: the null fails to arrest in response to UV and therefore dies (the usual checkpoint defect), but the T215A mutant arrests and fails to recover. It is proposed that T215 phosphorylation by Cdc2 is required to turn off the checkpoint system -- that is, repair of the lesions may not be sufficient. T215 phosphorylation by Cdc2 may be a part of a feedback loop to reestablish the cell cycle. A small population of Cdc2 that is active after the completion of DNA repair may phosphorylate Crb2, which subsequently reduces Chk1 kinase activity, thereby accelerating mitotic entry. Cdc2 may be an antagonist of Crb2, while Crb2 activates Chk1 for restraining Cdc2 (Esashi, 1999).

DNA replication in higher eukaryotes requires activation of a Cdk2 kinase by Cdc25A, a labile phosphatase subject to further destabilization upon genotoxic stress. A distinct, markedly stable form of Cdc25A, is described that plays a previously unrecognized role in mitosis. Mitotic stabilization of Cdc25A reflects its phosphorylation on Ser17 and Ser115 by cyclin B-Cdk1, modifications required to uncouple Cdc25A from its ubiquitin-proteasome-mediated turnover. Cdc25A binds and activates cyclin B-Cdk1, accelerates cell division when overexpressed, and its downregulation by RNA interference (RNAi) delays mitotic entry. DNA damage-induced G2 arrest, in contrast, is accompanied by proteasome-dependent destruction of Cdc25A, and ectopic Cdc25A abrogates the G2 checkpoint. Thus, phosphorylation-mediated switches among three differentially stable forms ensure distinct thresholds, and thereby distinct roles for Cdc25A in multiple cell cycle transitions and checkpoints (Mailand, 2002).

Arguably the most unexpected finding of this study was that entry into mitosis is accompanied by an abrupt switch, which converts Cdc25A from a labile to a stable protein. Hence, while acceleration of the basal Cdc25A protein turnover participates in response to genotoxic stress or stalled replication in interphase, uncoupling of Cdc25A from the ubiquitin-proteasome-dependent degradation plays an important role once the cells become committed to undergo cell division. Whereas destruction of the phosphatase guards against premature mitosis of cells with an incompletely replicated and/or damaged genome, stabilization of Cdc25A ensures that even in the absence of de novo mRNA synthesis, Cdc25A is abundant and active during initial stages of mitotis. Both reduction and extension of the Cdc25A protein half-life rely on phosphorylations within its N-terminal regulatory region. It has been shown that in S-phase cells, phosphorylation of Ser123 by the ATM-Chk2 kinase cascade (see loki) is required to induce degradation of Cdc25A in response to ionizing radiation. Such a mechanism may also operate in the DNA damage-induced G2 checkpoint. The mitotic stabilization of Cdc25A also reflects site-specific phosphorylation. Both Ser17 and Ser115 fulfill the minimum requirements for a CDK consensus site, and phosphorylation of Ser17 and Ser115 is mediated by cyclin B-Cdk1 in vitro, and is detectable in vivo only after entry into mitosis when the cyclin B-Cdk1 complex becomes physiologically active. Although the integrity of Ser17 and Ser115 appears crucial for Cdc25A stabilization, the data do not exclude the potential involvement of other residues. Thus, while concomitant mutation of Ser17 and Ser115 does confer marked instability in mitosis, the protein turnover of such a Cdc25A(2A) mutant could still be accelerated moderately by roscovitine, and the Ser17/Ser115-deficient protein still undergoes a partial mitosis-associated electrophoretic mobility shift. This indicates that cyclin B-Cdk1 might phosphorylate more residues than those identified by mass spectrometry analysis. Moreover, the fact that the endogenous Cdc25A protein remains partly shifted in roscovitine-treated mitotic cells suggests that other kinases (such as Plk1), specifically activated at the G2/M transition, assist in inducing and/or maintaining the stability of Cdc25A (Mailand, 2002).

A mechanism for controlled breakage of under-replicated chromosomes during mitosis

While DNA replication and mitosis occur in a sequential manner, precisely how cells maintain their temporal separation and order remains elusive. This study unveils a double-negative feedback loop between replication intermediates and an M-phase-specific structure-selective endonuclease, MUS81-SLX4 (see Drosophila Mus81 and Mus312), which renders DNA replication and mitosis mutually exclusive. MUS81 nuclease is constitutively active throughout the cell cycle (see Drosophila cell cycle) but requires association with SLX4 for efficient substrate targeting. To preclude toxic processing of replicating chromosomes, WEE1 (see Drosophila Wee1) kinase restrains CDK1 (see Drosophila Cdk1) and PLK1 (see Drosophila polo)-mediated MUS81-SLX4 assembly during S phase. Accordingly, WEE1 inhibition triggers widespread nucleolytic breakage of replication intermediates, halting DNA replication and leading to chromosome pulverization. Unexpectedly, premature entry into mitosis-licensed by unrestrained CDK1 activity during S phase-requires MUS81-SLX4, which inhibits DNA replication. This suggests that ongoing replication assists WEE1 in delaying entry into M phase and, indirectly, in preventing MUS81-SLX4 assembly. Conversely, MUS81-SLX4 activation during mitosis promotes targeted resolution of persistent replication intermediates, which safeguards chromosome segregation (Duda, 2016).

Table of contents

cdc2: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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