Requirement for phosphorylation of cyclin B1 for Xenopus oocyte maturation.

Abstract

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 Bi and B2, both of which are phosphorylated.In this study, we have investigated the biological significance of this phosphorylation for Xenopus cyclin Bi during meiotic maturation. We have used a combination of sitedirected 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 Bi 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 Bi 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 Bi is not required for cdc2 kinase activity, for binding to cdc2 protein, for stability of cyclin Bi before GVBD, or for destruction of cyclin Bi 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 Bi. These data confirm that phosphorylation, as mimicked by Ser->Glu mutations, confers enhanced biological activity to cyclin Bi. Possible roles of cyclin Bi phosphorylation are discussed that might account for the increased biological activity of the quintuple Glu mutant.

ΔMEKK3:ER* activation induces a p38α/β2-dependent cell cycle arrest at the G2 checkpoint

“…In the absence of 4-HT, cyclin B1 accumulated as expected when the cells passed through G 2 and into mitosis ( Figure 6a). Furthermore, a slower migrating form of cyclin B1 was observed after 15 h ( Figure 6a) and most likely reflects cyclin B1 phosphorylation (Li et al, 1995;Hagting et al, 1999). However, activation of DMEKK3:ER* when cells were released from the aphidicolin block prevented the accumulation of cyclin B1 protein which was normally observed after 10 -15 h. Indeed, cyclin B1 levels actually declined to below control levels, and were nearly undetectable 25 h To examine the impact of loss of cyclin A and B on the kinase activities of CDK2 and CDK1, the active complexes were immunoprecipitated using antibodies against cyclin A and cyclin B, and their ability to phosphorylate histone H1 assayed.…”

Constitutive activation of the shh–ptc1 pathway by a patched1 mutation identified in BCC

“…These nuclear-targeted cyclin B1 derivatives contain the five Ser phosphorylation sites in the human CRS domain (S116, S126, S128, S133 and S147) mutated to Ala or Glu, to mimic phosphorylation states of cyclin B1 (Hagting et al, 1999). Xenopus cyclin B1 contains four regulatory phosphoserine sites within the CRS (Li et al, 1995). The expression of NLS-B1 hs-E enhances Gli1 activity to about sixfold over mocktransfected cells (Figure 5b).…”

“…In addition, cyclin B1 is regulated by phosphorylation during G2 phase. Specifically, the cytoplasmic retention signal (CRS) domain of cyclin B1 is phosphorylated on four Ser residues, resulting in nuclear localization of MPF and access to critical nuclear substrates for G2-M progression (Li et al, 1995(Li et al, , 1997Borgne et al, 1999;Hagting et al, 1999).…”

On cyclins, oocytes, and eggs

“…By mutagenesis of putative cdc2 kinase and MAP kinase phosphorylation sites in Xenopus, cyclins B1 (Ser-94 and Ser-96) and B2 (Ser-90); Izumi and Maller, (1991) showed that phosphorylation of Xenopus cyclins B1 and B2 is not required for cyclin degradation, induction of mitosis, or induction of oocyte maturation. Intriguingly, another group showed by mutagenesis of Ser-2, 94, 96, 101, and 113 of cyclin B1 that, although cyclin B1 phosphorylation 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, it is required for Xenopus oocyte maturation (Li et al, 1995). Cyclin B1 phosphorylation regulates the biological activity of the Fig.…”