The RRASK motif in Xenopus cyclin B2 is required for the substrate recognition of Cdc25C by the cyclin B-Cdc2 complex

The FLRRXSK sequence is conserved in the second cyclin box fold of B-type cyclins. We show that this conserved sequence in Xenopus cyclin B2, termed the RRASK motif, is required for the substrate recognition by the cyclin B-Cdc2 complex of Cdc25C. Mutations to charged residues of the RRASK motif of cyclin B2 abolished its ability to activate Cdc2 kinase without affecting its capacity to bind to Cdc2. Cdc2 bound to the cyclin B2 RRASK mutant was not dephosphorylated by Cdc25C, and as a result, the complex was inactive. The cyclin B2 RRASK mutants can form a complex with the constitutively active Cdc2, but a resulting active complex did not phosphorylate a preferred substrate Cdc25C in vitro, although it can phosphorylate the non-specific substrate histone H1. The RRASK mutations prevented the interaction of Cdc25C with the cyclin B2-Cdc2 complex. Consistently, the RRASK mutants neither induced germinal vesicle breakdown in Xenopus oocyte maturation nor activated in vivo Cdc2 kinase during the cell cycle in mitotic extracts. These results suggest that the RRASK motif in Xenopus cyclin B2 plays an important role in defining the substrate specificity of the cyclin B-Cdc2 complex.     

The N-terminal helix of Xenopus cyclins A and B contributes to binding specificity of the cyclin-CDK complex

Mitotic cyclins A and B contain a conserved N-terminal helix upstream of the cyclin box fold that contributes to a significant interface between cyclin and cyclin-dependent kinase (CDK). To address its contribution on cyclin-CDK interaction, we have constructed mutants in conserved residues of the N-terminal helix of Xenopus cyclins B2 and A1. The mutants showed altered binding affinities to Cdc2 and/or Cdk2. We also screened for mutations in the C-terminal lobe of CDK that exhibited different binding affinities for the cyclin-CDK complex. These mutations were at residues that interact with the cyclin N-terminal helix motif. The cyclin N-terminal helix mutations have a significant effect on the interaction between the cyclin-CDK complex and specific substrates, Xenopus Cdc6 and Cdc25C. These results suggest that the N-terminal helix of mitotic cyclins is required for specific interactions with CDKs and that to interact with CDK, specific substrates Cdc6 and Cdc25C require the CDK to be associated with a cyclin. The interaction between the cyclin N-terminal helix and the CDK C-terminal lobe may contribute to binding specificity of the cyclin-CDK complex.     

A Cdc2-sensitive interaction of the UbL domain of XDRP1S with cyclin B mediates the degradation of cyclin B in Xenopus egg extracts

The yeast UbL-UBA protein Dsk2 is thought to act as a shuttle protein that delivers polyubiquitinated proteins to the proteasome. Previously, we identified Xenopus Dsk2-related protein, XDRP1, as a cyclin A-interacting protein. Using Xenopus egg extracts, we further characterized its two isoforms, XDRP1L and XDRP1S, with respect to cyclin binding and its degradation. Polyubiquitinated cyclins bound to the UBA domain of XDRP1L and XDRP1S, whereas monomeric cyclins A and B bound to the UbL domain of XDRP1S but not to XDRP1L. Binding of XDRP1S with monomeric cyclins was affected by a Cdc2-mediated phosphorylation of either the XDRP1S UbL domain or cyclins. Degradation of cyclin B was also prevented by XDRP1S in a Cdc2-sensitive manner. Loss of the XDRP1S-cyclin interaction allowed cyclins to be degraded in calcium-treated CSF extracts. These results suggest that the shuttling pathway via the UbL-UBA protein XDRP1 participates in degradation of mitotic cyclins in Xenopus eggs.     

