P34cdc2 Kinase Activity Research Articles

An Accumulation of p34cdc2at the End of Mouse Oocyte Growth Correlates with the Acquisition of Meiotic Competence

Growing incompetent mouse oocytes released from follicular cells are unable to spontaneously resume meiosisin vitro.To identify the reasons for meiotic incompetence in these cells, the levels of p34cdc2/cyclin B kinase and p42MAPKbetween incompetent and competent oocytes were compared. p34cdc2was present at very low levels in incompetent oocytes and accumulated abruptly at the time of meiotic competence acquisition. By contrast, cyclin B and p42MAPKwere present at similar concentrations in both types of oocytes. Okadaic acid induced centrosome phosphorylation and meiotic reinitiation in incompetent oocytes, without inducing an increase in p34cdc2concentration. However, the p34cdc2present in incompetent oocytes was activated and all events following germinal vesicle breakdown were induced up to the formation of a metaphase I spindle including p42MAPKactivation, sustained increase in p34cdc2kinase activity, and translational activation of a dormant mRNA. We suggest that a threshold level of p34cdc2has to be reached for meiotic reinitiation to be spontaneously triggered: competence is restricted at a point preceding MPF activation. Whatever the mechanism involved in this restriction point, i.e., subthreshold concentration of p34cdc2and/or lack of an activator or presence of an inhibitor, it is bypassed by okadaic acid. Downstream of this point meiosis progresses up to metaphase I, even though p34cdc2concentration remains low.

B-type cyclins regulate G1 progression in fission yeast in opposition to the p25rum1 cdk inhibitor.

The onset of S phase in fission yeast is regulated at Start, the point of commitment to the mitotic cell cycle. The p34cdc2 kinase is essential for G1 progression past Start, but until now its regulation has been poorly understood. Here we show that the cig2/cyc17 B-type cyclin has an important role in G1 progression, and demonstrate that p34cdc2 kinase activity is periodically associated with cig2 in G1. Cells lacking cig2 are defective in G1 progression, and this is particularly clear in small cells that must regulate Start with respect to cell size. We also find that the cig1 B-type cyclin can promote G1 progression. Whilst p25rum1 can inhibit cig2/cdc2 activity in vitro, and may transiently inhibit this complex in vivo, cig1 is regulated independently of p25rum1. Since cig1/cdc2 kinase activity peaks in mitotic cells, and decreases after mitosis with similar kinetics to cdc13-associated kinase activity, we suggest that cig2 is likely to be the principal fission yeast G1 cyclin. cig2 protein levels accumulate in G1 cells, and we propose that p25rum1 may transiently inhibit cig2-associated p34cdc2 activity until the critical cell size required for Start is reached.

A single fission yeast mitotic cyclin B p34cdc2 kinase promotes both S-phase and mitosis in the absence of G1 cyclins.

Deletion of the fission yeast mitotic B-type cyclin gene cdc13 causes cells to undergo successive rounds of DNA replication. We have used a strain which expresses cdc13 conditionally to investigate re-replication. Activity of Start genes cdc2 and cdc10 is necessary and p34cdc2 kinase is active in re-replicating cells. We tested to see whether other cyclins were required for re-replication using cdc13delta. Further deletion of cig1 and puc1 had no effect, but deletion of cig2/cyc17 caused a severe delay in re-replication. Deletion of cig1 and cig2/cyc17 together abolished re-replication completely and cells arrested in G1. This, and analysis of the temperature sensitive cdc13-117 mutant, suggests that cdc13 can effectively substitute for the G1 cyclin activity of cig2/cyc17. We have characterized p56cdc13 activity and find evidence that in the absence of G1 cyclins, S-phase is delayed until the mitotic p34cdc2-p56cdc13 kinase is sufficiently active. These data suggest that a single oscillation of p34cdc2 kinase activity provided by a single B-type cyclin can promote ordered progression into both DNA replication and mitosis, and that the level of cyclin-dependent kinase activity may act as a master regulator dictating whether cells undergo S-phase or mitosis.

