Cyclin B Eukaryotic cells have evolved regulatory mechanisms to ensure the strict alternation of DNA replication and mitosis. Recent work has suggested that the mitotic form of cyclin-dependent kinase (cdc2/cyclin B) has a role in preventing re-replication of the genome before mitosis. Several proteins have been proposed as linking DNA replication to cyclin/cdc2 dynamics. A Xenopus homolog of S. pombe cdc21 has been characterized as a new member of the MCM family of proteins (See the DNA replication site for details). The cdc21 protein exhibits cell-cycle dependent chromatin binding and phosphorylation in association with S-phase control. Cdc21 binds to decondensing chromatin at the end of mitosis, localizing to numerous foci that form prior to reconstitution of the nuclear membrane. Cdc21 remains bound to chromatin during the initiation of DNA replication and is displaced as the DNA replication forks progress. These subnuclear changes in localization correlate with cell-cycle-regulated changes in phosphorylation. Cdc21 binds to chromatin in an underphosphorylated state, but in early S phase the nuclear localized cdc21 is partially phosphorylated before it is displaced from the chromatin. Cytoplasmic cdc21 remains underphosphorylated but at the beginning of mitosis the entire pool of cdc21 is hyperphosphorylated, possibly by the cdc2/cyclin B kinase dimer. These properties identify Xenopus cdc21 as a possible component of the DNA licensing factor (Coué, 1996).
The mitotic cyclin-dependent kinase affects DNA replication by inhibiting the accumulation and function of cdc18, a critical regulator of S-phase entry in S. pombe. The ruml+ gene efficiently suppresses the lethality of a conditional cdc18 mutant. Conversely, deletion of ruml+ increases the severity of the cdc18 mutant phenotype, resulting in inappropriate cell division and a rapid loss of viability. Biochemical experiments indicate that Ruml potently inhibits cdc2 phosphorylation by directly interacting with the cdc2/Cyclin B complex. Overexpression of Ruml under conditions that promote re-replication of the genome induces a striking accumulation of cdc18 protein by a largely post-transcriptional mechanism. This work links a potent inhibitor of cdc2 kinase to a key protein required for the initiation of DNA replication and strongly suggest that inhibition of cdc18 by cyclin-dependent kinases plays an important role in ensuring that the genome is duplicated precisely once each cell cycle (Jallepalli, 1996).
During the cell cycle, a checkpoint prevents the initiation of mitosis until S-phase is completed. The molecular mechanism may involve the RCC1 protein, which catalyses guanine nucleotide exchange on the Ras-related nuclear protein Ran (or TC4). Genetic studies have suggested that RCC1 may be involved in sensing the replication state of DNA and controlling the activation of cdc2/cyclin B protein kinase through Ran. Direct biochemical evidence has been found for the post-translational control of cdc2/cyclin B activation by Ran. In a cell-free system of concentrated Xenopus egg extracts supplemented with nuclei, a mutant form of Ran (T24N) analogous to dominant inactive mutants of other Ras-related GTPases inhibits cdc2/Cyclin B activation in the presence of replicating nuclear DNA. This role for Ran is mediated through control of the tyrosine phosphorylation state of cdc2. When extracts are supplemented with RCC1 protein prior to addition of Ran T24N, inhibition of cdc2/Cyclin B by Ran T24N is relieved. This suggests that Ran T24N may act in a dominant manner by sequestering RCC1 in an inactive form. In the light of these results, it is proposed that generation of the GTP-bound form of Ran is required for cdc2/Cyclin B activation and entry into mitosis when this process is coupled to the progression of S-phase (Clarke, 1995).
A 60-kD protein called NAP1/SET (in the same family with but not to be confused with Drosophila Nucleosome assembly protein 1) has been purified form Xenopus, S. cerevisiae, and Drosophila. The yeast protein is known to function in nucleosome assembly. Members of the NAP/SET family of proteins interact specifically with B-type cyclins. This interaction is highly conserved during evolution: NAP1/SET interacts with cyclins B1 and B2, but not with cyclin A; the S. cerevisiae homolog interacts with B-type cyclins Clb2 but not Clb3. The yeast NAP1/SET protein is cytoplasmic. Genetic experiments in budding yeast indicate that NAP1/SET plays an important role in the function of Clb2, while biochemical experiments demonstrate that purifed NAP1 can be phosphorylated by cyclin B/p34cdc2 kinase complexes but not cyclin A/p34cdc2 kinase complexes. These results suggest that NAP1/SET is a protein involved in the specific functions of cyclin B/p34cdc2 kinase complexes (Kellogg, 1995a).
NAP1/SET is a 60-kD protein that interacts specifically with mitotic cyclins in budding yeast and frogs. The yeast B-type mitotic cyclin Clb2 is unable to carry out its full range of functions without NAP1, even though Clb2/p34CDC28-associated kinase activity rises to normal levels. In the absence of NAP1/SET, Clb2 is unable to efficiently induce mitotic events, and cells undergo a prolonged delay at the short spindle stage with normal levels of Clb2/p34CDC28 kinase activity. NAP1/SET is also required for the ability of Clb2 to induce the switch from polar to isotropic bud growth. As a result, polar bud growth continues during mitosis, giving rise to highly elongated cells. These experiments also suggest that NAP1/SET is required for the ability of the Clb2/p34CDC28 kinase complex to amplify its own production, and that NAP1/SET plays a role in regulation of microtubule dynamics during mitosis. Together, these results demonstrate that NAP1/SET is required for the normal function of the activated Clb2/p34CDC28 kinase complex, and provide a step towards understanding how cyclin-dependent kinase complexes induce specific events during the cell cycle (Kellogg, 1995b).
Passage through mitosis is required to reset replication origins for the subsequent S phase. During mitosis,the proper separation and segregation of sister chromatids is coordinated by a series of biochemical reactions involving cyclin-dependent kinases (CDKs), the anaphase promoting complex or cyclosome (APC/C), and a mitotic exit network including Cdc5, 14, and 15 coordinates. Previous studies have shown that inhibition of Clb/Cdc28p kinase by overexpression of the Clb/Cdc28 inhibitor Sic1 drives pre-RC formation. Subsequent repression of Sic1p expression allows reactivation of Clb/Cdc28p kinase and induces rereplication in G2/M arrested cells. Cyclin B/CDK inactivation can drive origin resetting in either early S phase or mitosis. This origin resetting occurs efficiently in the absence of APC/C function and mitotic exit network function. It is concluded that CDK inactivation is the single essential event in mitosis required to allow pre-RC assembly for the next cell cycle. The clear implication of these results is that some component or components of the pre-RC are phosphorylated and inactivated by Clb/Cdc28p kinase (Noton, 2000).
A replication competent (RC) complex has been isolated from calf thymus, containing DNA polymerase alpha, DNA polymerase delta and replication factor C. The RC complex has now been isolated from
nuclear extracts of synchronized HeLa cells; the complex contains DNA replication proteins associated with cell-cycle regulation factors like
cyclin A, cyclin B1, Cdk2 and Cdk1. In addition, it contains a kinase activity and DNA polymerase activities able to switch from a
distributive to a processive mode of DNA synthesis, which is dependent on proliferating cell nuclear antigen. In vivo cross-linking of
proteins to DNA in synchronized HeLa cells demonstrates the association of this complex to chromatin. There is a dynamic association of cyclins/Cdks with the RC
complex during the cell cycle. Indeed, cyclin A and Cdk2 associates with the complex in S phase, and cyclin B1 and Cdk1 are present exclusively in G2/M phase,
suggesting that the activity, as well the localization, of the RC complex might be regulated by specific cyclin/Cdk complexes (Frouin, 2002).
These results suggest the presence
of two complexes: (1) one bound to the chromatin that contains replication proteins, cyclin A and no Cdks; and (2) a soluble complex in the nucleus containing the
same replicative proteins as the chromatin bound complexes, except PCNA, which is absent. This DNA-unbound complex is associated with Cdk2/cyclin A in S
phase and Cdk1/cyclin A and B1 in G2 phase. This complex displays a kinase activity that is due to Cdks. Cdk/cyclins are known to phosphorylate several DNA replication proteins, such as SV40 T antigen, RP-A, pol alpha, pol delta and PCNA. Accordingly, Cdk-dependent phosphorylation of different proteins has been detected within the complex. Cdk-dependent phosphorylation of DNA replication proteins appears to have a
regulatory role. For example, cyclin A/Cdk2 has been shown to inhibit the replication activity of human pol alpha primase in an SV40 initiation assay,
whereas the activities of pol alpha and the tightly associated primase were not impaired in simpler in vitro assays. In addition to the role in modulating the activity of DNA replication enzymes, the results seem to suggest a role for cyclin/Cdk complexes in regulating the association of replication complexes to chromatin during the cell cycle. It could be that a stable association of cyclin A to replication complexes during S phase has the role of recruiting Cdk2, which in turn can regulate the dynamic association of the replication proteins to the chromatin. This might represent an example of intra-phase regulation, perhaps correlated to a different timing of origin firing. Cyclin A could have an 'informational' role, independent of its association to a Cdk. The appearance of Cdk1/cyclin B1 associated with replication complexes
in G2/M, concomitantly with the disappearance of Cdk2, could reflect an interphase regulatory mechanism, which prevents re-binding of replication complexes to
chromatin during G2/M phase. This hypothesis fits well with the so-called Cdk-driven 'replication switch' model, which predicts that Cdk activity serves both to activate initiation complexes and to inhibit further initiation complex assembly (Frouin, 2002).
Wee kinase inhibits cell division by phosphorylating cdc2, the kinase dimerization partner of Cyclins A and B. In a cell cycle, the mouse wee1 kinase is phosphorylated at M-phase. An in vitro study using a mitotic extract has revealed that phosphorylation occurs in the N-terminal domain, a domain absent from the human wee1 kinase. This results in kinase inactivation. The activity of the wee1 kinase is reduced by phosphorylation with the mitotic extract that contains cdc2-Cyclin B kinase (Honda, 1995).
Tyrosine phosphorylation of cdc2, the cyclin dependent kinase, occurs on Tyr15, a residue located within the ATP binding site of the protein: this is required for maintaining the inactivity of the cdc2-Cyclin B dimer until DNA replication is completed. At the end of G2, the activation of the cdc25 phosphatase (String in Drosophila) causes cdc2 dephosphorylation and the activation of the histone H1 kinase activity (Krek, 1991).
