Sequential Dephosphorylation of p34cdc2 on Thr-14 and Tyr-15 at the Prophase/Metaphase Transition

doi:10.1074/jbc.271.44.27847

Sequential Dephosphorylation of p34^cdc2 on Thr-14 and Tyr-15 at the Prophase/Metaphase Transition*

Annie Borgne^‡ and
Laurent Meijer

From the Centre National de la Recherche Scientifique, Station Biologique, BP 74, 29682 Roscoff cedex, France

^‡To whom correspondence should be addressed. Tel.: (33) 98 29 23 23; Fax: (33) 98 29 23 42; E-mail: borgne{at}sb-roscoff.fr.

Abstract

The G₂-M transition of the cell cycle is triggered by the p34^cdc2/cyclin B kinase. During the prophase/metaphase transition, the inactive, Thr-14/Tyr-15 phosphorylated form of p34^cdc2 (T^P-Y^P) is modified to an active, Thr-14/Tyr-15 dephosphorylated form (T-Y) by the cdc25 dual-specificity phosphatase. Using highly synchronized starfish oocytes as a cellular model, we show that dephosphorylation in vivo and in vitro occurs in two steps: Thr-14 dephosphorylation precedes Tyr-15 dephosphorylation. The transient intermediate form (T-Y^P), which can be obtained in vitro by treatment of T^P-Y^P by protein phosphatase 2A, displays low but significant kinase activity. These results raise the possibility that the intermediate form T-Y^P may be involved in the autocatalytic amplification of the p34^cdc2/cyclin B complex through phosphorylation/activation of the cdc25 phosphatase and phosphorylation/inactivation of the wee1 kinase.

Footnotes

↵* This research was supported by Grant ARC 6268 from the Association pour la Recherche sur le Cancer (to L. M.) and grants from Groupement des Entreprises Françaises dans la Lutte contre le Cancer (to L. M.) and the Conseil Régional de Bretagne (to L. M.). 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.
↵¹ The abbreviations used are:

cdk

cyclin-dependent kinase

MPF

M phase-promoting factor

1-MeAde

1-methyladenine

MOPS

4-morpholinepropanesulfonic acid

PP2A

protein phosphatase 2A

GST

glutathione S-transferase

DTT

dithiothreitol

PAGE

polyacrylamide gel electrophoresis

T^P-Y^P

Thr-14 and Tyr-15 phosphorylated form of p34^cdc2

T^P-Y

Thr-14 phosphorylated, Tyr-15 dephosphorylated form of p34^cdc2

T-Y^P

Thr-14 dephosphorylated, Tyr-15 phosphorylated form of p34^cdc2

T-Y

Thr-14 and Tyr-15 dephosphorylated form of p34^cdc2.
↵²L. Meijer, unpublished results.
- Received March 28, 1996.
- Revision received August 2, 1996.