A dual-specificity phosphatase Cdc25B is an unstable protein and triggers p34(cdc2)/cyclin B activation in hamster BHK21 cells arrested with hydroxyurea.
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.
Nishijima
H
H
Department of Molecular Biology, Graduate School of Medical Science, Kyushu University, Fukuoka 812-82, Japan.
Nishitani
H
H
Seki
T
T
Nishimoto
T
T
D000818:Animals; D016203:CDC2 Protein Kinase / Q000378:metabolism*; D002453:Cell Cycle / Q000187:drug effects / Q000502:physiology*; D018797:Cell Cycle Proteins / Q000378:metabolism*; D002460:Cell Line; D002474:Cell-Free System; D003001:Cloning, Molecular; D006224:Cricetinae; D016213:Cyclins / Q000378:metabolism*; D003513:Cycloheximide / Q000494:pharmacology; D004789:Enzyme Activation; D006918:Hydroxyurea / Q000494:pharmacology*; D007668:Kidney; D007700:Kinetics; D008938:Mitosis; D010749:Phosphoprotein Phosphatases / Q000378:metabolism*; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism; D017027:Protein Tyrosine Phosphatases / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D016196:S Phase; D015398:Signal Transduction; D020687:cdc25 Phosphatases
D018797:Cell Cycle Proteins; D016213:Cyclins; D011994:Recombinant Proteins; D003513:Cycloheximide; D011494:Protein Kinases; C102333:histone H1 kinase; D016203:CDC2 Protein Kinase; D010749:Phosphoprotein Phosphatases; D017027:Protein Tyrosine Phosphatases; D020687:cdc25 Phosphatases; D006918:Hydroxyurea