Cdc25 regulates entry into mitosis by regulating the activation of cyclin B/cdc2. In humans, at least two cdc25 isoforms have roles in controlling the G2/M transition. Two cdc25B splice variants, cdc25B2 and cdc25B3, are capable of activating cyclin A/cdk2 and cyclin B/cdc2, but the mitotic hyperphosphorylation of these proteins increases their activity toward only cyclin B1/cdc2. Cdc25C has only very low activity in its unphosphorylated form; following hyperphosphorylation it will efficiently catalyze the activation of only cyclin B/cdc2. This is reflected by the in vivo activity of the immunoprecipitated cdc25B and cdc25C from interphase and mitotic HeLa cells. The increased activity of the hyperphosphorylated cdc25s toward cyclin B1/cdc2 is in large part due to increased binding of this substrate. The substrate specificity, activities, and timing of the hyperphosphorylation of cdc25B and cdc25C during G2 and M suggest that these two mitotic cdc25 isoforms are activated by different kinases and perform different functions during progression through G2 into mitosis (Gabrielli, 1997).
Cell cycle arrest in G1 in response to ionizing radiation or senescence is believed to be provoked by inactivation of G1 cyclin-cyclin-dependent kinases (Cdks) by the Cdk inhibitor p21(see Drosophila Dacapo). In addition to exerting negative control of the G1/S phase transition, p21 may play a role at the onset of mitosis. In nontransformed fibroblasts, p21 transiently reaccumulates in the nucleus near the G2/M-phase boundary, concomitant with cyclin B1 nuclear translocation, and associates with a fraction of cyclin A-Cdk and cyclin B1-Cdk complexes. Premitotic nuclear accumulation of cyclin B1 is not detectable in cells with low p21 levels, such as fibroblasts expressing the viral human papillomavirus type 16 E6 oncoprotein, which functionally inactivates p53, or in tumor-derived cells. Synchronized E6-expressing fibroblasts show accelerated entry into mitosis, when compared to wild-type cells, and exhibit higher cyclin A- and cyclin B1-associated kinase activities. Primary embryonic fibroblasts derived from p21-/- mice have significantly reduced numbers of premitotic cells with nuclear cyclin B1. These data suggest that p21 promotes a transient pause late in G2 that may contribute to the implementation of late cell cycle checkpoint controls (Dulic, 1998).
A human homolog of Xenopus Eg5, a kinesin-related motor protein, is implicated in the assembly and dynamics of the mitotic spindle. Microinjection of antibodies against human Eg5 (HsEg5) blocks centrosome migration and causes HeLa cells to arrest in mitosis with monoastral microtubule arrays. Furthermore, an evolutionarily conserved cdc2 phosphorylation site (Thr-927) in HsEg5 is phosphorylated specifically during mitosis in HeLa cells and in vitro by p34cdc2/Cyclin B. Mutation of Thr-927 to nonphosphorylatable residues prevents HsEg5 from binding to centrosomes, indicating that phosphorylation controls the association of this motor with the spindle apparatus. These results indicate that HsEg5 is required for establishing a bipolar spindle and that p34cdc2 protein kinase directly regulates its localization (Blangy, 1995).
The p53 tumor suppressor controls multiple cell cycle checkpoints regulating the mammalian response to DNA damage. To identify the mechanism by which p53 regulates G2, a human ovarian cell has been derived that undergoes p53-dependent G2 arrest at 32 degrees C. p53 prevents G2/M transition by decreasing intracellular levels of cyclin B1 protein. To identify a mechanism by which p53 could cause a decrease in cyclin B1 protein and mRNA, it was determined whether p53 could regulate the activity of the cyclin B1 promoter. To this end, cultured cells were obtained with a CAT reporter construct containing 1,050 bp of the human cyclin B1 promoter, and reporter activity was then measured. This promoter contains the cis-DNA sequences necessary for cell cycle-specific transcription of the cyclin B1 gene. p53 has been shown to regulate the levels of cyclin B1 promoter-driven gene expression. The mechanism by which p53 regulates cyclin B1 promoter activity has yet to be determined. p53 could prevent cyclin B1 transcription by binding to and preventing the function of cyclin B1-specific transcription factors or p53 could interact directly with cyclin B1 promoter DNA. There is no obvious p53 consensus-binding site in the cyclin B1 promoter, suggesting that p53 regulates B1 transcription without direct interaction with promoter DNA. The cyclin B1 promoter lacks a consensus TATA box, and p53 is known to repress the activity of other TATA-less and TATA-containing promoters by interacting with the TATA box-binding protein (TBP) and preventing transcriptional initiation. However, there may be TBP-independent mechanisms for p53-mediated transcriptional repression; for example, p53 binds to and interferes with the functions of the SP1- and CCAAT-binding proteins. Transcription of the human cyclin B1 gene is activated by USF and NF-Y proteins and can be repressed by MyoD. p53 could regulate cyclin B1 promoter activity directly or indirectly through any or all of these transcription factors. The ability of p53 to control mitotic initiation by regulating intracellular cyclin B1 levels suggests that the cyclin B-dependent G2 checkpoint has a role in preventing neoplastic transformation (Innocente, 1999).
Cyclin B mRNA stored in immature zebrafish oocytes is translationally activated upon the stimulation of 17alpha,20beta-dihydroxy-4-pregnen-3-one (17alpha,20beta-DP), an event prerequisite for initiating oocyte maturation in this species. Localization of cyclin B mRNA was investigated in zebrafish oocytes. Cyclin B mRNA was found to be exclusively localized as an aggregation along the cytoplasm at the animal pole of full-grown immature oocytes. When oocytes are treated with 17alpha,20beta-DP, a meshwork of microfilaments in the oocyte cortex disappears and the aggregation of cyclin B mRNA disperses just prior to the initiation of cyclin B synthesis and germinal vesicle breakdown (GVBD). Cytochalasin B, but not nocodazole or taxol, deform the aggregation of cyclin B mRNA, indicating the involvement of microfilaments in organizing this form. Like 17alpha,20beta-DP, cytochalasin B induces both complete dispersion of the aggregation and translational activation of cyclin B mRNA, forcing the oocytes to undergo GVBD without 17alpha,20beta-DP. Conversely, disturbance of the aggregation of cyclin B mRNA with a low concentration of cytochalasin B inhibits 17alpha,20beta-DP-induced GVBD. These results suggest that the direct change in cyclin B mRNA from the aggregated form to the dispersed form is responsible for translational activation of the mRNA during zebrafish oocyte maturation (Kondo, 2001).
During oocyte maturation, cyclin B1 mRNA is translationally activated by cytoplasmic polyadenylation. This process is dependent on cytoplasmic polyadenylation elements (CPEs) in the 3' untranslated region (UTR) of the mRNA. To determine whether a titratable factor might be involved in the initial translational repression (masking) of this mRNA, high levels of cyclin B1 3' UTR were injected into oocytes. While this treatment had no effect on the poly(A) tail length of endogenous cyclin B1 mRNA, it induces cyclin B1 synthesis. A mutational analysis reveals that the most efficient unmasking element in the cyclin 3' UTR is the CPE. However, other U-rich sequences that resemble the CPE in structure, but which do not bind the CPE-binding polyadenylation factor CPEB (Drosophila homolog: Orb), fail to induce unmasking. When fused to the chloramphenical acetyl transferase (CAT) coding region, the cyclin B1 3' UTR inhibits CAT translation in injected oocytes. In addition, a synthetic 3' UTR containing multiple copies of the CPE also inhibits translation, and do so in a dose-dependent manner. Furthermore, efficient CPE-mediated masking requires cap-dependent translation. During the normal course of progesterone-induced maturation, cytoplasmic polyadenylation is necessary for mRNA unmasking. A model to explain how cyclin B1 mRNA masking and unmasking could be regulated by the CPE is presented (de Moor, 1999).
Translational control is prominent during meiotic maturation and early development. A mode of translational repression in Xenopus laevis oocytes has been investigated, with a focus placed on the mRNA encoding cyclin B1. Translation of cyclin B1 mRNA is relatively inactive in the oocyte and increases dramatically during meiotic maturation. It has been shown, by injection of synthetic mRNAs, that the cis-acting sequences responsible for repression of cyclin B1 mRNA reside within its 3'UTR. Repression can be saturated by increasing the concentration of reporter mRNA injected, suggesting that the cyclin B1 3'UTR sequences provide a binding site for a trans-acting repressor. The sequences that direct repression overlap and include U-rich sequence cytoplasmic polyadenylation elements (CPEs). These are sequences known to promote cytoplasmic polyadenylation. However, the presence of a CPE per se appears insufficient to cause repression, as other mRNAs that contain CPEs are not translationally repressed. Relief of repression and cytoplasmic polyadenylation are intimately linked. Repressing elements do not override the stimulatory effect of a long poly(A) tail, and polyadenylation of cyclin B1 mRNA is required for its translational recruitment. These results suggest that translational recruitment of endogenous cyclin B1 mRNA is a collaborative effect of derepression and poly(A) addition (Barkoff, 2000).
Several models for the regulation of cyclin B1 mRNA can be considered. In one, maturation causes both relief of repression (e.g., inactivation of the repressor) and activation of cytoplasmic polyadenylation. These events occur simultaneously, but are independent. In a second model, meiotic maturation causes relief of repression, which is a prerequisite for the acquisition of the polyadenylation machinery to the mRNA. In a third model, meiotic maturation promotes polyadenylation of the mRNA, which in turn causes relief of repression. Both the second and the third models suggest competition for overlapping 3'UTR binding sites; polyadenylation could displace or modify a repressor or removal of the repressor could allow the polyadenylation machinery to access the mRNA. Several lines of evidence support the third model: (1) poly(A) function is not significantly prevented by the presence of repressing elements in oocytes; (2) repression is maintained on mRNAs that are not polyadenylated after maturation. This suggests that polyadenylation, and not another maturation-specific event, is required. Finally, polyadenylation is necessary to recruit cyclin B1 mRNA onto polysomes (Barkoff, 2000).
One simple molecular incarnation of the third model follows. Prior to meiotic maturation the mRNA has a short poly(A) tail and is repressed by the binding of a repressor to its 3'UTR. This repressor may include a polyadenylation-inactive form of cytoplasmic polyadenylation element binding protein (CPEB), on its own or bound to additional repressor proteins. The cytoplasmic polyadenylation machinery is not active at this stage. During meiotic maturation, the polyadenylation apparatus is activated, enabling polyadenylation factors to bind to the RNA, in part through AAUAAA. The relevant polyadenylation factors may be a cytoplasmic form of CPSF, a polyadenylation-competent form, CPEB, or a combination of the two. The mRNA is then polyadenylated and efficiently translated. Either the binding of the polyadenylation machinery or the enhanced translational activity that results displaces or modifies the repressor allowing maximal translation. Support for a dual role for CPEB in repression and activation has emerged recently: p82, a clam CPEB homolog, acts as both a translational repressor in the oocyte and a polyadenylation factor during maturation. The mechanisms by which repression is maintained and relieved are key issues for future research (Barkoff, 2000).
