p21ras-induced meiotic maturation of Xenopus oocytes in the absence of protein synthesis: MPF activation is preceded by activation of MAP and S6 kinases.
Microinjection of transforming p21ras into Xenopus oocytes caused a time-dependent increase in the level of total cell protein phosphorylation that culminated with germinal vesicle breakdown (GVBD). The same pattern of phosphorylation was observed in oocytes matured by either progesterone or insulin. Treatment with cycloheximide (CHX) completely blocked both GVBD and the associated de novo phosphorylations induced by the hormones, but did not abolish p21ras-induced maturation nor the occurrence of associated maturation promoting factor (MPF)-dependent and -independent phosphorylations. Thus, induction of GVBD by p21ras in the absence of protein synthesis correlated with the activation of cytosolic MPF-associated kinase activity similar in specificity on exogenous (histone H1) and endogenous (47 kDa and a 42 kDa proteins) substrates to the MPF activity of hormonally-matured oocytes. The injection of p21ras in the presence of CHX caused also activation of other kinase(s) proceeding MPF activation which were responsible for the phosphorylation of endogenous substrates including a 41 kDa protein and a 92 kDa protein kinase that comigrated, respectively, with bands recognized specifically by antibodies to MAP2 kinase and S6 kinase. The phosphorylation of those bands correlated also with the activation of cytosolic kinases acting specifically on myelin basic protein (MBP) and a S6-derived peptide as substrates. These results indicate that, in the absence of protein synthesis, p21ras is able to activate phosphorylation events leading to GVBD and suggest that this oncoprotein can participate in at least two separate pathways of MPF activation. We propose that the activation of MAP/MBP kinases and S6 kinases is an early effect of p21ras oncoproteins.
Laboratory of Cellular and Molecular Biology, NCI, National Institutes of Health, Bethesda, Maryland 20892.
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Nebreda
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Porras
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Santos
D000818:Animals; D003513:Cycloheximide / Q000494:pharmacology; D004789:Enzyme Activation; D005260:Female; D016200:Maturation-Promoting Factor / Q000378:metabolism*; D008540:Meiosis / Q000187:drug effects*; D008845:Microinjections; D019950:Mitogen-Activated Protein Kinase 1; D009865:Oocytes / Q000502:physiology; D010766:Phosphorylation; D006656:Protamine Kinase / Q000032:analysis; D014176:Protein Biosynthesis; D017346:Protein Serine-Threonine Kinases / Q000032:analysis*; D011505:Protein-Tyrosine Kinases / Q000032:analysis*; D016283:Proto-Oncogene Proteins p21(ras) / Q000494:pharmacology*; D019893:Ribosomal Protein S6 Kinases; D014982:Xenopus laevis
D003513:Cycloheximide; D011505:Protein-Tyrosine Kinases; D006656:Protamine Kinase; D017346:Protein Serine-Threonine Kinases; D019893:Ribosomal Protein S6 Kinases; D016200:Maturation-Promoting Factor; D019950:Mitogen-Activated Protein Kinase 1; D016283:Proto-Oncogene Proteins p21(ras)