Intra-M phase-promoting factor phosphorylation of cyclin B at the prophase/metaphase transition

Activation of Cdc2-cyclin B (or M phase-promoting factor (MPF)) at the prop hase/metaphase transition proceeds in two steps: dephosphorylation of Cdc2 and phosphorylation of cyclin B, We here investigated the regulation of cyc lin B phosphorylation using the starfish oocyte model. Cyclin B phosphoryla tion is not required for Cdc2 kinase activity; both the prophase complex de phosphorylated on Cdc2 with Cdc25 and the metaphase complex dephosphorylate d on cyclin B with protein phosphatase 2A display high kinase activities. A n in vitro assay of cyclin B kinase activity closely mimics in vivo phospho rylation as shown by phosphopeptide maps of in vivo and in vitro phosphoryl ated cyclin B. We demonstrate that Cdc2 itself is the cyclin B kinase; cycl in B phosphorylation requires Cdc2 activity both in vivo (sensitivity to vi tamin K-3, a Cdc25 inhibitor) and in vitro (copurification with Cdc2-cyclin B, requirement of Cdc2 dephosphorylation, and sensitivity to chemical inhi bitors of cyclin-dependent kinases), Furthermore, cyclin B phosphorylation occurs as an intra-M phase-promoting factor reaction as shown by the follow ing: 1) active Cdc2 is unable to phosphorylate cyclin B associated to phosp horylated Cdc2, and 2) cyclin B phosphorylation is insensitive to enzyme/su bstrate dilution. We conclude that, at the prophase/metaphase transition, c yclin B is mostly phosphorylated by its own associated Cdc2 subunit.