The involvement of Ras in the activation of multiple early signaling pathways is well understood, but it is less clear how the various Ras effectors interact with the cell cycle machinery to cause G(1) progression. Ras-mediated activation of extracellular-regulated kinase/mitogen-activated protein kinase has been implicated in cyclin D(1) up-regulation, but there is little extracellular-regulated kinase activity during the later stages of G(1), when cyclin D(1) expression becomes maximal, implying that other effector pathways may also be important in cyclin D1 induction. The involvement of Ras effectors from the phosphatidylinositol (PI) 3-kinase and Ral-GDS families in G1 progression has been addressed and this involvement is compared to that of the Raf/mitogen-activated protein kinase pathway. PI 3-kinase activity is required for the expression of endogenous cyclin D1 and for S phase entry following serum stimulation of quiescent NIH 3T3 fibroblasts. Activated PI 3-kinase induces cyclin D1 transcription and E2F activity, at least in part mediated by the serine/threonine kinase Akt/PKB, and to a lesser extent the Rho family GTPase Rac. In addition, both activated Ral-GDS-like factor and Raf stimulate cyclin D1 transcription and E2F activity and act in synergy with PI 3-kinase. Therefore, multiple cooperating pathways mediate the effects of Ras on progression through the cell cycle (Gille, 1999).
A combination of site-directed mutagenesis and phosphopeptide-mapping has identified serine residues 2, 94, 96, 101, and 113 as presumptive phosphorylation sites of Xenopus Cyclin B. Together these sites account for all Cyclin B1 phosphorylation in oocytes before germinal vesicle breakdown (GVBD). Phosphorylation of Cyclin B1 appears to be required for Xenopus oocyte maturation. Furthermore, partial phosphorylation of these five sites is sufficient to meet this requirement. Phosphorylation of Cyclin B1 is not required for cdc2 kinase activity, for binding to cdc2 protein, for stability of Cyclin B1 before GVBD, or for destruction of cyclin B1 either after GVBD or after egg activation. These data confirm that phosphorylation confers enhanced biological activity to cyclin B1 (Li, 1995).
Mutations in the S. cerevisiae gene mad1 (mitotic arrest deficient) inactivate the spindle assembly checkpoint. The checkpoint functions to prevent cells from initiating anaphase until the spindle has been fully assembled. Mad1 protein become hyperphosphorylated when cells are arrested in mitosis. Mad1 appears to lie in the middle of the pathway for transducing spindle defects into a metaphase block (Hardwick, 1995).
Inactivation of cyclin B-Cdc2 kinase at the exit from M phase depends on the specific proteolysis of the cyclin B subunit, whereas the Cdc2 subunit remains present at nearly constant levels throughout the cell cycle. It is unknown how Cdc2 escapes degradation when cyclin B is destroyed. In Xenopus egg extracts that reproduce the exit from M phase in vitro, dissociation of the cyclin B-Cdc2 complex occurs under conditions where cyclin B is tethered to the 26S proteasome but not yet degraded. The dephosphorylation of Thr 161 on Cdc2 is unlikely to be necessary for the dissociation of the two subunits. However, the dissociation is dependent on the presence of a functional destruction box in cyclin B. Cyclin B ubiquitination is also, by itself, not sufficient for separation of Cdc2 and cyclin B. The 26S proteasome, but not the 20S proteasome, is capable of dissociating the two subunits. These results indicate that the cyclin B and Cdc2 subunits are separated by the proteasome through a mechanism that precedes proteolysis of cyclin B and is independent of proteolysis. As a result, cyclin B levels decrease on exit from M phase but Cdc2 levels remain constant (Nishiyama, 2000).
Because previous studies have demonstrated that the phosphorylation of Thr 161 on Cdc2 increases the stability of the cyclin B-Cdc2 complex, one might have anticipated that the dephosphorylation of Thr 161 might contribute to its dissociation. However, contrary to this premise, these results suggest a possibility that dephosphorylation on Thr 161 is not necessary for dissociation of the cyclin B-Cdc2 complex at exit from M phase. This possibility is consistent with the previous report that okadaic acid prevents both the dephosphorylation of Cdc2 and the drop in H1 kinase activity, but not cyclin B degradation. The fact that simply reversing the step that stabilizes the cyclin B-Cdc2 complex does not cause dissociation of the complex once it has formed, provides support for the notion that a positive-acting mechanism releases the two subunits at the end of M phase (Nishiyama, 2000).
The present results indicate that a functional destruction box is required for dissociation of cyclin B-Cdc2. Considering that the destruction box in cyclin B is known to be involved in polyubiquitination of cyclin B, one could postulate that polyubiquitination of cyclin B itself might cause the release of Cdc2 from cyclin B. In accordance with this notion, it has been suggested that the polyubiquitin chain helps to unfold the target proteins. However, in the present study polyubiquitination is not by itself sufficient to dissociate Cdc2 from cyclin B. It is not known to what extent cyclin B is unfolded as a result of ubiquitination. What then is the role of the destruction box in the dissociation of cyclin B-Cdc2? Although the possibility that the destruction box contributes to a function other than polyubiquitination of cyclin B, it is reasonable that polyubiquitination of cyclin B is a way for targeting cyclin B-Cdc2 to the dissociation because the proteasome is required for this dissociation in the present study and because a multi-ubiquitin chain is a well-known targeting signal to the proteasome. Thus, polyubiquitination of cyclin B is likely to target the complex to the proteasome where Cdc2 is released from cyclin B. Whether or not ubiquitination also plays a role in facilitating the disassembly of the complex by the proteasome remains to be resolved (Nishiyama, 2000).
How does the proteasome dissociate Cdc2 from cyclin B? The 19S regulatory particle is composed of two subcomplexes, the 'base' and the 'lid'. The base is located proximal and the lid distal to the 20S core particle. The lid is essential for the recognition, and possibly binding, of polyubiquitinated substrate proteins, whereas the base is likely to promote substrate unfolding through its six distinct AAA (ATPases associated with a variety of cellular activities)-type ATPase components in a chaperone-like manner that is independent of polyubiquitin chains. In addition, the 20S proteolytic core is composed of two alpha- and two beta-rings. The alpha-rings discriminate between unfolded and folded proteins, and the beta-rings, which constitute the central catalytic core, can cleave substrates proteolytically. On the basis of the above and the results presented in this paper, the following model for degradation of cyclin B: Initially, polyubiquitin chains of cyclin B that have been added by the APC/C tether the cyclin B-Cdc2 complex to the lid of the 19S particle. Then, because of a chaperone-like function of the base of the 19S particle with the aid of the alpha-rings in the 20S component, the tethered cyclin B-Cdc2 complex is unfolded. Consequently, Cdc2 dissociates from cyclin B, while cyclin B is translocated into the catalytic core of the 20S component. Lastly, cyclin B is degraded within the lumen of the 20S component by the proteolytic activity of the beta-rings. At present, however, it cannot be discriminated whether Cdc2 is passively separated from cyclin B as a consequence of cyclin B unfolding or whether Cdc2 is actively dissociated from the cyclin B-Cdc2 complex by a chaperone-like activity of the proteasome components (Nishiyama, 2000).
A model is presented for release of Cdc2 from cyclin B by the 26S proteasome. (1) Initially, cyclin B complexed with Cdc2 is polyubiquitinated by an APC/C-dependent pathway at exit from M phase. (2) Next, the lid of the 19S regulatory particle recognizes and tethers the polyubiquitinated cyclin B that remains associated with Cdc2. (3) The cyclin B-Cdc2 complex is unfolded and dissociated by the chaperone-like activity of the base of the 19S regulatory particle with the aid of the alpha ring of the 20S proteasome. The translocation of unfolded cyclin B to the 20S proteasome may be required for further unfolding of cyclin B, and both processes may be coupled to one another. (4) Finally, the unfolded cyclin B is translocated and degraded in the hollow center (beta rings of the 20S proteasome) in which all catalytic sites are located (Nishiyama, 2000).
Cdc20 (Drosophila homolog: Fizzy), an activator of the anaphase-promoting complex (APC), is also required for the exit from mitosis in Saccharomyces cerevisiae. During mitosis, both the inactivation of Cdc28-Clb2 kinase and the degradation of mitotic cyclin Clb2 occur in two steps. The first phase of Clb2 proteolysis, which commences at the metaphase-to-anaphase transition when Clb2 abundance is high, is dependent on Cdc20. The second wave of Clb2 destruction in telophase requires activation of the Cdc20 homolog, Hct1/Cdh1 (Drosophila homolog: Fizzy related). The first phase of Clb2 destruction, which lowers the Cdc28-Clb2 kinase activity, is a prerequisite for the second. Thus, Clb2 proteolysis is not solely mediated by Hct1 as generally believed; instead, it requires a sequential action of both Cdc20 and Hct1 (Yeong, 2000).
Ubiquitin-dependent proteolytic events play a critical role in driving eukaryotic cells through mitosis. Both chromosome segregation and the final exit from M phase, the two landmark events of mitosis, require a key ubiquitin ligase (or E3 enzyme) known as the anaphase-promoting complex (APC or cyclosome). The APC promotes chromosome separation by marking, via ubiquitination, the anaphase inhibitor Pds1 for destruction by the 26S proteasome. The proteolytic degradation of Pds1 liberates its binding partner Esp1, which in turn causes the cleavage of the cohesin subunit Scc1 allowing sister chromatid separation. Later, the APC mediates the exit of cells from mitosis by targeting for proteolysis the mitotic cyclins, such as Clb2. In the budding yeast, APC is composed of at least 12 different subunits including Cdc16, Cdc23, Cdc26, and Cdc27 proteins. To ubiquitinate various mitotic targets, the APC requires additional proteins, namely Cdc20 and its homolog Hct1/Cdh1. Although these WD40 repeat-containing proteins are not permanent members of the core APC, their increased binding to APC during the cell cycle correlates well with the increase in APC activity. Therefore, Cdc20 and Hct1 are called the activators of APC (hence the terms APCCdc20 and APCHct1), though the nature of this activation remains elusive. Like Cdc20 and Hct1 in budding yeast, pairs of homologous proteins have also been identified in Schizosaccharomyces pombe, Drosophila (Fizzy
and Fizzy related), Xenopus, and humans (Yeong, 2000 and references therein).
