S and G2 Phase Roles for Cdk2 Revealed by Inducible Expression of a Dominant-Negative Mutant in Human Cells
Open Access
- 1 April 2001
- journal article
- Published by Taylor & Francis Ltd in Molecular and Cellular Biology
- Vol. 21 (8), 2755-2766
- https://doi.org/10.1128/mcb.21.8.2755-2766.2001
Abstract
Cyclin-dependent kinase 2 (Cdk2) is essential for initiation of DNA synthesis in higher eukaryotes. Biochemical studies inXenopus 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 G2arrest 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.Keywords
This publication has 74 references indexed in Scilit:
- Activation of the DNA Replication Checkpoint Through RNA Synthesis by PrimaseScience, 2000
- Chfr defines a mitotic stress checkpoint that delays entry into metaphaseNature, 2000
- Centrosomal and Cytoplasmic Cdc2/Cyclin B1 Activation Precedes Nuclear Mitotic EventsExperimental Cell Research, 2000
- Requirement of the Prolyl Isomerase Pin1 for the Replication CheckpointScience, 2000
- pRB phosphorylation mutants reveal role of pRB in regulating S phase completion by a mechanism independent of E2FOncogene, 1998
- Cancer Cell CyclesScience, 1996
- Differential regulation of E2F transactivation by cyclin/cdk2 complexes.Journal of Bone and Joint Surgery, 1994
- Negative regulation of the growth-promoting transcription factor E2F-1 by a stably bound cyclin A-dependent protein kinaseCell, 1994
- Isolation of the human cdk2 gene that encodes the cyclin A- and adenovirus E1A-associated p33 kinaseNature, 1991
- Structure of a Peptide Inhibitor Bound to the Catalytic Subunit of Cyclic Adenosine Monophosphate-Dependent Protein KinaseScience, 1991