Activation of p34 cdc2 kinase around the meiotic resumption in bovine oocytes cultured in vitro

The p34 cdc2 kinase has been identified as a protein factor that is a regulator of meiotic maturation in mammalian oocytes. To investigate the regulatory function of the meiotic resumption in bovine oocytes cultured in vitro, the changes in the phosphorylation states of p34 cdc2 kinase and the histone H1 kinase activity were examined around germinal vesicle breakdown (GVBD). All bovine oocytes just after isolation from their follicles were arrested at the germinal vesicle (GV) stage, and these extracts exhibited two (upper and lower) bands of p34 cdc2 kinase on SDS-PAGE followed by immunoblotting with an antibody against C-terminal peptide of p34 cdc2. When these oocytes were cultured for 24 h in a medium supplemented with 100 μg/ml genistein, tyrosine phosphorylation inhibitor, GVBD was induced in 85% of oocytes, indicating that the upper band of p34 cdc2 kinase in bovine oocytes at the GV stage was already fully phosphorylated tyrosine residue prior to culture. Another (middle) band of p34 cdc2 kinase between the upper and lower bands appeared in the extracts of the oocytes cultured for 4 h, and significant activation of the histone H1 kinase was found in these oocytes (67 ± 18 fmol/ h/ oocyte) as compared to that in oocytes cultured for 0 h (46 ± 11 fmol/ h/ oocyte). The staining intensity of the middle band and the activity of the histone H1 kinase were further increased after the initiation of GVBD at 6 h of culture, but the quantitative changes of upper and lower bands were not detected throughout the 12 h of culture. Thus, it is concluded that the dephosphorylation of p34 cdc2 kinase followed by activation of the histone H1 kinase after the onset of culture plays a key role in the resumption of meiosis in bovine oocytes.

The antitumor drug fostriecin induces vimentin hyperphosphorylation and intermediate filament reorganization.

Fostriecin is an antitumor drug in phase I clinical trials. We have recently shown that it is a potent inhibitor of protein phosphatases 1 and 2A in vitro, a property not previously described for an antitumor drug. We have investigated its effects on protein phosphorylation in baby hamster kidney cells. Fostriecin strongly stimulated the phosphorylation of a single protein, which we identified as the intermediate filament vimentin. Fostriecin also caused rounding of the cells and a reorganization of the vimentin filaments. These effects are similar to those of the known protein phosphatase 1 and 2A inhibitors okadaic acid and calyculin A, which are also tumor promoters. Fostriecin induced vimentin hyperphosphorylation mostly at two sites, which were sensitive to staurosporine and could be phosphorylated by protein kinase C in vitro. Fostriecin-induced vimentin hyperphosphorylation also occurred in cells that lack p34cdc2 kinase activity. These results suggest that protein kinase C plays a direct or indirect role in vimentin hyperphosphorylation during exposure to fostriecin. The results also provide strong evidence that fostriecin inhibits protein phosphatases 1 and 2A in vivo and raise the possibility that it may have tumor-promoting activity.

Calcium requirements during mitotic cdc2 kinase activation and cyclin degradation in Xenopus egg extracts.

Activation of p34cdc2 kinase is essential for entry into mitosis while subsequent deactivation and cyclin degradation are associated with exit. In Xenopus embryos, both of these phases are regulated by post-translation modifications and occur spontaneously on incubation of extracts prepared late in the first cell cycle. Even though high levels of calcium buffer were initially used to prepare these extracts, we found that free calcium levels in them remained in the observed physiological range (200-500 nM). Further addition of calcium buffers only slightly reduced free calcium levels, but inhibited histone H1 (cdc2A) kinase deactivation and cyclin degradation. Higher buffer concentrations slowed the kinase activation phase. Reducing the free buffer concentration by premixing with calcium reversed the effects of the buffer, indicating that the inhibitory effects arose from the calcium-chelating properties of the buffer rather than non-specific side effects. Furthermore, additions of calcium buffer at the end of the H1 kinase activation phase did not prevent deactivation. From these results, and the order of effectiveness of different calcium buffers in disrupting the H1 kinase cycle, we suggest that local transient increases in free calcium influence the rate of cdc2 kinase activation and are required to initiate the pathway leading to cyclin degradation and kinase inactivation in mitotic cell cycles.

Induction of A- and D-type cyclins and cdc2 kinase activity during recovery from short-term hyperoxic lung injury

