Requirement of mos^Xe Protein Kinase for Meiotic Maturation of Xenopus Oocytes Induced by a cdc2 Mutant Lacking Regulatory Phosphorylation Sites

Abstract

The p34^cdc2 protein kinase is a component of maturation-promoting factor, the master regulator of the cell cycle in all eukaryotes. The activity of p34^cdc2 is itself tightly regulated by phosphorylation and dephosphorylation. Predicted regulatory phosphorylation sites of Xenopus p34^cdc2 were mutated in vitro, and in vitro-transcribed RNAs were injected into Xenopus oocytes. The cdc2 single mutants Thr-14→Ala and Tyr-15→Phe did not induce germinal vesicle breakdown (GVBD) upon microinjection into oocytes. In contrast, the cdc2 double mutant Ala-14/Phe-15 did induce GVBD. Both the Ala-14 and Ala-14/Phe-15 p34^cdc2 mutants were shown to coimmunoprecipitate cyclin B1 and to phosphorylate histone H1 in immune complex kinase assays. Microinjection of antisense oligonucleotides to c-mos^Xe was used to demonstrate the role of mos protein synthesis in the induction of GVBD by the Ala-14/Phe-15 cdc2 mutant. Thr-161 was also mutated. p34^cdc2 single mutants Ala-161 and GIu-161 and triple mutants Ala-14/Phe-15/Ala-161 and Ala-14/Phe-15/Glu-161 failed to induce GVBD in oocytes and showed a decreased binding to cyclin Bl in coimmunoprecipitations. Each of the cdc2 mutants was also assayed by coinjection with cyclin B1 or c-mos^Xe RNA into oocytes. Several of the cdc2 mutants were found to affect the kinetics of cyclin Bl and/or mos-induced GVBD upon coinjection, although none affected the rate of progesterone-induced maturation. We demonstrate here the significance of Thr-14, Tyr-15, and Thr-161 of p34^cdc2 in Xenopus oocyte maturation. In addition, these results suggest a regulatory role for mos^Xe in induction of oocyte maturation by the cdc2 mutant Ala-14/Phe-15.