Pre-MPF is absent in immature oocytes of fishes and amphibians except Xenopus

Maturation-promoting factor (MPF), a final trigger for initiating oocyte maturation, is activated in the oocyte cytoplasm, in response to maturation-inducing hormone (MIH) secreted from follicle cells surrounding the oocyte. MPF consists of cdc2 and cyclin B. We investigated the state of cdc2 and cyclin B in immature and mature oocytes of fishes (carp, catfish and lamprey) and amphibians ( Xenopus, frog [ Rana ] and toad [ Bufo ]) using monoclonal antibodies raised against mouse cdc2, which also recognize fish and amphibian cdc2, and monoclonal antibodies against goldfish cyclin B1 and polyclonal antibodies against Xenopus cyclins B1 and B2. Anti-cdc2 and anti-cyclin B immunoblotting of oocyte extracts fractionated by gel filtration chromatography showed that immature oocytes from all of these species with the exception of Xenopus contained only monomeric cdc2. Cyclin B-bound inactive cdc2 (pre-MPF) was present only in immature Xenopus oocytes. Cdc2-cyclin B complex was, however, found in mature oocytes from all the species examined. After the oocyte is induced to mature by MIH, cdc2 should therefore bind to cyclin B in all of these species, except Xenopus. These results suggest that the complex formation of cdc2 and cyclin B in response to MIH stimulation at the oocyte surface is a critical step for initiating oocyte maturation in fishes and amphibians, with the exception of Xenopus , in which pre-MPF already exists in immature oocytes and only its chemical modification is required for MPF activation.     

Activation of Cdc2 kinase during meiotic maturation of axolotl oocyte

Activity of Cdc2, the universal inducer of mitosis, is regulated by phosphorylation and binding to cyclin B. Comparative studies using oocytes from several amphibian species have shown that different mechanisms allow Cdc2 activation and entry into first meiotic division. In Xenopus, immature oocytes stockpile pre-M-phase promoting factor (MPF) composed of Cdc2-cyclin B complexes maintained inactive by Thr14 and Tyr15 phosphorylation of Cdc2. Activation of MPF relies on the conversion of pre-MPF into MPF by Cdc2 dephosphorylation, implying a positive feedback loop known as MPF auto-amplification. On the contrary, it has been proposed that pre-MPF is absent in immature oocyte and that MPF activation depends on cyclin synthesis in some fishes and other amphibians. We demonstrate here that MPF activation in the axolotl oocyte, an urodele amphibian, is achieved through mechanisms resembling partly those found in Xenopus oocyte. Pre-MPF is present in axolotl immature oocyte and is activated during meiotic maturation. However, monomeric Cdc2 is expressed in large excess over pre-MPF, and pre-MPF activation by Cdc2 dephosphorylation takes place progressively and not abruptly as in Xenopus oocyte. The intracellular compartmentalization as well as the low level of pre-MPF in axolotl oocyte could account for the differences in oocyte MPF activation in both species.     

Phosphorylation of Xenopus cyclins B1 and B2 is not required for cell cycle transitions.

The cdc2 kinase and B-type cyclins are known to be components of maturation- or M-phase-promoting factor (MPF). Phosphorylation of cyclin B has been reported previously and may regulate entry into and exit from mitosis and meiosis. To investigate the role of cyclin B phosphorylation, we replaced putative cdc2 kinase phosphorylation sites in Xenopus cyclins B1 and B2 by using oligonucleotide site-directed mutagenesis. We found that Ser-90 of cyclin B2 and Ser-94 or Ser-96 of cyclin B1 are the main phosphorylation sites both in functional Xenopus egg extracts and after phosphorylation with purified MPF in vitro. Microtubule-associated protein (MAP) kinase from Xenopus eggs phosphorylated cyclin B1 significantly at Ser-94 or Ser-96, whereas it was largely inactive against cyclin B2. The substitutions that ablated phosphorylation at these sites, however, resulted in no functional differences between mutant and wild-type cyclin, as judged by the kinetics of M-phase degradation, induction of mitosis in egg extracts, or induction of oocyte maturation. These results indicate that the phosphorylation of Xenopus B-type cyclins by cdc2 kinase or MAP kinase is not required for the hallmark functions of cyclin.     