Hyperoxia causes a reproducible pattern of lung injury and repair in rodents, in which proliferation of alveolar epithelial cells (AEC) and fibroblasts is observed during recovery. We postulated that if quiescent cells are stimulated to reenter the cell cycle, then cyclin expression and cyclin-dependent protein kinase activity would be reactivated in AEC during the repair process after hyperoxic lung injury. To test this hypothesis, we exposed adult rats to short-term hyperoxia, followed by recovery for various times in room air. Cellular proliferation in vivo was confirmed by 1) flow cytometric analysis of DNA content (FACS) of freshly isolated AEC and 2) immunohistochemistry of proliferating cell nuclear antigen (PCNA) and bromodeoxyuridine (BrdU) incorporation into DNA on lung sections. The percentage of freshly isolated AEC in S phase and G2/M phase on FACS analysis increased twofold to a maximum of 16.5%, after 48 h in 100% oxygen and 48 h recovery in air. Cyclins A and D and p34cdc2 protein expression were also increased during the recovery period; while p33cdk2 and p34cdk4 increased only slightly. p34cdc2 histone H1 kinase activity, both in whole lung and in AEC, decreased initially after 48 h in oxygen. However, a marked increase in p34cdc2 kinase activity was observed at 48 h recovery in whole lung and returned to baseline by 72 h. In isolated and cultured AEC, p34cdc2 kinase activity was maximal at 24 h of recovery in air. We conclude that cyclins A and D and p34cdc2 protein expression and p34cdc2 kinase activity are increased in vivo during recovery from hyperoxic lung injury in both adult rat lungs and in AEC isolated from these lungs. We speculate that the induction of cyclin-dependent protein kinase activity is a key event in mediating the proliferative cellular repair response to lung injury.

Analysis of the mechanism(s) of metaphase I arrest in maturing mouse oocytes.

Fully grown mouse oocytes are normally competent to progress from prophase I to metaphase II without interruption. However, growing mouse oocytes initially become only partially competent to undergo meiotic maturation. Meiotic maturation in these oocytes does not progress beyond metaphase I. In contrast to the oocytes of most strains of mice, most oocytes of strain LT/Sv mice become arrested at metaphase I even when they are fully grown. The initiation of oocyte maturation is correlated with an increase in p34cdc2 kinase activity that continues to rise until metaphase I. The transition into anaphase I is normally correlated with a decrease in p34cdc2 kinase activity. This study demonstrated that metaphase I arrest in both partially competent growing oocytes and fully grown LT/Sv oocytes is correlated with a sustained elevation of p34cdc2 kinase activity. In fact, p34cdc2 activity continued to increase during the time when activity normally decreased. In normally maturing oocytes, some, but not all, of the cyclin B, the regulatory protein associated with p34cdc2, became degraded in oocytes that entered anaphase I. In contrast, the amount of cyclin B present in the metaphase I-arrested oocytes continued to increase at the time when it was being degraded in normal oocytes progressing to metaphase II. These results suggest that the progression of meiosis is arrested at metaphase I in both groups of oocytes because of continued p34cdc2 kinase activity sustained, at least in part, by restricted degradation of cyclin B.(ABSTRACT TRUNCATED AT 250 WORDS)

Granzymes: exogenous porteinases that induce target cell apoptosis

Cytotoxic lymphocytes mediate immunity against viruses and surveillance against neoplastic transformation. They kill target cells by multiple mechanisms, but utilize a pore-forming protein, perforin, and a family of serine proteinases as their principal means of inflicting cell death. Recent studies have demonstrated that perforin and serine proteinases synergistically trigger an endogenous pathway of apoptosis resulting in dissolution of the target cell nuclear membrane and DNA fragmentation. These changes may be secondary to inappropriate activation of p34 cdc2 kinase and the subsequent derangement of cell cycle control. As discussed by Mark Smyth and Joseph Trapani, the immediate molecular targets of perforin/granzyme-mediated apoptosis are still unclear, though candidate molecules with homology to cell death gene products from primitive organisms are currently under close scrutiny.

Oocyte activation and passage through the metaphase/anaphase transition of the meiotic cell cycle is blocked in clams by inhibitors of HMG-CoA reductase activity.

Cell cycle progression for postembryonic cells requires the activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-R), the enzyme which catalyzes the production of the isoprenoid precursor, mevalonate. In this study, we examine the requirements of HMG-R activity for cell cycle progression during the meiotic and early mitotic divisions using oocytes and dividing embryos from the surf clam, Spisula solidissima. Using two different inhibitors of HMG-R, we find that the activity of this enzyme appears to be required at three distinct points of the cell cycle during meiosis. Depending on the stage at which these inhibitors are added to synchronous clam cultures, a reversible cell cycle block is triggered at the time of activation or at metaphase of either meiosis I or II, whereas there is not block to the mitotic cell cycle. Inhibition of HMG-R activity in activated oocytes does not affect the transient activation of p42MAPK but results in a block at metaphase of meiosis I that is accompanied by the stabilization of cyclins A and B and p34cdc2 kinase activity. Our results suggest that metabolites from the mevalonate biosynthetic pathway can act to influence the process of activation, as well as the events later in the cell cycle that lead to cyclin proteolysis and the exit from M phase during clam meiosis.

