Hyperphosphorylation of the N-terminal Domain of Cdc25 Regulates Activity toward Cyclin B1/Cdc2 But Not Cyclin A/Cdk2

doi:10.1074/jbc.272.45.28607

Hyperphosphorylation of the N-terminal Domain of Cdc25 Regulates Activity toward Cyclin B1/Cdc2 But Not Cyclin A/Cdk2*

From the Queensland Cancer Fund Research Laboratories and Joint Oncology Program, Queensland Institute of Medical Research, P. O. Royal Brisbane Hospital, Queensland, Australia 4029

Abstract

Cdc25 regulates entry into mitosis by regulating the activation of cyclin B/cdc2. In humans, at least two cdc25 isoforms have roles in controlling the G₂/M transition. Here we show, using bacterially expressed recombinant proteins, that two cdc25B splice variants, cdc25B2 and cdc25B3, are capable of activating cyclin A/cdk2 and cyclin B/cdc2, but that mitotic hyperphosphorylation of these proteins increases their activity toward only cyclin B1/cdc2. Cdc25C has only very low activity in its unphosphorylated form, and following hyperphosphorylation it will efficiently catalyze the activation of only cyclin B/cdc2. This was reflected by the in vivo activity of the immunoprecipitated cdc25B and cdc25C from interphase and mitotic HeLa cells. The increased activity of the hyperphosphorylated cdc25s toward cyclin B1/cdc2 was in large part due to increased binding of this substrate. The substrate specificity, activities, and timing of the hyperphosphorylation of cdc25B and cdc25C during G₂ and M suggest that these two mitotic cdc25 isoforms are activated by different kinases and perform different functions during progression through G₂ into mitosis.

Footnotes

↵* This work was supported in part by the National Health and Medical Research Council of Australia and the Queensland Cancer Fund.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵‡ Supported by the Fiuczek Trust. To whom correspondence should be addressed. Tel.: 61-7-3362-0304; Fax: 61-7-3362-0107; E-mail:brianG{at}qimr.edu.au.
↵1 The abbreviations used are: cdk, cyclin-dependent kinase; GST, glutathioneS-transferase; DTT, dithiothreitol; PAGE, polyacrylamide gel electrophoresis.
↵2 A. K. McCormack, C. P. C. De Souza, I. D. Tonks, J. M. Clark, N. K. Hayward, K. A. O. Ellem and B. G. Gabrielli, submitted for publication.
- Received June 10, 1997.
- Revision received August 11, 1997.