Either cyclin B1 or B2 is necessary and sufficient for inducing germinal vesicle breakdown during frog (Rana japonica) oocyte maturation

Oocyte maturation is finally triggered by the maturation-promoting factor (MPF), which consists of Cdc2 and cyclin B. We have cloned cDNAs encoding frog (Rana japonica) cyclins B1 and B2 and produced antibodies against their products. Using the antibodies, we investigated changes in protein states and levels of Cdc2 and cyclins B1 and B2 during oocyte maturation. In immature oocytes, all Cdc2 was a monomeric unphosphorylated inactive 35 kDa form and neither cyclin B1 nor cyclin B2 was present. Mature oocytes contained the MPF complex consisting of an active 34 kDa Cdc2 phosphorylated on threonine161 and a 49 kDa cyclin B1 or a 51 kDa cyclin B2. After progesterone stimulation, both cyclins B1 and B2 were synthesized from their stored mRNAs and bound to the preexisting 35 kDa Cdc2. The binding of Cdc2 with cyclin B and its activation probably through the phosphorylation on threonine161 occurred at almost the same time, in accordance with an electrophoretic mobility shift of Cdc2 from 35 to 34 kDa. Microinjection into immature oocytes of cyclin B1 or B2 mRNA alone, or a mixture of them, induced germinal vesicle breakdown (GVBD) with similar dose-dependence. When the translation of endogenous mRNAs of both cyclins B1 and B2 was inhibited with antisense RNAs, progesterone failed to induce GVBD in the oocytes, but the inhibition of only one of the two was unable to inhibit the progesterone-induced GVBD. These results indicate that either cyclin B1 or B2 is necessary and sufficient for inducing GVBD during Rana oocyte maturation. Mol. Reprod. Dev. 50:499–509, 1998. © 1998 Wiley-Liss, Inc.     

Comparative study of the molecular mechanisms of oocyte maturation in amphibians

Maturation-promoting factor (MPF), a complex of Cdc2 and cyclin B, is the final inducer of oocyte maturation. Its activity is controlled by inhibitory phosphorylation of Cdc2 on Tyr15/Thr14 and activating phosphorylation on Thr161. Full-grown immature oocytes of the African clawed frog Xenopus laevis contain inactive MPF (pre-MPF) that comprises cyclin B-bound Cdc2 phosphorylated on Tyr15/Thr14 and Thr161. The synthesis of Mos, but not cyclin B, after stimulation by the maturation-inducing steroid progesterone, is believed to be necessary for initiating Xenopus oocyte maturation through Tyr15/Thr14 dephosphorylation of pre-MPF. In contrast, amphibians other than Xenopus (and also fishes) employ a different mechanism. Full-grown immature oocytes of these species contain monomeric Cdc2 but not cyclin B. MPF is formed after hormonal stimulation by binding of the newly produced cyclin B to the pre-existing Cdc2 and is immediately activated through Thr161 phosphorylation. Mos/MAP kinase is neither necessary nor sufficient for initiating maturation in fishes and amphibians except for Xenopus. We propose a new model of MPF formation and activation during oocyte maturation that is applicable to all amphibians (as well as fishes), based on a novel concept that pre-MPF is an artificial molecule that is not essential for inducing oocyte maturation.     

Requirement for phosphorylation of cyclin B1 for Xenopus oocyte maturation.

Maturation-promoting factor, consisting of cdc2 protein kinase and a regulatory B-type cyclin, is a universal regulator of meiosis and mitosis in eukaryotes. In Xenopus, there are two subtypes of B-type cyclins, designated B1 and B2, both of which are phosphorylated. In this study, we have investigated the biological significance of this phosphorylation for Xenopus cyclin B1 during meiotic maturation. We have used a combination of site-directed mutagenesis and phosphopeptide-mapping to identify serine residues 2, 94, 96, 101, and 113 as presumptive phosphorylation sites, and together these sites account for all cyclin B1 phosphorylation in oocytes before germinal vesicle breakdown (GVBD). Single Ser-->Ala mutants as well as multiple site mutants have been constructed and characterized. Phosphorylation of cyclin B1 appears to be required for Xenopus oocyte maturation, based on the significantly diminished ability of the quintuple Ala mutant to induce 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 after GVBD or after egg activation. A quintuple Glu mutant was also constructed, with serine residues 2, 94, 96, 101, and 113 mutated to Glu. In contrast to the quintuple Ala mutant, the quintuple Glu mutant was able to induce oocyte maturation efficiently, and with more rapid kinetics than wild-type cyclin B1. These data confirm that phosphorylation, as mimicked by Ser-->Glu mutations, confers enhanced biological activity to cyclin B1. Possible roles of cyclin B1 phosphorylation are discussed that might account for the increased biological activity of the quintuple Glu mutant.     