Chromosome condensation induced by fostriecin does not require p34cdc2 kinase activity and histone H1 hyperphosphorylation, but is associated with enhanced histone H2A and H3 phosphorylation.

Chromosome condensation at mitosis correlates with the activation of p34cdc2 kinase, the hyperphosphorylation of histone H1 and the phosphorylation of histone H3. Chromosome condensation can also be induced by treating interphase cells with the protein phosphatase 1 and 2A inhibitors okadaic acid and fostriecin. Mouse mammary tumour FT210 cells grow normally at 32 degrees C, but at 39 degrees C they lose p34cdc2 kinase activity and arrest in G2 because of a temperature-sensitive lesion in the cdc2 gene. The treatment of these G2-arrested FT210 cells with fostriecin or okadaic acid resulted in full chromosome condensation in the absence of p34cdc2 kinase activity or histone H1 hyperphosphorylation. However, phosphorylation of histones H2A and H3 was strongly stimulated, partly through inhibition of histone H2A and H3 phosphatases, and cyclins A and B were degraded. The cells were unable to complete mitosis and divide. In the presence of the protein kinase inhibitor starosporine, the addition of fostriecin did not induce histone phosphorylation and chromosome condensation. The results show that chromosome condensation can take place without either the histone H1 hyperphosphorylation or the p34cdc2 kinase activity normally associated with mitosis, although it requires a staurosporine-sensitive protein kinase activity. The results further suggest that protein phosphatases 1 and 2A may be important in regulating chromosome condensation by restricting the level of histone phosphorylation during interphase, thereby preventing premature chromosome condensation.

Growth Condition-Induced Precocious Activation of p34cdc2 Kinase Inhibits the Expression of Developmental Competence

We examined the effect of growth conditions upon mitotic p34cdc2 kinase and developmental competence in Acanthamoeba castellanii. At G2/M of the cell cycle p34cdc2 kinase activity peaks in level, and p34cdc2 is transiently in a complex with newly synthesized cyclin B and phosphorylated on tyrosine (pre-MPF). Developmental competence peaks in level slightly preceeding p34cdc2 kinase activation and pre-MPF accumulation. Under adverse growth conditions p34cdc2 kinase activation and transient pre-MPF accumulation occur at a reduced G2 phase length and smaller cell size. Developmental competence is not expressed during the shortened G2 phase. Cycloheximide inhibits the precocious p34cdc2 kinase activation and both the precocious accumulation of cyclin B and tyrosine phosphorylation on p34cdc2. Nocodazole inhibits the precocious p34cdc2 kinase activation as well; however, it does not affect the precocious accumulation of pre-MPF. Developmental competence increases in level during artificially elongated G2 phases. The results indicate that, first p34cdc2 kinase activation requires cyclin B in a complex with p34cdc2 and the presence of intact microtubuli, second establishment of competence requires a certain length of G2, and third the regulation of pre-MPF accumulation, and thus of cyclin B expression, plays a role in the relationship among growth condition, cell cycle progression, and expression of the developmental program.

Nerve growth factor regulates the expression and activity of p33cdk2 and p34cdc2 kinases in PC12 pheochromocytoma cells.

In the absence of serum, nerve growth factor (NGF) promotes the survival and differentiation of the PC12 pheochromocytoma cell line. In the presence of serum, NGF acts primarily as a differentiation factor and negative regulator of cell cycling. To investigate NGF control of cell cycling, we have analyzed the regulation of cyclin dependent kinases during PC12 cell differentiation. NGF treatment leads to a reduction in the steady-state protein levels of p33cdk2 and p34cdc2, two key regulators of cell cycle progression. The decrease in p33cdk2 and p34cdc2 coincides with a decrease in the enzymatic activity of cyclinA-p34cdc2, cyclinB-p34cdc2, cyclinE-p33cdk2, and cyclinA-p33cdk2 kinases. The decline in p33cdk2 and p34cdc2 kinase activity in response to NGF is accelerated in cells that over-express the p140trk NGF receptor, suggesting that the timing of the down- regulation is dependent on the level of p140trk and the strength of the NGF signal. The level of cyclin A, a regulatory subunit of p33cdk2 and p34cdc2, is relatively constant during PC12 differentiation. Nevertheless, the DNA binding activity of the cyclinA-associated transcription factor E2F/DP decreases. Thus, NGF down-regulates the activity of cyclin dependent kinases and cyclin-transcription factor complexes during PC12 differentiation.