It is generally believed that Cdc20 and Hct1 determine the substrate specificity of APC such that the activation by Cdc20 leads to Pds1 destruction at the onset of anaphase while the Hct1-activated APC targets Clb2 for proteolysis, thus facilitating the departure of cells from mitosis. It is noteworthy that this substrate specificity is seen in cells arrested in S phase, but not in M phase, during which both Clb2 and Pds1 are actually degraded. Nevertheless, these studies provide a simple scheme for APC-driven progression through mitosis. However, contrary to this model, it has been reported that in the budding yeast, Cdc20 function is essential not only for chromosome segregation but also for the final exit from mitosis (Yeong, 2000 and references therein).
One of the crucial aspects of progression through mitosis is that Hct1-mediated Clb2 proteolysis occurs only after APCCdc20 consigns Pds1 for destruction. What is the mechanism that ensures the correct timing of these events? Recently, it has been shown that, while phosphorylation of Hct1 by the mitotic kinase (CDC28) renders it inactive, dephosphorylation by Cdc14 restores its activity. Cdc14, a dual specificity phosphatase, itself appears to be spatially regulated in that it is sequestered to the nucleolar RENT complex during G1 to anaphase. During late anaphase, Cdc14 is dispersed from the RENT complex in a Tem1-dependent manner resulting in the activation of Hct1, which in turn triggers Clb2 proteolysis. Hence, it is the antagonistic action of the mitotic kinase and the Cdc14 phosphatase on Hct1 that may determine the correct timing of Clb2 proteolysis. However, this scenario requires that, by the time cells reach late telophase, the balance is decisively tipped in favor of net dephosphorylation of Hct1. This would be achievable if the rate of Cdc14-mediated dephosphorylation were inherently higher than phosphorylation by the mitotic kinase Cdc28-Clb. Alternatively, since Cdc20 is also required for mitotic exit, it is conceivable that APCCdc20 causes destruction of a protein that normally inhibits Hct1 and thereby paves the way for rapid activation of Hct1 (Yeong, 2000 and references therein).
During mitosis, Clb2 degradation, and hence the inactivation of the Cdc28-Clb2 kinase, occurs in a biphasic manner. The first phase commences during anaphase and requires Cdc20 function. This leads to an initial lowering of the mitotic kinase activity that eventually results in, perhaps by facilitating Hct1 activation, the second phase of Clb2 proteolysis in late telophase. These results suggest that Cdc20 is not a substrate-specific activator of APC that only promotes Pds1 degradation but that its function is also essential for the rapid destruction of Clb2 during mitosis (Yeong, 2000).
The proposed scheme for the destruction of Clb2 is a clear departure from the purely deterministic model according to which Cdc20 and Hct1 are the substrate-specific activators of the APC. It is surmised that the overlapping specificity of Cdc20 toward Pds1, Clb2, and Clb5 destruction is not merely a 'slippage in the system'; instead, this flexibility plays an important physiological role in fine tuning the timing of Clb2 degradation. The kinetic intertwining of this kind between various cellular reactions may be widely used by cells to sharply define the timing of some of the key events during cell division (Yeong, 2000).
Cell division is driven by cyclin-B-dependent kinase and anaphase-promoting complex (APC)-mediated proteolysis. Continuing transcription of E2F target genes beyond the G1/S transition is required for coordinating S-phase progression with cell division. Using an in vivo assay to measure protein stability in real time during the cell cycle, it has been shown that repression of E2F activity or inhibition of cyclin-A-dependent kinase in S phase triggers the destruction of cyclin B1 through the re-assembly of APC, the ubiquitin ligase that is essential for mitotic cyclin proteolysis, with its activatory subunit Cdh1. Phosphorylation-deficient mutant Cdh1 or immunodepletion of cyclin A results in assembly of active Cdh1-APC even in S-phase cells. These results implicate an E2F-dependent, cyclin A/Cdk2-mediated phosphorylation of Cdh1 in the timely accumulation of cyclin B1 and the coordination of cell-cycle progression during the G-1 phase post-restriction point period (Lukas, 1999).
Two specific components are required for the ubiquitination of mitotic cyclins: E2-C, a cyclin-selective ubiquitin carrier protein that is constitutively active during the cell cycle, and E3-C, a cyclin-selective ubiquitin ligase that purifies as part of the cyclosome, an approximately 1500-kDa complex, active only near the end of mitosis. The cyclosome has been separated from cdc2, its ultimate upstream activator. The mitotic, active form of the cyclosome can be inactivated by incubation with a partially purified, endogenous okadaic acid-sensitive phosphatase; addition of cdc2 restores activity to the cyclosome after a lag that resembles the lag seen in intact cells and in crude extracts. These results demonstrate that activity of cyclin-ubiquitin ligase is controlled by reversible phosphorylation of the cyclosome complex (Lahav-Baratz, 1995).
Immediately before the transition from metaphase to anaphase, the protein kinase activity of maturation or M-phase promoting factor (MPF) is inactivated by a mechanism that involves the degradation of its regulatory subunit, cyclin B. The availability of biologically active goldfish cyclin B produced in Escherichia coli and purified goldfish proteasomes (a nonlysosomal large protease) has allowed the role of proteasomes in the regulation of cyclin degradation to be examined for the first time. The 26S proteasome (but not the 20S proteasome) digests recombinant 49-kD cyclin B at lysine 57 (K57), producing a 42-kD truncated form. The 42-kD cyclin is also produced by the digestion of native cyclin B, forming a complex with cdc2, a catalytic subunit of MPF; a fragment transiently appears during cyclin degradation when eggs are released from metaphase II arrest by egg activation. Mutant cyclin at K57 is resistant to both digestion by the 26S proteasome and degradation at metaphase/anaphase transition in Xenopus egg extracts. The results of this study indicate that the destruction of cyclin B is initiated by the ATP-dependent and ubiquitin-independent proteolytic activity of 26S proteasome through the first cutting in the NH2 terminus of cyclin (at K57 in the case of goldfish cyclin B). It is surmised that this cut allows the cyclin to be ubiquitinated for further destruction by ubiquitin-dependent activity of the 26S proteasome that leads to MPF inactivation (Tokumoto, 1997).
The initiation of anaphase and exit from mitosis depend on the anaphase-promoting complex (APC), which mediates the ubiquitin-dependent proteolysis of anaphase-inhibiting proteins and mitotic cyclins. An investigation was carried out using Xenopus egg extracts to see if protein phosphatases are required for mitotic APC activation. In Xenopus egg extracts, APC activation occurs normally in the presence of protein phosphatase 1 inhibitors, suggesting that the anaphase defects caused by protein phosphatase 1 mutation in several organisms are not due to a failure to activate the APC. Contrary to this, the initiation of mitotic cyclin B proteolysis is prevented by inhibitors of protein phosphatase 2A, such as okadaic acid. Okadaic acid induces an activity that inhibits cyclin B ubiquitination. This activity is referred to as inhibitor of mitotic proteolysis because it also prevents the degradation of other APC substrates. A similar activity exists in extracts of Xenopus eggs that are arrested at the second meiotic metaphase by the cytostatic factor activity of the protein kinase mos. In Xenopus eggs, the initiation of anaphase II may therefore be prevented by an inhibitor of APC-dependent ubiquitination (Vorlaufer, 1998).
Cell cycle-specific proteolysis is critical for proper execution of mitosis in all eukaryotes. Ubiquitination and subsequent proteolysis of the mitotic regulators Clb2 and Pds1 depend on the cyclosome/APC and the 26S proteasome. Components of the cell cycle machinery in yeast, specifically the cell cycle regulatory cyclin-dependent kinase Cdc28 and a conserved associated protein Cks1/Suc1, interact genetically, physically, and functionally with components of the 26S proteasome. A mutation in Cdc28 (cdc28-1N) that interferes with Cks1 binding, or inactivation of Cks1 itself, confers stabilization of Clb2, the principal mitotic B-type cyclin in budding yeast. Surprisingly, Clb2-ubiquitination in vivo and in vitro is not affected by mutations in cks1, indicating that Cks1 is not essential for cyclosome/APC activity. However, mutant Cks1 proteins no longer physically interact with the proteasome, suggesting that Cks1 is required for some aspect of proteasome function during M-phase-specific proteolysis. Evidence is provided that Cks1 function is required for degradation of the anaphase inhibitor Pds1. Stabilization of Pds1 is partially responsible for the metaphase arrest phenotype of cks1 mutants because deletion of PDS1 partially relieves the metaphase block in these mutants (Kaiser, 1999).
Perhaps a clue to Cks1 function lies in the observation that more cdc28-1N is found in immunocomplexes with proteasome subunits than wild-type Cdc28. Similar observations have also been made in in vitro experiments, in which portions of the 19S proteasome cap have been reconstituted. Taking into account both differential binding of Cdc28-1N and Cdc28 to the proteasome and the independent association of Cks1 with the proteasome, it is suggested that Cks1 may function as a recycling factor for Cdc28. After proteolysis of Clb2, Cks1 binding to Cdc28 would release the kinase subunit from a putative receptor on the proteasome and allow new Cdc28/Clb2 complexes to bind. A defect in Cdc28/Cks1 complex formation would disrupt the recycling cycle and result in a blocked receptor leading to defects in Clb2 proteolysis. Consistent with this idea, Cdc28-1N confers a partial dominant-negative phenotype when overexpressed. This model is largely hypothetical and a more direct mechanism of regulation of proteasome activity by Cdc28/Cks1 is equally plausible (Kaiser, 1999).
The proteolysis of key regulatory proteins is thought to control progress through mitosis. The centromeres of misaligned chromosomes prevent the multisubunit ubiquitin ligase called anaphase promoting complex (APC) from degrading mitotic regulators by sequestering an essential adaptor protein, Cdc20/Slp1, through a multiprotein complex composed of the MAD and BUB proteins. Cyclin B1 degradation has been analyzed in real time; degradation begins as soon as the last chromosome aligns on the metaphase plate, just after the spindle-assembly checkpoint is inactivated. At this point, cyclin B1 staining disappears from the spindle poles and from the chromosomes. Cyclin B1 destruction can subsequently be inactivated throughout metaphase if the spindle checkpoint is reimposed, and this correlates with the reappearance of cyclin B1 on the spindle poles and the chromosomes. These results provide a temporal and spatial link between the spindle-assembly checkpoint and ubiquitin-mediated proteolysis (Clute, 1999).
