Tyrosine Dephosphorylation Of P34cdc2 Research Articles

Tyrosine phosphorylation of p34cdc2 in metaphase II-arrested pig oocytes results in pronucleus formation without chromosome segregation

At the G2-M boundary, maturation-promoting factor (MPF) activation is usually induced in one or both of two ways; tyrosine dephosphorylation of p34cdc2 or synthesis of cyclin B according to cell type and species. At the end of M-phase, however, MPF inactivation is normally triggered only by cyclin degradation. We investigated whether tyrosine phosphorylation of p34cdc2 can inactivate MPF and what kinds of events are induced in pig metaphase II (MII)-arrested oocytes. First, cyclin B1 in MII-arrested oocytes is degraded upon fertilization. Second, when MII oocytes were treated with vanadate, an inhibitor of tyrosine phosphatases, they were released from MII arrest, but MPF was inactivated by further tyrosine phosphorylation of p34cdc2 rather than cyclin B1 degradation. The vanadate-induced exit from M-phase is distinct from normal M-phase exit, which is accompanied by cyclin B1 degradation; the former lacks both sister chromatid separation and second polar body emission. Vanadate itself has no inhibitory effect on chromosome segregation since calcium ionophore induced chromosome segregation in the presence of vanadate. Furthermore, when MII oocytes were treated with olomoucine, an inhibitor of cyclin-dependent kinases, they exited from MII arrest in a manner similar to vanadate-treated MII oocytes. Finally, we propose that MPF inactivation by tyrosine phosphorylation of p34cdc2 enables MII oocytes to form an interphase nucleus, but not to segregate sister chromatid due to the absence of the mechanisms required to trigger sister chromatid separation such as anaphase-promoting complex (APC)-mediated proteolysis, on the signaling pathway from intracellular Ca2+ increase to MPF inactivation. Mol. Reprod. Dev. 52:107–116, 1999. © 1999 Wiley-Liss, Inc.

Tyrosine phosphorylation of p34cdc2 in metaphase II‐arrested pig oocytes results in pronucleus formation without chromosome segregation

At the G2-M boundary, maturation-promoting factor (MPF) activation is usually induced in one or both of two ways; tyrosine dephosphorylation of p34cdc2 or synthesis of cyclin B according to cell type and species. At the end of M-phase, however, MPF inactivation is normally triggered only by cyclin degradation. We investigated whether tyrosine phosphorylation of p34cdc2 can inactivate MPF and what kinds of events are induced in pig metaphase II (MII)-arrested oocytes. First, cyclin B1 in MII-arrested oocytes is degraded upon fertilization. Second, when MII oocytes were treated with vanadate, an inhibitor of tyrosine phosphatases, they were released from MII arrest, but MPF was inactivated by further tyrosine phosphorylation of p34cdc2 rather than cyclin B1 degradation. The vanadate-induced exit from M-phase is distinct from normal M-phase exit, which is accompanied by cyclin B1 degradation; the former lacks both sister chromatid separation and second polar body emission. Vanadate itself has no inhibitory effect on chromosome segregation since calcium ionophore induced chromosome segregation in the presence of vanadate. Furthermore, when MII oocytes were treated with olomoucine, an inhibitor of cyclin-dependent kinases, they exited from MII arrest in a manner similar to vanadate-treated MII oocytes. Finally, we propose that MPF inactivation by tyrosine phosphorylation of p34cdc2 enables MII oocytes to form an interphase nucleus, but not to segregate sister chromatid due to the absence of the mechanisms required to trigger sister chromatid separation such as anaphase-promoting complex (APC)-mediated proteolysis, on the signaling pathway from intracellular Ca2+ increase to MPF inactivation. Mol. Reprod. Dev. 52:107–116, 1999. © 1999 Wiley-Liss, Inc.