Oncogenic ras stimulates a 96-kDa histone H2b kinase activity in activated Xenopus egg extracts. Correlation with the suppression of p34cdc2 kinase.

We previously showed that purified, bacterially expressed oncogenic human rasH protein blocks or delays the progression of the embryonic cell cycle into M-phase in activated Xenopus egg extracts. This block correlates with the suppression of the activation of p34cdc2 kinase (Pan, B.-T., Chen, C.-T., and Lin, S.-M. (1994) J. Biol. Chem. 269, 5968-5975). In an attempt to identify kinases that are involved in mediating the effect of oncogenic Ras on the cell cycle, we assayed aliquots of activated Xenopus egg extracts, which were incubated at 25 degrees C for various times in the absence and presence of oncogenic Ras, for their kinase activities toward a calf thymus histone fraction (Hf1). We find that the suppression of the histone H1 kinase activity of p34cdc2 by oncogenic Ras correlates with a simultaneous stimulation of a histone H2b serine kinase activity. Using a histone H2b in-the-gel kinase assay, we further show that the stimulated histone H2b kinase activity is attributed mainly to a 96-kDa kinase and slightly to p42mapk. Although Xenopus p90rsk is also activated by oncogenic Ras, we demonstrate that activated p90rsk is not responsible for the 96-kDa histone H2b kinase activity. The identity of the 96-kDa kinase remains unclear. Our data suggests that the 96-kDa kinase may be involved in mediating the effect of oncogenic Ras on the embryonic cell cycle of Xenopus.

Premature chromatin condensation upon accumulation of NIMA.

The NIMA protein kinase of Aspergillus nidulans is required for the G2/M transition of the cell cycle. Mutants lacking NIMA arrest without morphological characteristics of mitosis, but they do contain an activated p37nimX kinase (the Aspergillus homologue of p34cdc2). To gain a better understanding of NIMA function we have investigated the effects of expressing various NIMA constructs in Aspergillus, fission yeast and human cells. Our experiments have shown that the instability of the NIMA protein requires sequences in the non-catalytic C-terminus of the protein. Removal of this domain results in a stable protein that, once accumulated, promotes a lethal premature condensation of chromatin without any other aspects of mitosis. Similar effects were also observed in fission yeast and human cells accumulating Aspergillus NIMA. This phenotype is independent of cell cycle progression and does not require p34cdc2 kinase activity. As gain of NIMA function by accumulation results in premature chromatin condensation, and loss of NIMA function results in an inability to enter mitosis, we propose that NIMA functions in G2 to promote the condensation of chromatin normally associated with entry into mitosis.

Expression of N-terminally truncated cyclin B in the Drosophila larval brain leads to mitotic delay at late anaphase

We have introduced an N-terminally truncated form of cyclin B into the Drosophila germ-line downstream of the yeast upstream activator that responds to GAL4. When such lines of flies are crossed to lines in which GAL4 is expressed in imaginal discs and larval brain, the majority of the resulting progeny die at the late pupal stage of development. Very rarely (< 0.1% of progeny) adults emerge that have a mutant phenotype typical of flies with mutations in genes required for the cell cycle; they have rough eyes, deformed wings, abnormal bristles, and die within hours of emergence. The brains of third instar larval progeny show an abnormally high proportion of mitotic cells containing overcondensed chromatids that have undergone anaphase separation, together with cells that cannot be assigned to a particular mitotic stage. Immunostaining indicates that these anaphase cells contain moderate levels of cyclin B, suggesting that persistent p34cdc2 kinase activity can prevent progression from anaphase into telophase.

Inhibition of Human B Lymphocyte Cell Cycle Progression and Differentiation by Rapamycin

In this study, we have analyzed the effects of the immunosuppressive agent rapamycin on the activation of highly purified normal human B lymphocytes. When the polyclonal activators Staphylococcus aureus (SA) and soluble CD40 ligand (CD40L) were used to stimulate B cells, rapamycin inhibited both interleukin 2 (IL2)-dependent and -independent proliferation, as well as IL2-dependent differentiation into antibody-secreting cells. Cell cycle analysis indicated that rapamycin inhibited the progression of SA+IL2-stimulated B cells past the mid-G1 phase of the cell cycle. To begin to identify rapamycin-sensitive signaling events essential for B cell activation, we examined the effects of rapamycin on p34cdc2 and p33cdk2 kinase activities. SA+IL2 stimulation induced the activation of both cyclin-dependent kinases. Of interest, rapamycin abrogated the activation of both p34cdc2 and p33cdk2. Our results indicate therefore that rapamycin inhibits a number of SA- and CD40L-inducible events that may be necessary for both entry into S phase and for permitting subsequent B cell differentiation. These studies emphasize the utility of this drug as a tool to begin to dissect the activation pathways utilized by human B cells, as well as to provide implications for the therapeutic use of rapamycin in vivo.