Thus the initiation of cyclin B1 proteolysis correlates with the inactivation of the spindle-assembly checkpoint and lends support to the theory that the checkpoint keeps the multisubunit ubiquitin ligase [called anaphase promoting complex (APC)] inactive in prometaphase of normal mitosis. Moreover, the finding that reimposing the checkpoint after chromosome alignment is able to inactivate cyclin B1 proteolysis within 1 min shows that the activity of the APC is highly responsive to the state of the chromosomes, at least until sister chromatid separation. The current model of how the checkpoint regulates the APC proposes that the tetrameric form of Mad2, a component of the MAD-BUB complex on the centromere, sequesters proteins of the Cdc20/Fizzy family that are essential elements of APC-mediated proteolysis. If this is also how the checkpoint inactivates proteolysis after chromosome alignment, rapid relocalization of Cdc20 family members to the centromeres, and possibly to the spindle poles, should be detectable after treating metaphase cells with taxol. Mad2 only reappears on a subset of kinetochores in these cells; therefore these should be the ones that sequester Cdc20 (Clute, 1999).
The changes in the staining pattern of cyclin B1-GFP before and after its destruction begins suggest that the APC may primarily be active on the chromosomes and, perhaps, the spindle poles. This agrees with immunofluorescence data showing that components of the APC, such as the human homologs of budding yeast Cdc16 (APC6) and Cdc27 (APC3)33 and of Aspergillus BIME (APC1)34, are localized to the spindle poles and/or centromeres. Some of the components involved in the spindle-assembly checkpoint (MAD and BUB proteins) are also at the centromere. Moreover, there are data suggesting that the control of anaphase is restricted to the area of the spindle. In a cell with two spindles, anaphase in one spindle can only be inhibited by unaligned chromosomes in the other if the two sets of microtubules interact. Thus, there may be a role for microtubule-associated motors in regulating the proteolysis of cyclin B1. However, once one spindle initiates anaphase, the second spindle will begin anaphase 9 min later, even if it contains unaligned chromosomes. One possible explanation is that the APC becomes refractory to the spindle checkpoint after anaphase, perhaps through the exchange of Cdc20 for the related Hct1 protein (Clute, 1999).
These data indicate that there is a dynamic component to cyclin B1 destruction. There is an increase in the fluorescence on the spindle when the spindle-assembly checkpoint is reimposed in metaphase. Cyclin B1 is displaced from the chromosomes and the spindle poles when the APC is activated, and reassociates as soon as the APC is inactivated. However, this change in localization is not directly due to destruction because it is not reversed by MG132, a proteasome inhibitor. Thus, ubiquitin conjugation itself may cause cyclin B1 to dissociate from the mitotic apparatus. Lastly, a mutant of cyclin B1 with an altered destruction box does not associate with the chromosomes, possibly because it cannot recognize a component of the destruction machinery that is bound to the chromosomes (Clute, 1999).
Mitosis requires activity of the cyclin B cyclin-dependent kinase 1 (cdc2) heterodimer. Exit from mitosis depends on the inactivation of the complex by the degradation of cyclin B. Cdk2 is also active during mitosis. In Xenopus egg extracts, cdk2 is primarily in complex with cyclin E, which is stable. At the end of mitosis, downregulation of cdk2-cyclin E activity is accompanied by inhibitory phosphorylation of cdk2. Cdk2-cyclin E activity maintains cdk1-cyclin B during mitosis. At mitosis exit, cdk2 is inactivated prior to cdk1. The loss of cdk2 activity follows and depends upon an increase in protein kinase A (PKA) activity. Prematurely inactivating cdk2 advances the time of cyclin B degradation and cdk1 inactivation. Blocking PKA, instead, stabilizes cdk2 activity and inhibits cyclin B degradation and cdk1 inactivation. The stabilization of cdk1-cyclin B is also induced by a mutant cdk2-cyclin E complex that is resistant to inhibitory phosphorylation. P21-Cip1, which inhibits both wild-type and mutant cdk2-cyclin E, reverses mitotic arrest under either condition. These findings indicate that the proteolysis-independent downregulation of cdk2 activity at the end of mitosis depends on PKA and is required to activate the proteolysis cascade that leads to mitosis exit (D'Angiolella, 2001).
In the early embryonic cell cycle, exit from M phase is immediately followed by entry into S phase without an intervening gap phase. To understand the regulatory mechanisms for the cell cycle transition from M to S phase, dependence on Cdc2 inactivation of cell-cycle events occurring during the M-S transition period was examined using Xenopus egg extracts in which the extent of Cdc2 inactivation at M phase exit was quantitatively controlled. The results demonstrate that the occurance of MCM binding and the initiation of DNA replication of nuclear chromatin depends on the decrease of Cdc2 activity to critical levels. Similarly, it was found that Cdc2 inhibitory phosphorylation and cyclin B degradation are turned on and off, respectively, depending on the decrease in Cdc2 activity. However, sensitivity of these processes to Cdc2 activity was different, with the turning-on of Cdc2 inhibitory phosphorylation occurring at higher Cdc2 activity levels than the turning-off of cyclin B degradation. This means that, when cyclin B degradation ceases at M phase exit, Cdc2 inhibitory phosphorylation is necessarily activated. In the presence of constitutive synthesis of cyclin B, this condition favors the occurrence of the Cdc2 inactivation period after M phase exit, thereby ensuring progression through S phase. Thus, M phase exit and S phase entry are coordinately regulated by the Cdc2 activity level in the early embryonic cell cycle (Iwabuchi, 2002).
The anaphase promoting complex/cyclosome (APC/C), activated by fzy and fzr (fizzy and fizzy-related), degrades cell cycle proteins that carry RXXL or KEN destruction boxes (d-boxes). APC/C substrates regulate sequential events and must be degraded in the correct order during mitosis and G1. How d-boxes determine APC/Cfzy/APC/Cfzr specificity and degradation timing was studied. Cyclin B1 has an RXXL box and is degraded by both APC/Cfzy and APC/Cfzr; fzy has a KEN box and is degraded by APC/Cfzr only. The degradation of substrates with swapped d-boxes was characterized. Cyclin B1 with KEN is degraded by APC/Cfzr only. Fzy with RXXL can be degraded by APC/Cfzy and APC/Cfzr. Interestingly, APC/Cfzy-but not APC/Cfzr-specific degradation is highly dependent on the location of RXXL. Degradation of tagged substrates was studied in real time and it was observed that APC/Cfzr is activated in early G1. These observations demonstrate how d-box specificities of APC/Cfzy and APC/Cfzr, and the successive activation of APC/Cby fzy and fzr, establish the temporal degradation pattern. These observations can explain further why some endogenous RXXL substrates are degraded by APC/Cfzy, while others are restricted to APC/Cfzr (Zur, 2002).
More than a dozen different groups of proteins are degraded by the APC/C pathway, including mitotic A and B type cyclins, fzy, securin, E2-C, polo kinase, nek2A, hsl1, cdc6 and geminin. While all these proteins are degraded by the APC/C, they start to be degraded at different time points, such as prometaphase for cyclin A and nek2A, metaphase for cyclin B1, securin and xkid, and G1 for cdc6. APC/C substrates carry conserved motifs, so-called destruction boxes (d-boxes), which are required for their degradation. The cyclin B1 d-box (RTALGDIGN) is crudely shared by many of the other APC/C substrates. The arginine (R) and the leucine (L) are conserved in almost all substrates except in pim1, where arginine is replaced by lysine, and in cyclin B3, where leucine is replaced by phenylalanine. The asparagine (N) at position 9 is conserved in a subset of substrates and is required for the degradation of cyclin B1 in Xenopus extracts. Other residues of this RXXL box are much less conserved and it is virtually impossible to identify such a box merely by its sequence. However, the APC/C is evidently able to identify real RXXL boxes because not every protein that carries an RXXL is degraded. Moreover, fine differences in this box can contribute to changes in degradation, as is the case for cyclins A and B. An important recent advance is the identification of the KEN box as a targeting signal of some APC/C substrates. The discovery of this motif explained how vertebrate fzy, which lacks an RXXL box, is targeted for degradation by the APC/C. This box also plays a role in the degradation of substrates that do have an RXXL box, such as securin, clb2, hsl1 and nek2A. However, the KEN motif is also abundant in many proteins that are not APC/C substrates (Zur, 2002 and references therein).
The APC/C is activated by two WD repeat proteins: fzy/cdc20 and fzr/cdh1/hct1. In yeast, these two proteins confer some substrate specificity on the APC/C: pds1 is ubiquitylated by APC/Ccdc20, and clb2 by APC/Ccdh1. A similar specificity has been suggested in mammalian cells, and it was shown that fzy is ubiquitylated by APC/Cfzr only. Fzy and fzr directly bind different APC/C substrates in vitro. Fzy is restricted to substrates that have an RXXL box, and fzr binds both RXXL and KEN box substrates (Zur, 2002 and references therein).
In order to study the signal specificity of the RXXL and KEN boxes, artificial motifs were inserted into known substrates and their degradation was studied in vivo. The degradation of cyclin B1 with a mutated RXXL box can be restored by the insertion of an artificial KEN box close to the N-terminus of the mutated RXXL box. The location of this KEN is critical for its capacity to support degradation. Strikingly, cyclin B1 with a KEN box and a mutated RXXL box is ubiquitylated in vitro and degraded in vivo by APC/Cfzr only. This is in contrast to cyclin B1 with a wild-type RXXL box, which is degraded by both APC/Cfzy and APC/Cfzr (Zur, 2002).
The degradation of fzy, which is targeted for degradation by a KEN box and is an APC/Cfzr-specific substrate, was studied. Mutation of the KEN box stabilizes fzy, and its degradation can be restored by the insertion of an RXXL box. Following the replacement of KEN with RXXL, fzy is targeted by APC/Cfzy, as well as by APC/Cfzr. RXXL inserted anywhere into the N-terminus of fzy can support APC/Cfzr-specific degradation. Degradation by APC/Cfzy is, however, highly dependent on the location of the RXXL, suggesting that flanking sequences or conformation influence the APC/Cfzr/APC/Cfzy specificity of the RXXL box. This could explain why certain RXXL substrates are degraded by APC/Cfzr only while others are degraded by both APC/Cfzr and APC/Cfzy (Zur, 2002)
The degradation of green fluorescent protein (GFP)-tagged versions of APC/Cfzy and APC/Cfzr-specific substrates was studied in real time. APC/Cfzy-specific degradation starts upon sister chromatid separation, and APC/Cfzr-specific degradation starts in early G1 (Zur, 2002).