Intracellular Regulation Mechanism of Porcine Oocyte Maturation

In the present study, a regulation mechanism of porcine oocyte maturation was examined with special focus on maturation promoting factor (MPF), a crucial regulator of cell division cycle composed of catalytic subunit, p34cdc2 and regulatory subunit, cyclin B, and Mitogen-activated protein kinase (MAPK). At first, an in vitro maturation system using porcine follicular fluid as maturation medium was established in order to mature the oocytes physiologically normal. The oocytes in the germinal vesicle stage (GV) had a significant amount of p34cdc2 and MAPK, which concentrations were not changed throughout maturation. An extremely small amount of cyclin B was present in GV oocytes and cyclin B was not synthesised during GV. The extremely small amount of cyclin B associated p34cdc2 was inactivated by tyrosine phosphorylation and MAPK is also inactive during GV. Germinal vesicle breakdown (GVBD) was induced by tyrosine dephosphorylation of p34cdc2. After GVBD, cyclin B synthesis was started and the amount of cyclin B associated p34cdc2 and MPF activity were increased dramatically. MAPK was also activated by phosphorylation and maintained the high activity until the second metaphase (Met2). MPF activity was decreased transiently after the first metaphase (Merl) then reactivated at Met2 without tyrosine dephosphorylation. The amount of cyclin B associated p34cdc2 in Met2 was greater than that in Metl and most of them were stored as pre-MPF. These results suggest that the regulation of porcine oocyte maturation is essentially similar with other reported species but have some modifications specific for porcine oocytes.

Maintenance of Meiotic Arrest by a Phosphorylated p34cdc2 is Independent of Cyclic Adenosine 3′,5′-Monophosphate

The meiotic division in oocytes is arrested in the G2 phase of the cell cycle. Resumption of meiosis, also known as oocyte maturation, entails a G2 to M transition and is associated with a drop in intraoocyte concentrations of cAMP. Recent studies imply that tyrosine dephosphorylation of p34cdc2 is a prerequisite for entry into the M-phase of the cell cycle. Our study was designed to test the involvement of protein tyrosine phosphatase (PTPase)-regulated dephosphorylation of p34cdc2 in resumption of meiosis in rat oocytes and to explore the possible control of this event by the intraoocyte concentrations of cAMP. Isolated rat oocytes undergoing meiotic maturation spontaneously in vitro served as our experimental model. We found that sodium metavanadate, an inhibitor of PTPase, reversibly blocked the spontaneous maturation in vitro of rat oocytes (ED50 = 0.26 mM). We further demonstrated that the vanadate-sensitive event is completed by 2 h after reinitiation of meiosis. Immunoblot analysis using specific antiphosphotyrosine antibodies revealed that vanadate caused accumulation of phosphotyrosine on a 34-kDa protein, also recognized by anti-p34cdc2 antibodies. The phosphorylated form of p34cdc2 was also detected in oocytes arrested in the G2 phase by the phosphodiesterase inhibitor isobutyl methylxanthine (IBMX). Intraoocyte concentrations of cAMP in vanadate-inhibited oocytes were similar to those in oocytes that resumed meiosis spontaneously in vitro and lower than those in oocytes maintained in meiotic arrest by IBMX (0.73 +/- 0.08, 0.84 +/- 0.09, and 1.42 +/- 0.3 fmol/oocyte, respectively) [corrected]. We conclude that a PTPase that regulates the phosphorylation state of p34cdc2 participates in the control of meiosis in rat oocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Activation of p34 cdc2 kinase by cyclin is negatively regulated by cyclic Amp-dependent protein kinase in Xenopus oocytes

Microinjection of a bacterially expressed stable Δ90 sea urchin cyclin B into Xenopus prophase oocytes, in absence or presence of cycloheximide, provokes the activation of histone H1 kinase and the tyrosine dephosphorylation of p34 cdc2 Unexpectedly, when prophase oocytes are submitted to a treatment known to elevate the intracellular cAMP level (3-isobutyl-1-methylxanthine and cholera toxin), Δ90 cyclin has no effect and the oocytes remain blocked in prophase. This inhibition is reverted by the microinjection of the inhibitor of cAMP-dependent protein kinase. When Δ90 cyclin is microinjected into oocytes depleted of endogenous cyclins (cycloheximide-treated metaphase I) and in the presence of a high intracellular concentration of cAMP, p34 cdc2 kinase is tyrosine rephosphorylated. Altogether, our results indicate that in Xenopus oocyte, cAMP-dependent protein kinase (A-kinase) controls the formation of the cyclin B/p34 cdc2 complex which remains inactive and tyrosine phosphorylated.