Thr-161 phosphorylation of monomeric Cdc2. Regulation by protein phosphatase 2C in Xenopus oocytes

Fully grown Xenopus oocyte is arrested at prophase I of meiosis. Re-entry into meiosis depends on the activation of MPF (M-phase promoting factor or cyclin B.Cdc2 complex), triggered by progesterone. The prophase-arrested oocyte contains a store of Cdc2. Most of the protein is present as a monomer whereas a minor fraction, called pre-MPF, is found to be associated with cyclin B. Activation of Cdc2 depends on two key events: cyclin binding and an activating phosphorylation on Thr-161 residue located in the T-loop. To get new insights into the regulation of Thr-161 phosphorylation of Cdc2, monomeric Cdc2 was isolated from prophase oocytes. Based on its activation upon cyclin addition and detection by an antibody directed specifically against Cdc2 phosphorylated on Thr-161, we show for the first time that the prophase oocyte contains a significant amount of monomeric Cdc2 phosphorylated on Thr-161. PP2C, a Mg2+-dependent phosphatase, negatively controls Thr-161 phosphorylation of Cdc2. The unexpected presence of a population of free Cdc2 already phosphorylated on Thr-161 could contribute to the generation of the Cdc2 kinase activity threshold required to initiate MPF amplification.     

Cell cycle-regulated degradation of Xenopus cyclin B2 requires binding to p34cdc2.

The protein kinase activity of the cell cycle regulator p34cdc2 is inactivated when the mitotic cyclin to which it is bound is degraded. The amino (N)-terminus of mitotic cyclins includes a conserved "destruction box" sequence that is essential for degradation. Although the N-terminus of sea urchin cyclin B confer cell cycle-regulated degradation to a fusion protein, a truncated protein containing only the N-terminus of Xenopus cyclin B2, including the destruction box, is stable under conditions where full length molecules are degraded. In an attempt to identify regions of cyclin B2, other than the destruction box, involved in degradation, the stability of proteins encoded by C-terminal deletion mutants of cyclin B2 was examined in Xenopus egg extracts. Truncated cyclin with only the first 90 amino acids was stable, but other C-terminal deletions lacking between 14 and 187 amino acids were unstable and were degraded by a mechanism that was neither cell cycle regulated nor dependent upon the destruction box. None of the C-terminal deletion mutants bound p34cdc2. To investigate whether the binding of p34cdc2 is required for cell cycle-regulated degradation, the behavior of proteins encoded by a series of full length Xenopus cyclin B2 cDNA with point mutations in conserved amino acids in the p34cdc2-binding domain was examined. All of the point mutants failed to form stable complexes with p34cdc, and their degradation was markedly reduced compared to wild-type cyclin. Similar results were obtained when the mutant cyclins were synthesized in reticulocyte lysates and when cyclin mRNA was translated directly in a Xenopus egg extract. These results indicate that mutations that interfere with p34cdc2 binding also interfere with cyclin destruction, suggesting that p34cdc2 binding is required for the cell cycle-regulated destruction of Xenopus cyclin B2.     

Regulation of Cdc25C Activity During the Meiotic G2/M Transition

The Cdc25C phosphatase is a key activator of Cdc2/cyclin B that controls M-phase entry in eukaryotic cells. Here we discuss the regulation of Cdc25C by phosphorylation during the meiotic maturation of Xenopus oocytes. In G2 arrested oocytes, Cdc25C is phosphorylated on Ser287 and associated with 14-3-3 proteins. Entry of the oocytes into M-phase of meiosis is triggered by progesterone, which activates a signaling pathway leading to the dephosphorylation of Ser287, probably mediated by the PP1 phosphatase. The activation of Cdc25C during oocyte maturation correlates also with its phosphorylation on multiple sites. These phosphorylations involve several signaling pathways, including Polo kinases and MAP kinases, and might require also the inhibition of the PP2A phosphatase. Finally, Cdc25C is further phosphorylated by its substrate Cdc2/cyclin B, as part of an auto-amplification loop that ensures the high Cdc2/cyclin B activity level required to drive the oocyte through the meiotic cell cycle.     