These results show that d-box type and location determine APC/Cfzy and APC/Cfzr specificity, and that fzy and fzr sequentially activate the APC/C. This specificity could thus form the basis of the ordered degradation of APC/C substrates during the different stages of mitosis and G1 (Zur, 2002).
Chromosome separation during the cell-cycle transition from metaphase to anaphase requires the
proteolytic destruction of anaphase inhibitors such as Pds1. pds1 mutants exhibit
precocious dissociation of sister chromatids in the presence of these microtubule inhibitors. pds1 mutants are defective in anaphase arrest that normally is imposed by a
spindle-damage checkpoint. Proteolysis of Pds1 is mediated by a
ubiquitin-protein ligase, the anaphase-promoting complex (APC) or cyclosome. The APC is also
necessary for the ubiquitin-dependent degradation of mitotic cyclins in late telophase as cells exit
mitosis. Although phosphorylation seems to be involved, it is not clear what activates the
APC at the onset of anaphase. In Saccharomyces cerevisiae, chromosome segregation also requires
the CDC20 gene, whose product contains WD40 repeats. The functional
relationship between the APC and the Cdc20 protein has been investigated. Evidence is presented that strongly suggests that
Cdc20 is an essential regulator of APC-dependent proteolysis such that in the absence of Cdc20, cells
are unable to degrade either Pds1 at the onset of anaphase or the mitotic cyclin Clb2 during telophase.
This notion is consistent with observations that Cdc20 is localized in the nucleus and
co-immunoprecipitates with an APC component, Cdc23 (Lim, 1998).
Elaborate regulatory mechanisms might control the precise progression
of mitosis. Ubiquitin-dependent proteolysis of Cut2/Pds1, which
triggers sister chromatid separation, and Cyclin B are required for sister chromatid separation and exit from mitosis, respectively.
Anaphase-promoting complex/cyclosome (APC) specifically ubiquitinates Cut2/Pds1 at metaphase-anaphase transition, and
ubiquitinates Cyclin B in late mitosis and G1 phase.
Recent genetic and biochemical analyses in yeast, Drosophila, and Xenopus have indicated that APC is activated by the WD-repeat proteins,
Cdc20/p55CDC/Fizzy (Cdc20) and Cdh1/Hct1/Fizzy-related (Cdh1: Drosophila homolog Fizzy related) in a substrate-specific manner,
whereas APC is inactivated by a spindle assembly checkpoint through Mad2. Whereas Mad1, Mad2, Mad3/Bub1, and Bub3 suppress
Cdc20-dependent APC activation, Bub2 localized in the spindle pole body regulates Cdh1-dependent APC activation. Furthermore, Cdc14, whose activity is regulated by Bub2/Byr4 and RENT complex, dephosphorylates Cdh1/Hct1 and inactivates APC, Therefore, APC
activity is regulated by at least four distinct mechanisms: activation and inactivation by phosphorylation and dephosphorylation of APC itself; activation by the binding
of substrate-specific activators Cdc20 and Cdh1 to APC; suppression of APC activity by the spindle assembly checkpoint through Mad family and Bub family, and
regulation of APC activity by Bub2/RENT complex system. However, the precise regulatory mechanism of substrate-specific activation of mammalian APC with the
right timing through these complicated mechanisms remains to be elucidated.
The binding of the activators Cdc20 and Cdh1 and
the inhibitor Mad2 to APC regulate APC activity. Also the phosphorylation of Cdc20 and Cdh1 by Cdc2-Cyclin B (MPF) and phosphorylation of APC by Polo-like kinase
and cAMP-dependent protein kinase regulate APC activity. The cooperation of the phosphorylation/dephosphorylation and the
regulatory factors in regulation of APC activity may thus control the precise progression of mitosis (Kotani, 1999).
Thus, phosphorylation of Cdc20 is required for Cdc20-dependent APC activation at least
in vitro. It has been reported that Cdc20 is expressed during G2 phase and mitosis and binds to APC in early mitotic stages. Cdc20 is clearly phosphorylated
during mitosis in HeLa cells, and Cdc20 is phosphorylated by MPF in Xenopus embryos. Actually, the possible
Cdc2 phosphorylation site is conserved in budding yeast Cdc20, fission yeast Slp1, and mammalian Cdc20. Therefore, it is most likely that Cdc20 is indeed
phosphorylated during mitosis in vivo. Taken together, it is concluded that Cdc20 activates APC when it is phosphorylated by MPF. The activation of APC can be blocked by the binding of Mad2 to pCdc20. Therefore, at least two events may be required to activate APC at
metaphase-anaphase transition: release of Mad2 from pCdc20 after spindle assembly checkpoint is released, and phosphorylation of Cdc20 bound to APC by MPF
or binding of pCdc20 to APC. Furthermore, at least in vitro APC activation can be suppressed by PKA,
which phosphorylates two APC subunits, APC1 and APC3. Thus, dephosphorylation of PKA phosphorylation sites on APC by a specific
phosphatase yet unidentified, which may be PP1, might also be required for the onset of anaphase (Kotani, 1999 and references).
Mad2 cannot inhibit Cdh1-induced APC activation, and pAPC (phosphorylated APC) can ubiquitinate Cyclin B even in the presence of Mad2. Thus Mad2
inhibits APC activation only through Cdc20. Further, the binding of Cdc20 or pCdc20 (phosphorylated pCdc20) to APC or pAPC was not affected by Mad2, indicating that
Mad2 inhibits the function of pCdc20 but not the binding of pCdc20 to APC. Whereas Mad1, Mad2, Mad3/Bub1, and Bub3
suppress Cdc20-dependent APC activation, Bub2 localized in the spindle pole body regulates Cdh1-dependent APC activation. Further detailed functional analyses of the Mad family and Bub family are required to clarify the molecular
mechanisms of spindle assembly checkpoint and of APC regulation (Kotani, 1999 and references).
It was clearly shown that pAPC can ubiquitinate Cyclin B but not Cut2 in vitro, but it remains unclear whether free pAPC actually exists during mitosis in vivo. It is
most likely that the majority of pAPC forms a complex with either pCdc20 or Cdh1 that ubiquitinates Cyclin B in vivo. Furthermore, phosphorylation of APC by
pPlk may not be essential for APC activation at the metaphase-anaphase transition, but phosphorylation of APC by
pPlk is required for complete Cyclin B ubiquitination in
later stages of mitosis (Kotani, 1999).
pCdc20 and Cdh1 (but neither Cdc20 nor pCdh1) activates APC in vitro, and the following switching mechanism from pCdc20 to Cdh1 could
be speculated. When the cells enter anaphase after Cut2/Pds1 is entirely degraded, the pAPC-pCdc20 complex steadily ubiquitinates Cyclin B and, consequently,
MPF activity decreases by the end of mitosis. The pCdc20 can be dephosphorylated by a specific phosphatase and released from pAPC or it may be degraded. When MPF activity is high, Cdh1 is phosphorylated and remains inactive, but when Cdc14 is
activated, Cdh1 is dephosphorylated by Cdc14 and binds to and activates APC.
Consequently, the pAPC-pCdc20 complex is replaced by the pAPC-Cdh1 (or APCp-Cdh1) complex in late mitosis. The pAPC-Cdh1 or APC-Cdh1 complex
further ubiquitinates Cyclin B in late mitosis and G1 phase. Thus, the switch mechanism from pCdc20 to Cdh1 may be dependent upon the MPF activity during
mitosis, and the MPF activity itself is controlled by the pCdc20- and Cdh1-dependent APC activity (Kotani, 1999 and references).
Cdc20 has been reported to activate ubiquitination of the factors regulating sister chromatid separation and Cdh1 promotes ubiquitination of mitotic cyclins. However, in Xenopus early embryos, Cdh1 is not expressed,
which is consistent with the observation that Cdh1 is not expressed before stage 13 in Drosophila embryos. It was also described that
Cdc20 regulates ubiquitination of both Cut2/Pds1 and Cyclin B in the early embryonic cell cycle. Ubiquitin-dependent proteolysis of Cyclin B begins at metaphase-anaphase transition in HeLa cells, which is consistent with the results obtained with
clam embryo. These findings are consistent with the observation that pCdc20 can activate ubiquitination not only of Cut2/Pds1, but also of
Cyclin B. Thus, pCdc20 alone may be enough for cells to go through mitosis without Cdh1. However, in the somatic cell cycle, Cdh1 in addition to pCdc20, may be
required for effective and complete ubiquitination of Cyclin B in later stages of mitosis and G1 phase (Kotani, 1999 and references).
This study has demonstrated that Cdh1 can be phosphorylated by MPF, and Cdh1 but not pCdh1 binds to and activates APC only after
dephosphorylation. These in vitro results are consistent with in vivo data in budding yeast that the dephosphorylated form of
Cdh1/Hct1 activates APC. Cdh1 is constantly expressed throughout the cell cycle, binds to APC in late mitosis and G1 phase, and is phosphorylated during mitosis in HeLa cells. Cdh1/Hct1 binding to APC is regulated by cyclin-dependent kinases.
Cdc14 dephosphorylates Cdh1/Hct1 and inactivates APC. Further, the activation of Cdc14 is regulated by Bub2/Byr4 and
RENT complex, although it remains unresolved whether the regulation by RENT complex
works in the mammalian system (Kotani, 1999 and references).
It is possible that Cdh1-APC complex activity is maintained until late G1 phase, while it might be diminished by the phosphorylation of Cdh1 with
Cdk2, Cdk4, or another unidentified specific kinase that is active at G1/S transition. It has been reported that the low level of Cyclin B is
translated even in G1 phase. Therefore, in the G1 phase, APC-Cdh1 complex may effectively ubiquitinate this newly translated Cyclin B to
avoid activation of MPF. The possible involvement of G1/S Cdks or other specific kinases in Cdh1-dependent APC inactivation merits further study (Kotani, 1999).
The current findings support the notion that pCdc20 but not Cdc20 activates APC, and that Cdh1 but not pCdh1 binds to and activates APC.
Therefore, phosphorylation and dephosphorylation of APC regulatory factors by MPF are critical for their binding to APC and/or APC activation. The MPF activity
itself is regulated by the pCdc20- and Cdh1-dependent APC activity, and this feedback control precisely regulates APC activity. Taken together, the APC activity is
regulated by the phosphorylation and dephosphorylation of APC and of the regulatory factors, Cdc20 and Cdh1, by MPF, Plk, PKA, and PP1, as well as by the
binding of positive and negative regulatory factors, Cdc20, Cdh1, and Mad2, to APC. These elaborate regulatory mechanisms might control the precise progression
of mitosis (Kotani, 1999).
