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Department of Molecular Biology, Graduate School of Medical Science, Kyushu University, Fukuoka 812-82, Japan
By incubating at 30°C 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 40°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 p34cdc2/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 p34cdc2/cyclin B activation.
When tsBN2 cells arrested in S phase were incubated at
40°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 p34cdc2/cyclin B. Consistently, the immunodepletion of Cdc25B from the extract inhibited
the activation of p34cdc2/cyclin B. Cdc25B was found to
be unstable (half-life < 30 min). Cdc25B, but not
Cdc25C, immunoprecipitated from the extract directly activated the p34cdc2/cyclin B of cycloheximide-treated
cells as well as that of nontreated cells, although
Cdc25C immunoprecipitated from the extract of mitotic cells activated the p34cdc2/cyclin B within the extract of cycloheximide-treated cells. Our data suggest
that Cdc25B made an initial activation of p34cdc2/cyclin
B, which initiates mitosis through the activation of
Cdc25C.
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