/Cyclin B Activation in Hamster BHK21 Cells Arrested with Hydroxyurea

By incubating at 30 8 C in the presence of an energy source, p34 cdc2 /cyclin B was activated in the extract prepared from a temperature-sensitive mutant, tsBN2, which prematurely enters mitosis at 40 8 C, the nonpermissive temperature (Nishimoto, T., E. Eilen, and C. Basilico. 1978. Cell . 15:475–483), and wild-type cells of the hamster BHK21 cell line arrested in S phase, without protein synthesis. Such an in vitro activation of p34 cdc2 /cyclin B, however, did not occur in the extract prepared from cells pretreated with protein synthesis inhibitor cycloheximide, although this extract still retained the ability to inhibit p34 cdc2 /cyclin B activation. When tsBN2 cells arrested in S phase were incubated at 40 8 C in the presence of cycloheximide, Cdc25B, but not Cdc25A and C, among a family of dual-specificity phosphatases, Cdc25, was lost coincidentally with the lack of the activation of p34 cdc2 /cyclin B. Consistently, the immunodepletion of Cdc25B from the extract inhibited the activation of p34 cdc2 /cyclin B. Cdc25B was found to be unstable (half-life , 30 min). Cdc25B, but not Cdc25C, immunoprecipitated from the extract directly activated the p34 cdc2 /cyclin B of cycloheximide-treated cells as well as that of nontreated cells, although Cdc25C immunoprecipitated from the extract of mitotic cells activated the p34 cdc2 /cyclin B within the extract of cycloheximide-treated cells. Our data suggest that Cdc25B made an initial activation of p34 cdc2 /cyclin B, which initiates mitosis through the activation of