In the presence of unattached/weakly attached kinetochores, the spindle assembly checkpoint (SAC) delays exit from mitosis by preventing the anaphase-promoting complex (APC)-mediated proteolysis of cyclin B, a regulatory subunit of cyclin-dependent kinase 1 (Cdk1). Like all checkpoints, the SAC does not arrest cells permanently, and escape from mitosis in the presence of an unsatisfied SAC requires that cyclin B/Cdk1 activity be inhibited. In yeast and likely Drosophila this occurs through an 'adaptation' process involving an inhibitory phosphorylation on Cdk1 and/or activation of a cyclin-dependent kinase inhibitor (Cdki). The mechanism that allows vertebrate cells to escape mitosis when the SAC cannot be satisfied is unknown. To explore this issue, fluorescence microscopy studies were conducted on rat kangaroo (PtK) and human (RPE1) cells dividing in the presence of nocodazole. In the absence of microtubules (MTs), escape from mitosis occurs in the presence of an active SAC and requires cyclin B destruction. cyclin B is progressively destroyed during the block by a proteasome-dependent mechanism. Thus, vertebrate cells do not adapt to the SAC. Rather, the data suggest that in normal cells, the SAC cannot prevent a slow but continuous degradation of cyclin B that ultimately drives the cell out of mitosis (Brito, 2006).
Cyclin-dependent kinase 1 (Cdk1) initiates mitosis and later activates the anaphase-promoting complex/cyclosome (APC/C) to destroy cyclins. Kinetochore-derived checkpoint signaling delays APC/C-dependent cyclin B destruction, and checkpoint-independent mechanisms cooperate to limit APC/C activity when kinetochores lack checkpoint components in early mitosis. The APC/C and cyclin B localize to the spindle and poles, but the significance and regulation of these populations remain unclear. This study describes a critical spindle pole-associated mechanism, called the END (Emi1/NuMA/dynein-dynactin) network, that spatially restricts APC/C activity in early mitosis. The APC/C inhibitor Emi1 binds the spindle-organizing NuMA/dynein-dynactin complex to anchor and inhibit the APC/C at spindle poles, and thereby limits destruction of spindle-associated cyclin B. Cyclin B/Cdk1 activity recruits the END network and establishes a positive feedback loop to stabilize spindle-associated cyclin B critical for spindle assembly. The organization of the APC/C on the spindle also provides a framework for understanding microtubule-dependent organization of protein destruction (Ban, 2007).
Progression through mitosis depends on the periodic accumulation and destruction of cyclins. Cyclin B accumulates and activates the cyclin-dependent kinase 1 (Cdk1) in mitosis to form mitosis-promoting factor (MPF). MPF drives chromosome reorganization and formation of the mitotic spindle. Later in mitosis, MPF downregulates its own activity by initiating the ubiquitination and destruction of cyclins by the anaphase-promoting complex/cyclosome (APC/C), an E3 ubiquitin ligase. The delay between activation of the APC/C by MPF at the beginning of mitosis and the destruction of cyclin B at mitotic exit is critical to allow sufficient time for the spindle to form and direct chromosome congression. Inhibition of the APC/C during this period is linked to kinetochore-dependent activation of spindle assembly checkpoint components, notably the APC/C inhibitors Mad2 and BubR1. However, the potential role for other APC/C inhibitors, including Emi1, in contributing to APC/C regulation in mitosis is poorly understood (Ban, 2007).
In S and G2 phases of vertebrate cells, Emi1 prevents the premature destruction of cyclins A and B, thereby allowing cells to progress into mitosis. As cells commit to mitosis at nuclear envelope breakdown (NEBD), the bulk of Emi1 is destroyed by the SCFβTRCP E3 ligase, thus permitting selective activation of the APC/C to degrade substrates in prometaphase, such as cyclin A. The APC/C is restrained from destroying cyclin B, in part by establishment of the spindle checkpoint. In early mitosis, however, checkpoint-independent mechanisms cooperate to restrain APC/C activity when kinetochore signaling is not yet fully established. Indeed, a population of Emi1 localizes to the spindle poles in early mitosis, suggesting that this inhibitor could function to regulate the APC/C during this critical period when spindles are forming (Ban, 2007).
MPF activation drives spindle assembly by initiating NEBD, which in turn permits Ran-GTP-dependent release of microtubule-organizing activities to nucleate microtubule asters. MPF activity also promotes microtubule organization by phosphorylating microtubule-associated proteins and motor proteins. Spindle microtubules are integrated and focused at spindle poles by several factors, including the minus end-directed dynein-dynactin motor complex and the spindle protein NuMA (nuclear mitotic apparatus), as a critical step in the formation of the bipolar spindle. Because MPF regulates both spindle assembly and APC/C activation, a mechanism coupling the processes would ensure proper timing of cyclin accumulation and destruction (Ban, 2007).
The mitotic spindle itself may organize and regulate APC/C and MPF activity, thus providing a link between the processes. Both cyclin B and the APC/C are localized to the spindle and poles in mitosis. Moreover, cyclin B appears to be destroyed at the poles and spindles at the metaphase-to-anaphase transition. These studies have suggested that the organization of MPF and the APC/C on the spindle potentially contribute to maintaining the spindle structure and facilitating cyclin destruction during mitotic exit. How these components are organized and regulated on the spindle is unclear (Ban, 2007).
This study, carried out in mammalian cells, describes an essential regulatory network that physically and functionally links the NuMA and dynein-dynactin spindle-organizing components to the APC/C and its associated inhibitor Emi1. A network of Emi1, NuMA, and dynein-dynactin (END) spatially regulates the APC/C on the mitotic spindle to prevent premature cyclin B destruction on the spindle. Stabilization of cyclin B/Cdk1 activity promotes NuMA-dependent assembly of microtubules at spindle poles and reinforces the recruitment of the END network to poles. It is proposed that the END network establishes a positive feedback loop in early mitosis that sustains localized cyclin B/Cdk1 activity on the spindle critical for maintaining spindle integrity (Ban, 2007).
BubR1 is an essential mitotic checkpoint protein with multiple functional domains. It has been implicated in mitotic checkpoint control, as an active kinase at unattached kinetochores, and as a cytosolic inhibitor of APC/C(Cdc20) activity, as well as in mitotic timing and stable chromosome-spindle attachment. Using BubR1-conditional knockout cells and BubR1 domain mutants, it was demonstrated that the N-terminal Cdc20 binding domain of BubR1 is essential for all of these functions, whereas its C-terminal Cdc20-binding domain, Bub3-binding domain, and kinase domain are not. The BubR1 N terminus binds to Cdc20 in a KEN box-dependent manner to inhibit APC/C activity in interphase, thereby allowing accumulation of cyclin B in G(2) phase prior to mitosis onset. Together, these results suggest that kinetochore-bound BubR1 is nonessential and that soluble BubR1 functions as a pseudosubstrate inhibitor of APC/C(Cdc20) during interphase to prevent unscheduled degradation of specific APC/C substrates (Malureanu, 2009).
A cyclin B variant derived from alternative splicing is produced in sea urchin oocytes and embryos. This splice variant protein lacks highly conserved sequences in the COOH terminus of the protein. It has been found strikingly abundant in growing oocytes and cells committed to differentiation during embryogenesis. Cyclin B splice variant (CBsv) protein associates weakly in the cell with Xenopus cdc2 and with budding yeast CDC28p. In contrast to classical cyclin B, CBsv very poorly complements a triple CLN deletion in budding yeast; its microinjection prevents an initial step in MPF activation, leading to an important delay in oocyte meiosis reinitiation. CBsv microinjection in fertilized eggs induces cell cycle delay and abnormal development. It is assumed that CBsv is produced in growing oocytes to keep them in prophase, and during embryogenesis, to slow down cell cycle in cells that will be committed to differentiation (Lozano, 1998).
Among the proteins whose synthesis and/or degradation is necessary for a proper progression through meiotic maturation, cyclin B appears to be one of the most important. The level of cyclin B1 and B2 synthesis were modulated during meiotic maturation of the mouse oocyte. Cyclin B1 or B2 mRNAs with poly(A) tails of different sizes and cyclin B1 or B2 antisense RNAs were used. Oocytes microinjected with cyclin B1 mRNA show two phenotypes: most are blocked in MI, while the others extrude the first polar body in advance when compared to controls. Moreover, these effects are correlated with the length of the poly(A) tail. Thus it seems that the rate of cyclin B1 translation controls the timing of the first meiotic M phase and the transition to anaphase I. Moreover, overexpression of cyclin B1 or B2 is able to bypass the dibutyryl cAMP-induced germinal vesicle block, but only the cyclin B1 mRNA-microinjected oocytes do not extrude their first polar body. Oocytes injected with the cyclin B1 antisense progress through the first meiotic M phase but extrudes the first polar body in advance and are unable to enter metaphase II. This suggests that inhibition of cyclin B1 synthesis only takes place at the end of the first meiotic M phase, most likely because the cyclin B1 mRNA was protected. The injection of cyclin B2 antisense RNA has no effect. The live observation of the synthesis and degradation of a cyclin B1-GFP chimera during meiotic maturation of the mouse oocyte demonstrates that degradation can only occur during a given period of time once it has started. Taken together, these data demonstrate that the rates of cyclin B synthesis and degradation determine the timing of the major events taking place during meiotic maturation of the mouse oocyte (Ledan, 2001).
Cyclin B1, the regulatory component of M phase-promoting factor (MPF), is degraded during the metaphase-anaphase transition in an anaphase-promoting complex/cyclosome (APC/C)-dependent process. MPF activity is stable in eggs, and a sperm-triggered Ca2+ signal is needed to promote cyclin degradation. In frogs, a single Ca2+ spike promotes cell cycle resumption, but, in mammals, the Ca2+ signal is more complex, consisting of a series of spikes that stop several hours after sperm fusion. Using dual imaging in mouse eggs, an examination was carried out to see how the Ca2+ signal generates cyclin B1 destruction using destructible and nondestructible GFP-tagged constructs. APC/C activity is present in unfertilized eggs, giving cyclin B1 a half-life of 1.15 ± 0.28 hr. However, APC/C-dependent cyclin degradation is elevated 6-fold when sperm raises cytosolic Ca2+ levels above 600 nM. This activation is transitory since cyclin B1 levels recover between Ca2+ spikes. For continued cyclin degradation at basal Ca2+ levels, multiple spikes are needed. APC/C-mediated degradation is observed until eggs have completed meiosis with the formation of pronuclei, and, at this time, Ca2+ spikes stop. Therefore, the physiological need for a repetitive Ca2+ signal in mammals is to ensure long-term cyclin destruction during a protracted exit from meiosis (Nixon, 2002).
