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 prophase/metaphase transition proceeds in two steps: dephosphorylation of Cdc2 and phosphorylation of cyclin B. We here investigated the regulation of cyclin B phosphorylation using the starfish oocyte model. Cyclin B phosphorylation is not required for Cdc2 kinase activity; both the prophase complex dephosphorylated on Cdc2 with Cdc25 and the metaphase complex dephosphorylated on cyclin B with protein phosphatase 2A display high kinase activities. An in vitro assay of cyclin B kinase activity closely mimics in vivo phosphorylation as shown by phosphopeptide maps of in vivo and in vitro phosphorylated cyclin B. We demonstrate that Cdc2 itself is the cyclin B kinase; cyclin B phosphorylation requires Cdc2 activity both in vivo (sensitivity to vitamin K3, a Cdc25 inhibitor) and in vitro (copurification with Cdc2-cyclin B, requirement of Cdc2 dephosphorylation, and sensitivity to chemical inhibitors of cyclin-dependent kinases). Furthermore, cyclin B phosphorylation occurs as an intra-M phase-promoting factor reaction as shown by the following: 1) active Cdc2 is unable to phosphorylate cyclin B associated to phosphorylated Cdc2, and 2) cyclin B phosphorylation is insensitive to enzyme/substrate dilution. We conclude that, at the prophase/metaphase transition, cyclin B is mostly phosphorylated by its own associated Cdc2 subunit.
Borgne
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A
Centre National de la Recherche Scientifique, Station Biologique, B. P. 74, 29682 Roscoff cedex, Bretagne, France.
Ostvold
A C
AC
Flament
S
S
Meijer
L
L
D000595:Amino Acid Sequence; D000818:Animals; D016203:CDC2 Protein Kinase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D019926:Cyclin B / Q000378:metabolism*; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D008677:Metaphase; D008969:Molecular Sequence Data; D009865:Oocytes / Q000166:cytology / Q000378:metabolism; D010449:Peptide Mapping; D010766:Phosphorylation; D011418:Prophase; D017386:Sequence Homology, Amino Acid; D013215:Starfish; D014982:Xenopus laevis
D019926:Cyclin B; D004791:Enzyme Inhibitors; D016203:CDC2 Protein Kinase