Protein phosphatase 2A regulates MPF activity and sister chromatid cohesion in budding yeast

Background Mitosis is regulated by MPF (maturation promoting factor), the active form of Cdc2/28–cyclin B complexes. Increasing levels of cyclin B abundance and the loss of inhibitory phosphates from Cdc2/28 drives cells into mitosis, whereas cyclin B destruction inactivates MPF and drives cells out of mitosis. Cells with defective spindles are arrested in mitosis by the spindle-assembly checkpoint, which prevents the destruction of mitotic cyclins and the inactivation of MPF. We have investigated the relationship between the spindle-assembly checkpoint, cyclin destruction, inhibitory phosphorylation of Cdc2/28, and exit from mitosis.Results The previously characterized budding yeast mad mutants lack the spindle-assembly checkpoint. Spindle depolymerization does not arrest them in mitosis because they cannot stabilize cyclin B. In contrast, a newly isolated mutant in the budding yeast CDC55 gene, which encodes a protein phosphatase 2A (PP2A) regulatory subunit, shows a different checkpoint defect. In the presence of a defective spindle, these cells separate their sister chromatids and leave mitosis without inducing cyclin B destruction. Despite the persistence of B-type cyclins, cdc55 mutant cells inactivate MPF. Two experiments show that this inactivation is due to inhibitory phosphorylation on Cdc28: phosphotyrosine accumulates on Cdc28 in cdc55Δ cells whose spindles have been depolymerized, and a cdc28 mutant that lacks inhibitory phosphorylation sites on Cdc28 allows spindle defects to arrest cdc55 mutants in mitosis with active MPF and unseparated sister chromatids.Conclusions We conclude that perturbations of protein phosphatase activity allow MPF to be inactivated by inhibitory phosphorylation instead of by cyclin destruction. Under these conditions, sister chromatid separation appears to be regulated by MPF activity rather than by protein degradation. We discuss the role of PP2A and Cdc28 phosphorylation in cell-cycle control, and the possibility that the novel mitotic exit pathway plays a role in adaptation to prolonged activation of the spindle-assembly checkpoint.     

Dependence of Mos-induced Cdc2 activation on MAP kinase function in a cell-free system.

The progression of G2-arrested Xenopus laevis oocytes into meiotic M-phase is accompanied by the nearly simultaneous activation of p42 MAP kinase and Cdc2/cyclin B. This timing raises the possibility that the activation of one kinase might depend upon the other. Here we have examined whether Cdc2 activation requires p42 MAP kinase function. We have reconstituted Mos-induced Cdc2 activation in cell-free Xenopus oocyte extracts, and have found that Mos-induced Cdc2 activation requires active p42 MAP kinase, is inhibited by a MAP kinase phosphatase and is independent of protein synthesis. These findings indicate that p42 MAP kinase is an essential component of the M phase trigger in this system.     

Phosphorylation independent activation of human cyclin-dependent kinase 2 by cyclin A in vitro.

p33cdk2 is a serine-threonine protein kinase that associates with cyclins A, D, and E and has been implicated in the control of the G1/S transition in mammalian cells. Recent evidence indicates that cyclin-dependent kinase 2 (Cdk2), like its homolog Cdc2, requires cyclin binding and phosphorylation (of threonine-160) for activation in vivo. However, the extent to which mechanistic details of the activation process are conserved between Cdc2 and Cdk2 is unknown. We have developed bacterial expression and purification systems for Cdk2 and cyclin A that allow mechanistic studies of the activation process to be performed in the absence of cell extracts. Recombinant Cdk2 is essentially inactive as a histone H1 kinase (< 4 x 10(-5) pmol phosphate transferred.min-1 x microgram-1 Cdk2). However, in the presence of equimolar cyclin A, the specific activity is approximately 16 pmol.mon-1 x microgram-1, 4 x 10(5)-fold higher than Cdk2 alone. Mutation of T160 in Cdk2 to either alanine or glutamic acid had little impact on the specific activity of the Cdk2/cyclin A complex: the activity of Cdk2T160E was indistinguishable from Cdk2, whereas that of Cdk2T160A was reduced by five-fold. To determine if the Cdk2/cyclin A complex could be activated further by phosphorylation of T160, complexes were treated with Cdc2 activating kinase (CAK), purified approximately 12,000-fold from Xenopus eggs. This treatment resulted in an 80-fold increase in specific activity. This specific activity is comparable with that of the Cdc2/cyclin B complex after complete activation by CAK (approximately 1600 pmol.mon-1 x microgram-1). Neither Cdk2T160A/cyclin A nor Cdk2T160E/cyclin A complexes were activated further by treatment with CAK. In striking contrast with cyclin A, cyclin B did not directly activate Cdk2. However, both Cdk2/cyclin A and Cdk2/cyclin B complexes display similar activity after activation by CAK. For the Cdk2/cyclin A complex, both cyclin binding and phosphorylation contribute significantly to activation, although the energetic contribution of cyclin A binding is greater than that of T160 phosphorylation by approximately 5 kcal/mol. The potential significance of direct activation of Cdk2 by cyclins with respect to regulation of cell cycle progression is discussed.     

