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© The Rockefeller University Press, 0021-9525/1997//1105 $5.00
The Journal of Cell Biology, Volume 138, Number 5, , 1997 1105-1116

Article

A Dual-Specificity Phosphatase Cdc25B Is an Unstable Protein and Triggers p34cdc2/Cyclin B Activation in Hamster BHK21 Cells Arrested with Hydroxyurea



Hitoshi Nishijima, Hideo Nishitani, Takashi Seki, and Takeharu Nishimoto

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.

Abbreviations used in this paper: CAK, CDK-activating kinase; CDK, cyclin-dependent kinase; HU, hydroxyurea; PCC, premature chromatin condensation; ts, temperature sensitive.

Please address all correspondence to Dr. Takeharu Nishimoto, Department of Molecular Biology, Graduate School of Medical Science, Kyushu University, Higashi-ku, 3-1-1, Maidashi, Fukuoka 812-82, Japan. Tel.: (81) 92-642-6175; Fax: (81) 92-642-61831; E-mail: tnishi{at}mailserver.med.kyushu-u.ac.jp

Hideo Nishitani's present address is Imperial Cancer Research Fund, Lincoln's Inn Fields, London WC2A 3PX, UK.

Takashi Seki's present address is Imperial Cancer Research Fund, Clare Hall Laboratories, South Mimms, Potters Bar, Hers EN6 3LD, UK.


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