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Molecular and Cellular Biology, April 2001, p. 2755-2766, Vol. 21, No. 8
0270-7306/01/$04.00+0   DOI: 10.1128/MCB.21.8.2755-2766.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

S and G2 Phase Roles for Cdk2 Revealed by Inducible Expression of a Dominant-Negative Mutant in Human Cells

Bing Hu,1,2,3 Jayashree Mitra,1,2,3 Sander van den Heuvel,4 and Greg H. Enders1,2,3,*

Departments of Medicine1 and Genetics2 and Cancer Center,3 University of Pennsylvania, Philadelphia, Pennsylvania, and Massachusetts General Hospital Cancer Center, Charlestown, Massachusetts4

Received 8 November 2000/Accepted 17 January 2001

Cyclin-dependent kinase 2 (Cdk2) is essential for initiation of DNA synthesis in higher eukaryotes. Biochemical studies in Xenopus egg extracts and microinjection studies in human cells have suggested an additional function for Cdk2 in activation of Cdk1 and entry into mitosis. To further examine the role of Cdk2 in human cells, we generated stable clones with inducible expression of wild-type and dominant-negative forms of the enzyme (Cdk2-wt and Cdk2-dn, respectively). Both exogenous proteins associated efficiently with endogenous cyclins. Cdk2-wt had no apparent effect on the cell division cycle, whereas Cdk2-dn inhibited progression through several distinct stages. Cdk2-dn induction could arrest cells at the G1/S transition, as previously observed in transient expression studies. However, under normal culture conditions, Cdk2-dn induction primarily arrested cells with S and G2/M DNA contents. Several observations suggested that the latter cells were in G2 phase, prior to the onset of mitosis: these cells contained uncondensed chromosomes, low levels of cyclin B-associated kinase activity, and high levels of tyrosine-phosphorylated Cdk1. Furthermore, Cdk2-dn did not delay progression through mitosis upon release of cells from a nocodazole block. Although the G2 arrest imposed by Cdk2-dn was similar to that imposed by the DNA damage checkpoint, the former was distinguished by its resistance to caffeine. These findings provide evidence for essential functions of Cdk2 during S and G2 phases of the mammalian cell cycle.

* Corresponding author. Mailing address: Penn/GI Division, 600 CRB, 415 Curie Blvd., Philadelphia, PA 19104-6144. Phone: (215) 898-0159. Fax: (215) 573-2024. E-mail address: endersgh{at}mail.med.upenn.edu.

Molecular and Cellular Biology, April 2001, p. 2755-2766, Vol. 21, No. 8
0270-7306/01/$04.00+0   DOI: 10.1128/MCB.21.8.2755-2766.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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