Combinatorial control of cyclin B1 nuclear trafficking through phosphorylation at multiple sites

Entry into mitosis is regulated by the Cdc2 kinase complexed to B-type cyclins. We and others recently reported that cyclin B1/Cdc2 complexes, which appear to be constitutively cytoplasmic during interphase, actually shuttle continually into and out of the nucleus, with the rate of nuclear export exceeding the import rate (). At the time of entry into mitosis, the import rate is increased, whereas the export rate is decreased, leading to rapid nuclear accumulation of Cdc2/cyclin B1. Although it has recently been reported that phosphorylation of 4 serines within cyclin B1 promotes the rapid nuclear translocation of Cdc2/cyclin B1 at G(2)/M, the role that individual phosphorylation sites play in this process has not been examined (, ). We report here that phosphorylation of a single serine residue (Ser(113) of Xenopus cyclin B1) abrogates nuclear export of cyclin B1. This serine lies directly within the cyclin B1 nuclear export sequence and, when phosphorylated, prevents binding of the nuclear export factor, CRM1. In contrast, analysis of phosphorylation site mutants suggests that coordinate phosphorylation of all 4 serines (94, 96, 101, and 113) is required for the accelerated nuclear import of cyclin B1/Cdc2 characteristic of G(2)/M. Additionally, binding of cyclin B1 to importin-beta, the factor known to be responsible for the slow interphase nuclear entry of cyclin B1, appears to be unaffected by the phosphorylation state of cyclin B. These data suggest that a distinct import factor must be recruited to enhance nuclear entry of Cdc2/cyclin B1 at the G(2)/M transition.     

The Polo-like Kinase Plx1 Is Required for Activation of the Phosphatase Cdc25C and Cyclin B-Cdc2 in Xenopus Oocytes

In the Xenopus oocyte system mitogen treatment triggers the G(2)/M transition by transiently inhibiting the cAMP-dependent protein kinase (PKA); subsequently, other signal transduction pathways are activated, including the mitogen-activated protein kinase (MAPK) and polo-like kinase pathways. To study the interactions between these pathways, we have utilized a cell-free oocyte extract that carries out the signaling events of oocyte maturation after addition of the heat-stable inhibitor of PKA, PKI. PKI stimulated the synthesis of Mos and activation of both the MAPK pathway and the Plx1/Cdc25C/cyclin B-Cdc2 pathway. Activation of the MAPK pathway alone by glutathione S-transferase (GST)-Mos did not lead to activation of Plx1 or cyclin B-Cdc2. Inhibition of the MAPK pathway in the extract by the MEK1 inhibitor U0126 delayed, but did not prevent, activation of the Plx1 pathway, and inhibition of Mos synthesis by cycloheximide had a similar effect, suggesting that MAPK activation is the only relevant function of Mos. Immunodepletion of Plx1 completely inhibited activation of Cdc25C and cyclin B-Cdc2 by PKI, indicating that Plx1 is necessary for Cdc25C activation. In extracts containing fully activated Plx1 and Cdc25C, inhibition of cyclin B-Cdc2 by p21(Cip1) had no significant effect on either the phosphorylation of Cdc25C or the activity of Plx1. These results demonstrate that maintenance of Plx1 and Cdc25C activity during mitosis does not require cyclin B-Cdc2 activity.     

