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 p34cdc2 kinase and the tyrosine dephosphorylation of p34cdc2 in the presence or absence of protein synthesis. The level of p34cdc2 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 p34cdc2, 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 p34cdc2/cyclin B complex through tyrosine phosphorylation of p34cdc2 during metaphase I.
Rime
H
H
Laboratoire de Physiologie de la Reproduction, INRA/URA CNRS 1449, Université Pierre-et-Marie-Curie, Paris.
Huchon
D
D
De Smedt
V
V
Thibier
C
C
Galaktionov
K
K
Jessus
C
C
Ozon
R
R
D000818:Animals; D016203:CDC2 Protein Kinase / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000008:administration & dosage* / Q000494:pharmacology*; D000242:Cyclic AMP / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D016213:Cyclins / Q000378:metabolism; D003513:Cycloheximide / Q000494:pharmacology; D005260:Female; D016200:Maturation-Promoting Factor / Q000378:metabolism*; D008540:Meiosis / Q000502:physiology*; D008677:Metaphase / Q000502:physiology*; D008845:Microinjections; D009865:Oocytes / Q000166:cytology*; D010749:Phosphoprotein Phosphatases / Q000008:administration & dosage* / Q000494:pharmacology* / Q000502:physiology*; D010766:Phosphorylation; D011418:Prophase / Q000187:drug effects; D011498:Protein Precursors / Q000378:metabolism; D011500:Protein Synthesis Inhibitors / Q000378:metabolism; D008941:Spindle Apparatus / Q000235:genetics; D013997:Time Factors; D014982:Xenopus laevis / Q000235:genetics*; D020687:cdc25 Phosphatases
D018797:Cell Cycle Proteins; D016213:Cyclins; D011498:Protein Precursors; D011500:Protein Synthesis Inhibitors; C067034:maturation-promoting factor precursor; D003513:Cycloheximide; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D016203:CDC2 Protein Kinase; D016200:Maturation-Promoting Factor; D010749:Phosphoprotein Phosphatases; D020687:cdc25 Phosphatases