ELIMINATION OF CDC2 PHOSPHORYLATION SITES IN THE CDC25 PHOSPHATASE BLOCKS INITIATION OF M-PHASE

The cdc25 phosphatase is a mitotic inducer that activates p34(cdc2) at the G2/M transition by dephosphorylation of Tyr15 in p34(cdc2). cdc25 itself is also regulated through periodic changes in its phosphorylation state. To elucidate the mechanism for induction of mitosis, phosphorylation of cdc25 has been investigated using recombinant proteins. cdc25 is phosphorylated by both cyclin A/p34(cdc2) and cyclin B/p34(cdc2) at similar sets of multiple sites in vitro. This phosphorylation retards its electrophoretical mobility and activates its ability to increase cyclin B/p34(cdc2) kinase activity three- to fourfold in vitro, as found for endogenous Xenopus cdc25 in M-phase extracts. The threonine and serine residues followed by proline that are conserved between Xenopus and human cdc25 have been mutated. Both the triple mutation of Thr48, Thr67, and Thr138 and the quintuple mutation of these three threonine residues plus Ser205 and Ser285, almost completely abolish the shift in electrophoretic mobility of cdc25 after incubation with M-phase extracts or phosphorylation by p34(cdc2). These mutations inhibit the activation of cdc25 by phosphorylation with p34 by 70 and 90%, respectively. At physiological concentrations these mutants cannot activate cyclin B/p34(cdc2) in cdc25-immunodepleted oocyte extracts, suggesting that a positive feedback loop between cdc2 and cdc25 is necessary for the full activation of cyclin B/p34(cdc2) that induces abrupt entry into mitosis in vivo.