MPF is activated in growing immature Xenopus oocytes in the absence of detectable tyrosine dephosphorylation of p34 cdc2

Tyrosine-phosphorylated p34 cdc2 and cyclin B2 are present and physically associated in small growing stage IV oocytes (800 μm in diameter) of Xenopus laevis. Microinjection of M-phase promoting factor (MPF) into stage IV oocytes induces germinal vesicle breakdown and the activation of the kinase activity of the p34 cdc2 /cyclin B2 complex measured on p13 suc1 beads. During the in vivo activation of MPF in stage IV oocytes, p34 cdc2 tyrosine dephosphorylation is not detectable, in contrast to stage VI oocytes. Addition of cycloheximide in MPF-injected stage IV oocytes induces neither the inhibition of histone H1 kinase activity nor the cyclin B2 degradation. Therefore, the activation mechanism of histone H1 kinase in stage IV oocytes does not require detectable tyrosine dephosphorylation of p34 cdc2 . It is suggested rather that the tyrosine phosphorylation of p34 cdc2 plays a role in inhibiting cyclin B2 degradation.

Antibodies to phosphotyrosine injected in Xenopus laevis oocytes modulate maturation induced by insulin/IGF-I

Xenopus oocytes carry IGF-I receptors, and undergo meiotic maturation in response to binding of IGF-I or insulin to the IGF-I receptor. Maturation is initiated upon activation of the IGF-I receptor tyrosine kinase and requires tyrosine dephosphorylation of p34 cdc2, the kinase component of maturation promoting factor (MPF). To further evaluate the role of tyrosine phosphorylation in the signalling pathway triggered by insulin/IGF-I, we have injected antibodies to phosphotyrosine into oocytes and examined their effects on oocyte maturation. Antibodies at a low concentration (40 ng/ oocyte, corresponding to a concentration of 40 μg/ml), enhanced specifically insulin-, but not progesterone-induced maturation. In contrast, at 150 ng/oocyte, the same antibodies decreased maturation induced by insulin, progesterone, or microinjected MPF. In cell-free systems, antibodies to phosphotyrosine recognized the oocyte IGF-I receptor and modulated its ligand-induced tyrosine kinase activity in a biphasic manner, with a stimulation at 40 μg/ml and an inhibition at higher concentrations. Moreover, antibodies at 150 ng/oocyte neutralized the kinase activity of a crude MPF extract. This neutralization was not accompanied by a rephosphorylation of p34 cdc2, but by a decrease in tyrosine phosphorylation of a 60-kDa protein, which was present in M phase extracts and undetectable in G2-arrested oocytes. Taken together, these results point to at least two levels of anti-phosphotyrosine antibody action: (i) the IGF-I receptor signalling system, and (ii) a regulatory step of MPF activation, which might be distinct of the well-documented inactivating phosphorylation of p34 cdc2.

Erbstatin and tyrphostins block protein-serine kinase activation and meiotic maturation of sea star oocytes

The effects of ten putative protein-tyrosine kinase inhibitors on the activation of protein-serine kinase and germinal vesicle breakdown (GVBD) in maturing sea star oocytes were investigated. Erbstatin and tyrphostins such as AG18 and AG125 blocked 1-methyladenine-induced GVBD in sea star oocytes with IC 50 values of less than 20 μM. Inhibition of the rate of GBVD was achieved even when these compounds were added up to 15 min after exposure of the oocytes to 1-methyladenine. The action of these substances on oocyte maturation was reversed by subsequent washing and culturing of the cells in natural sea water free of the inhibitors. Cell viability was maintained for at least 12 h in their presence, as assessed by Trypan blue dye exclusion. These inhibitors prevented the 1-methyladenine-induced activations of the histone H1 kinase p34 cdc2, the myelin basic protein kinase p44 mpk and a ribosomal S6 peptide kinase. Erbstatin, AG18 and AG125 prevented 1-methyladenine-induced tyrosine dephosphorylation of p34 cdc2, and they inhibited tyrosine phosphorylation of p44 mpk. These studies imply that activation of a protein-tyrosine kinase may be necessary for stimulation of p34 cdc2 in maturing sea star oocytes.