Ectopic Expression of Cdc25A Accelerates the G₁/S Transition and Leads to Premature Activation of Cyclin E- and Cyclin A-Dependent Kinases

Abstract

Human Cdc25 phosphatases play important roles in cell cycle regulation by removing inhibitory phosphates from tyrosine and threonine residues of cyclin-dependent kinases. Three human Cdc25 isoforms, A, B, and C, have been discovered. Cdc25B and Cdc25C play crucial roles at the G₂/M transition. In the present study, we have investigated the function of human Cdc25A phosphatase. Cell lines that express human Cdc25A in an inducible manner have been generated. Ectopic expression of Cdc25A accelerates the G₁/S-phase transition, indicating that Cdc25A controls an event(s) that is rate limiting for entry into S phase. Furthermore, we carried out a detailed analysis of the expression and activation of human Cdc25A. Activation of endogenous Cdc25A occurs during late G₁ phase and increases in S and G₂ phases. We further demonstrate that Cdc25A is activated at the same time as cyclin E- and cyclin A-dependent kinases. In vitro, Cdc25A dephosphorylates and activates the cyclin-Cdk complexes that are active during G₁. Overexpression of Cdc25A in the inducible system, however, leads to a premature activation of both cyclin E-Cdk2 and cyclin A-Cdk2 complexes, while no effect of cyclin D-dependent kinases is observed. Furthermore, Cdc25A overexpression induces a tyrosine dephosphorylation of Cdk2. These results suggest that Cdc25A is an important regulator of the G₁/S-phase transition and that cyclin E- and cyclin A-dependent kinases act as direct targets.

ACKNOWLEDGMENTS

We are grateful to Harald zur Hausen for his support, to Manfred Gossen and Herrman Bujard for pUHD 10-3, to Dalia Resnitzky and Steve Reed for providing the R12 cell line, and to Carol Murphy for the hygromycin resistance vector. We thank Tareg Bashir for technical advice and many discussions. Christiane Lammer is thanked for excellent technical assistance. We thank Martin Scheffner and the members of our lab for critically reading the manuscript.

This work was supported by a grant from the Deutsche Forschungsgemeinschaft (Ho1299/3-3).