Requirement for phosphorylation of cyclin B1 for Xenopus oocyte maturation.
Maturation-promoting factor, consisting of cdc2 protein kinase and a regulatory B-type cyclin, is a universal regulator of meiosis and mitosis in eukaryotes. In Xenopus, there are two subtypes of B-type cyclins, designated B1 and B2, both of which are phosphorylated. In this study, we have investigated the biological significance of this phosphorylation for Xenopus cyclin B1 during meiotic maturation. We have used a combination of site-directed mutagenesis and phosphopeptide-mapping to identify serine residues 2, 94, 96, 101, and 113 as presumptive phosphorylation sites, and together these sites account for all cyclin B1 phosphorylation in oocytes before germinal vesicle breakdown (GVBD). Single Ser-->Ala mutants as well as multiple site mutants have been constructed and characterized. Phosphorylation of cyclin B1 appears to be required for Xenopus oocyte maturation, based on the significantly diminished ability of the quintuple Ala mutant to induce oocyte maturation. Furthermore, partial phosphorylation of these five sites is sufficient to meet this requirement. Phosphorylation of cyclin B1 is not required for cdc2 kinase activity, for binding to cdc2 protein, for stability of cyclin B1 before GVBD, or for destruction of cyclin B1 after GVBD or after egg activation. A quintuple Glu mutant was also constructed, with serine residues 2, 94, 96, 101, and 113 mutated to Glu. In contrast to the quintuple Ala mutant, the quintuple Glu mutant was able to induce oocyte maturation efficiently, and with more rapid kinetics than wild-type cyclin B1. These data confirm that phosphorylation, as mimicked by Ser-->Glu mutations, confers enhanced biological activity to cyclin B1. Possible roles of cyclin B1 phosphorylation are discussed that might account for the increased biological activity of the quintuple Glu mutant.
Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla 92093-0322, USA.
J
J
Li
A N
AN
Meyer
D J
DJ
Donoghue
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D016203:CDC2 Protein Kinase / Q000378:metabolism; D019926:Cyclin B; D056744:Cyclin B1; D016213:Cyclins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000939:Epitopes / Q000378:metabolism; D005260:Female; D008540:Meiosis; D008845:Microinjections; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009866:Oogenesis; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D012333:RNA, Messenger / Q000235:genetics; D014982:Xenopus laevis / Q000502:physiology*
D019926:Cyclin B; D056744:Cyclin B1; D016213:Cyclins; D000939:Epitopes; D012333:RNA, Messenger; D016203:CDC2 Protein Kinase