On the synthesis and destruction of A- and B-type cyclins during oogenesis and meiotic maturation in Xenopus laevis

We have measured the levels of cyclin mRNAs and polypeptides during oogenesis, progesterone-induced oocyte maturation, and immediately after egg activation in the frog, Xenopus laevis. The mRNA for each cyclin is present at a constant level of approximately 5 x 10(7) molecules per oocyte from the earliest stages of oogenesis until after fertilization. The levels of polypeptides show more complex patterns of accumulation. The B-type cyclins are first detectable in stage IV and V oocytes. Cyclin B2 polypeptide is present at approximately 2 x 10(9) molecules (150 pg) per oocyte by stage VI. The amount increases after progesterone treatment, but returns to its previous level after GVBD and undergoes no further change until it is destroyed at fertilization. Cyclin B1 is present at 4 x 10(8) molecules per oocyte in stage VI oocytes, and rises steadily during maturation, ultimately reaching similar levels to cyclin B2 in unfertilized eggs. Unlike the B-type cyclins, cyclin A is barely detectable in stage VI oocytes, and only starts to be made in significant amounts after oocytes are exposed to progesterone. A portion of all the cyclins are destroyed after germinal vesicle breakdown (GVBD), and cyclins B1 and B2 also experience posttranslational modifications during oocyte maturation. Progesterone strongly stimulates both cyclin and p34cdc2 synthesis in these oocytes, but whereas cyclin synthesis continues in eggs and after fertilization, synthesis of p34cdc2 declines strongly after GVBD. The significance of these results is discussed in terms of the activation and inactivation of maturation-promoting factor.     

Differential regulation of Cdc2 and Cdk2 by RINGO and cyclins.

Cyclin-dependent kinases (Cdks) are key regulators of the eukaryotic cell division cycle. Cdk1 (Cdc2) and Cdk2 should be bound to regulatory subunits named cyclins as well as phosphorylated on a conserved Thr located in the T-loop for full enzymatic activity. Cdc2- and Cdk2-cyclin complexes can be inactivated by phosphorylation on the catalytic cleft-located Thr-14 and Tyr-15 residues or by association with inhibitory subunits such as p21(Cip1). We have recently identified a novel Cdc2 regulator named RINGO that plays an important role in the meiotic cell cycle of Xenopus oocytes. RINGO can bind and activate Cdc2 but has no sequence homology to cyclins. Here we report that, in contrast with Cdc2- cyclin complexes, the phosphorylation of Thr-161 is not required for full activation of Cdc2 by RINGO. We also show that RINGO can directly stimulate the kinase activity of Cdk2 independently of Thr-160 phosphorylation. Moreover, RINGO-bound Cdc2 and Cdk2 are both less susceptible to inhibition by p21(Cip1), whereas the Thr-14/Tyr-15 kinase Myt1 can negatively regulate the activity of Cdc2-RINGO with reduced efficiency. Our results indicate that Cdk-RINGO complexes may be active under conditions in which cyclin-bound Cdks are inhibited and can therefore play different regulatory roles.     

A Novel Yeast Screen for Mitotic Arrest Mutants Identifies DOC1, a New Gene Involved in Cyclin Proteolysis

B-type cyclins are rapidly degraded at the transition between metaphase and anaphase and their ubiquitin-mediated proteolysis is required for cells to exit mitosis. We used a novel enrichment to isolate new budding mutants that arrest the cell cycle in mitosis. Most of these mutants lie in the CDC16, CDC23, and CDC27 genes, which have already been shown to play a role in cyclin proteolysis and encode components of a 20S complex (called the cyclosome or anaphase promoting complex) that ubiquitinates mitotic cyclins. We show that mutations in CDC26 and a novel gene, DOC1, also prevent mitotic cyclin proteolysis. Mutants in either gene arrest as large budded cells with high levels of the major mitotic cyclin (Clb2) protein at 37°C and cannot degrade Clb2 in G₁-arrested cells. Cdc26 associates in vivo with Doc1, Cdc16, Cdc23, and Cdc27. In addition, the majority of Doc1 cosediments at 20S with Cdc27 in a sucrose gradient, indicating that Cdc26 and Doc1 are components of the anaphase promoting complex.