Sea urchins are members of a limited group of animals in which meiotic maturation of oocytes is completed prior to fertilization. This is different from oocytes of most animals such as mammals and amphibians in which fertilization reactivates an arrested meiotic cycle. Using a recently developed technique for in vitro maturation of sea urchin oocytes, the role was analyzed of cyclin B, the regulatory component of maturation-promoting factor, in the control of sea urchin oocyte meiotic induction and progression. Oocytes of the sea urchin Lytechinus variegatus accumulate significant amounts of cyclin B mRNA and protein during oogenesis. Cyclin B synthetic requirements in oocytes and early embryos was analyzed by inhibiting cyclin B synthesis with DNA and morpholino antisense oligonucleotides. Cyclin B synthesis is not necessary for the entry of G2-arrested oocytes into meiosis; however, it is required for the proper progression through meiotic divisions. Surprisingly, mature sea urchin eggs contain significant cyclin B protein following meiosis that serves as a maternal store for early cleavage divisions. Cyclin A can functionally substitute for cyclin B in early embryos but not in oocytes. These studies provide a foundation for understanding the mechanism of meiotic maturation independent of the zygotic cell cycle (Voronina, 2003).
Vertebrate oocytes proceed through meiosis I before undergoing a cytostatic factor (CSF)-mediated arrest at metaphase of meiosis II. Exit from MII arrest is stimulated by a sperm-induced increase in intracellular Ca2+. This increase in Ca2+ results in the destruction of cyclin B1, the regulatory subunit of cdk1 that leads to inactivation of maturation promoting factor (MPF) and egg activation. Progression through meiosis I also involves cyclin B1 destruction, but it is not known whether Ca2+ can activate the destruction machinery during MI. Ca2+-induced cyclin destruction was investigated in MI and MII by using a cyclin B1-GFP fusion protein and measurement of intracellular Ca2+. No evidence was found for a role for Ca2+ in MI since oocytes progress through MI in the absence of detectable Ca2+ transients. Furthermore, Ca2+ increases induced by photorelease of InsP3 stimulate a persistent destruction of cyclin B1-GFP in MII but not MI stage oocytes. In addition to a steady decrease in cyclin B1-GFP fluorescence, the increase in Ca2+ stimulated a transient decrease in fluorescence in both MI and MII stage oocytes. Similar transient decreases in fluorescence imposed on a more persistent fluorescence decrease were detected in cyclin-GFP-injected eggs undergoing fertilization-induced Ca2+ oscillations. The transient decreases in fluorescence were not a result of cyclin B1 destruction since transients persisted in the presence of a proteasome inhibitor and were detected in controls injected with eGFP and in untreated oocytes. It is concluded that increases in cytosolic Ca2+ induce transient changes in autofluorescence. Also, the pattern of cyclin B1 degradation at fertilization is not stepwise but exponential. Furthermore, this Ca2+-induced increase in degradation of cyclin B1 requires factors specific to mature oocytes, and that to overcome arrest at MII, Ca2+ acts to release the CSF-mediated brake on cyclin B1 destruction (Marangos, 2004).
In mitosis, the spindle checkpoint protein Mad2 averts aneuploidy by delaying anaphase onset until chromosomes align. Depletion of Mad2 in meiosis I mouse oocytes induces an increased incidence of aneuploidy. Proteolysis of cyclin B and securin commences earlier in Mad2-depleted oocytes, resulting in a shortened duration of meiosis I. Furthermore, overexpression of Mad2 inhibits homolog disjunction. It is concluded that Mad2 delays the onset of cyclin B and securin degradation and averts aneuploidy during meiosis I in mammalian oocytes. The data suggest a link between trisomies such as Down syndrome and defective oocyte spindle checkpoint function (Homer, 2005).
Vertebrate oocyte maturation is an extreme form of asymmetric cell division, producing a mature egg alongside a diminutive polar body. Critical to this process is the attachment of one spindle pole to the oocyte cortex prior to anaphase. This study reports that asymmetric spindle pole attachment and anaphase initiation are required for localized cortical activation of Cdc42, which in turn defines the surface of the impending polar body. The Cdc42 activity zone overlaps with dynamic F-actin and is circumscribed by a RhoA-based actomyosin contractile ring. During cytokinesis, constriction of the RhoA contractile ring is accompanied by Cdc42-mediated membrane outpocketing such that one spindle pole and one set of chromosomes are pulled into the Cdc42 enclosure. Unexpectedly, the guanine nucleotide exchange factor Ect2, which is necessary for contractile ring formation, does not colocalize with active RhoA. Polar body emission thus requires a classical RhoA contractile ring and Cdc42-mediated membrane protrusion (Zhang, 2008).
Cdc42 activation is first observed at the spindle-cortex contact site shortly (within a few minutes) following anaphase initiation, suggesting that activation of Cdc42 might be temporarily and mechanistically coupled to anaphase. Attempts were made to inhibit anaphase without altering spindle assembly or the perpendicular spindle attachment to the oocyte cortex. The role of cyclin B degradation and the resultant loss of Cdk1 activity during amphibian meiosis is controversial. While it has been reported that stabilization of Cdk1 activity via prevention of cyclin B destruction does not prevent polar body formation, it has also been reported that experimental stabilization of Cdk1 by other means does block polar body emission and that this blockade can be relieved by Cdk1 inhibition. The role of cyclin B degradation on polar body emission was reinvestigated. A truncated form of cyclin B1 (ΔN cyclin B1 was employed that lacks the destruction box required for APC-targeted degradation. Injection of ΔN cyclin B1 mRNA efficiently eliminated the transient inactivation of MPF seen in control oocytes. Oocytes injected with ΔN cyclin B1 underwent progesterone-induced GVBD indistinguishably from control oocytes. However, oocytes injected with ΔN cyclin B1 mRNA failed to emit the first polar body. Similarly, injection of methylubiquitin effectively inhibited cyclin B degradation and inhibited first polar body emission (Zhang, 2008).
Time-lapse imaging (or 4D imaging) experiments were carried to determine the effect of ΔN cyclin B1 on Cdc42 activation in live oocytes, using eGFP-wGBD, a GFP fusion protein containing the GTPase-binding domain of WASP (wGBD) that binds only active (GTP-bound) Cdc42. Control oocytes exhibited Cdc42 activation approximately 2 hr after GVBD, emitted the first polar body, and then arrested in metaphase II indefinitely. In contrast, ΔN cyclin B1-injected oocytes failed to activate Cdc42, and the spindle remained intact and attached to the cortex for an extended period of time. Similarly, methylubiquitin-injected oocytes exhibited intact metaphase I spindles that remained asymmetrically attached to the cortex, with no Cdc42 activation (Zhang, 2008).
Given the controversy regarding whether the transient degradation of cyclin B is required for homolog separation in Xenopus oocytes, chromosome dynamics were observed in live oocytes injected with ΔN cyclin B1 mRNA. Fluorescent antibodies against Xenopus Aurora B were used to track endogenous Aurora B, a chromosome passenger kinase. Fluorescent anti-Aur B faithfully tracked chromosomes through the metaphase I (00:10) to metaphase II (00:52) transition. In addition, fluorescent anti-Aur B also clearly marked the central spindle or spindle midzone (MZ) at anaphase/telophase (arrows, 00:20–00:24), and the midbody following polar body emission. These data are consistent with previous conclusions based on staining of fixed oocytes of other species. It is noteworthy that in contrast to mitosis in which Aurora B is completely transferred to the central spindle at anaphase, in the oocytes Aurora B persisted with the segregated chromosome homologs. Presumably, Aurora B signal persisted at anaphase I due to the cohesion of sister centromeres, which only resolve at anaphase II upon fertilization (Zhang, 2008).
Having established the utility of fluorescent anti-Aur B in tracking chromosomes in live oocytes, the fate of chromosomes was followed in oocytes injected with ΔN cyclin B1. Metaphase I spindles remained asymmetrically positioned against the animal pole cortex for an extended period of time, with no homolog separation. Similarly, oocytes injected with methylubiquitin did not separate chromosome homologs. These data clearly indicate that cyclin B degradation is required for anaphase initiation and for Cdc42 activation (Zhang, 2008).
Cellular differentiation is a complex process involving growth arrest, exit from the cell cycle, and expression of differentiated cell-type-specific functions. To identify small molecules promoting this process, a chemical library was screened by using a myeloid leukemic cell line that retains the potential to differentiate in culture. In the presence of a purine derivative, aminopurvalanol (AP), cells acquire phenotypic characteristics of differentiated macrophages and became arrested in the cell cycle with a 4N DNA content. AP also inhibits mitosis in Xenopus egg extracts, suggesting that it acts on an evolutionarily conserved cell cycle regulatory pathway. Affinity chromatography and biochemical reconstitution experiments with Xenopus egg extracts have identified cyclin-dependent kinase (CDK) 1-cyclin B as a target of the compound. Although AP potently inhibits immunoprecipitates of both human CDK1 and CDK2 from human leukemic cell extracts, these results indicate that the compound preferentially targets the G2/M-phase transition in vivo (Rosania, 1999).
A substantial amount of mitochondrial energy is required for cell-cycle progression. The mechanisms underlying the coordination of the mitochondrial respiration with cell-cycle progression, especially the G2/M transition, remain to be elucidated. This study shows that a fraction of cyclin B1/Cdk1 proteins localizes to the matrix of mitochondria and phosphorylates a cluster of mitochondrial proteins, including the complex I (CI) subunits in the respiratory chain. Cyclin B1/Cdk1-mediated CI phosphorylation enhances CI activity, whereas deficiency of such phosphorylation in each of the relevant CI subunits results in impairment of CI function. Mitochondria-targeted cyclin B1/Cdk1 increases mitochondrial respiration with enhanced oxygen consumption and ATP generation, which provides cells with efficient bioenergy for G2/M transition and shortens overall cell-cycle time. Thus, cyclin B1/Cdk1-mediated phosphorylation of mitochondrial substrates allows cells to sense and respond to increased energy demand for G2/M transition and, subsequently, to upregulate mitochondrial respiration for successful cell-cycle progression (Wang, 2014).
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