Abstract
By incubating at 30 degrees 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 degrees 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 degrees 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 Cdc25C.
Highlights
By incubating at 30ЊC in the presence of an energy source, p34cdc2/cyclin B was activated in the extract prepared from a temperature-sensitive mutant, tsBN2, which prematurely enters mitosis at 40ЊC, the nonpermissive temperature
The activation of p34cdc2/cyclin B occurs in the extract prepared from S phase-arrested hamster BHK21 cells without protein synthesis, but not in the extract of cells pretreated with protein synthesis inhibitor cycloheximide, which inhibits the in vivo activation of p34cdc2/cyclin B as well
It can be deduced that S phasearrested hamster BHK21 cells possess all the molecules required for the observed activation of p34cdc2/cyclin B and that either some molecules or protein modifications that are required for the activation of p34cdc2/cyclin B were lost by blocking protein synthesis
Summary
By incubating at 30ЊC in the presence of an energy source, p34cdc2/cyclin B was activated in the extract prepared from a temperature-sensitive mutant, tsBN2, which prematurely enters mitosis at 40ЊC, the nonpermissive temperature When tsBN2 cells arrested in S phase were incubated at 40ЊC in the presence of cycloheximide, Cdc25B, but not Cdc25A and C, among a family of dual-specificity phosphatases, Cdc, was lost coincidentally with the lack of the activation of p34cdc2/cyclin B. P34cdc is phosphorylated on Thr-14 and Tyr-15 to prevent the premature activation of p34cdc2/cyclin B (for review see by Dunphy, 1994). During S phase, the activity of Thr-14 and Tyr-15 kinase is held high, and the activity of Cdc is low. When DNA replication is completed, the balance between Thr-14 and Tyr-15 kinase and Cdc phosphatase is switched to activate p34cdc2/cyclin B. Cdc25C, which is phosphorylated and thereby activated by p34cdc2/cyclin B, induces the full activation of p34cdc by forming a positive feedback mutual activation loop
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