MPF components and meiotic competence in growing pig oocytes.

Growing pig oocytes (< or = 90 microns in diameter) are unable to resume meiosis in vitro. The objective of our present experiments has been to identify the reasons for meiotic competence in these cells. By comparing histone H1 kinase activity in growing and fully grown oocytes we demonstrate that incompetence is associated with an inability to activate H1 kinase in growing oocytes. Immunoblotting was used to determine whether this kinase inactivity resulted from a lack of either p34cdc2 protein or B-type cyclin. The results established that each of these cell cycle molecules are present in comparable amounts in both growing and fully grown oocytes. In the third series of studies experiments were carried out in an attempt to induce p34cdc2 activation during growth. Treatment with okadaic acid, an inhibitor of phosphatase 1 and 2A known to stimulate and accelerate the transition into M-phase of the meiotic cycle in a number of different species, was able to induce p34cdc2 kinase activity and facilitated the G2- to M-phase in growing oocytes. We conclude that although growing oocytes in pigs have sufficient key cell cycle components for the G2 to M transition, they remain incapable of converting these components to active maturation-promoting factor (MPF) until growth is virtually completed. Inhibition of phosphatase 1 or 2A induces the formation of active MPF during growth by an as yet unidentified pathway.

Oncogenic Ras blocks cell cycle progression and inhibits p34cdc2 kinase in activated Xenopus egg extracts.

The effect of purified, bacterially expressed human RasH proteins on embryonic cell cycle progression in activated Xenopus egg extracts was studied. Bacterially expressed human oncogenic RasH protein is able to block the progression of the Xenopus embryonic cell cycle into M-phase. In contrast, the corresponding normal human Ras protein is relatively ineffective when assayed in a like manner. The observed arresting activity can be blocked by the addition of Y13-259 anti-Ras monoclonal antibody but not by nonspecific IgG. Oncogenic Ras also induces unique morphological changes in the reconstituted nuclei; the nuclei appear to be enlarged, and the chromatin partially condenses into fiber-like structures. This induced arrest is associated with suppression of p34cdc2 kinase activity, indicating that the oncogenic Ras protein induces the arrest by inactivating p34cdc2. This inactivation by oncogenic Ras protein does not result from inhibition of the synthesis of cyclin B or of the binding of the newly synthesized cyclin B to the p34cdc2.

Microinjection of Cdc25 protein phosphatase into Xenopus prophase oocyte activates MPF and arrests meiosis at metaphase I

Microinjection of bacterially expressed human cdc25A protein into Xenopus prophase oocytes provokes the activation of p34 cdc2 kinase and the tyrosine dephosphorylation of p34 cdc2 in the presence or absence of protein synthesis. The level of p34 cdc2 kinase activity then drops in parallel with the degradation of cyclin B2 and finally increases again to stabilize at a high level. Cdc25 microinjection induces the assembly of a metaphase I spindle which is abnormally located in the deep cytoplasm. Moreover, oocytes arrest at the metaphase I stage and do not reach metaphase II even 10 h after cdc25 microinjection. The extended metaphase I period observed in cdc25-injected oocytes results from an equilibrium between degradation of cyclins and synthesis of new cyclins. This is in contrast with progesterone-stimulated oocytes where cyclin degradation is turned off when oocytes enter metaphase II. During metaphase I, the reactivation of MPF activity can be disrupted in two different ways: 1) cycloheximide, an inhibitor of protein synthesis, by preventing the synthesis of new cyclins, provokes the disappearance of MPF kinase activity and the reformation of a nucleus; 2) when the cAMP level is increased during the metaphase I period in cdc25-injected oocytes, MPF kinase activity drops following a rephosphorylation of tyrosine 15 of p34 cdc2, while the cyclin turn-over remains unaffected. Moreover, increasing the cAMP level in prophase oocytes totally prevents the action of cdc25. Our results indicate that in Xenopus oocytes, the PKA pathway negatively regulates the activation of MPF and the activity of p34 cdc2/cyclin B complex through tyrosine phosphorylation of p34 cdc2 during metaphase I.