Chk2/hCds1 functions as a DNA damage checkpoint in G1 by stabilizing p53

  1. Nabil H. Chehab1,2,
  2. Asra Malikzay1,
  3. Michael Appel1, and
  4. Thanos D. Halazonetis1,3,4
  1. 1Department of Molecular Genetics, The Wistar Institute, Philadelphia, Pennsylvania 19104 USA; 2Graduate Program in Biochemistry and, 3Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104 USA

Abstract

Chk2/hcds1, the human homolog of theSaccharomyces cerevisiae RAD53/SPK1 andSchizosaccharomyces pombe cds1 DNA damage checkpoint genes, encodes a protein kinase that is post-translationally modified after DNA damage. Like its yeast homologs, the Chk2/hCds1 protein phosphorylates Cdc25C in vitro, suggesting that it arrests cells in G2 in response to DNA damage. We expressed Chk2/hCds1 in human cells and analyzed their cell cycle profile. Wild-type, but not catalytically inactive, Chk2/hCds1 led to G1 arrest after DNA damage. The arrest was inhibited by cotransfection of a dominant-negative p53 mutant, indicating that Chk2/hCds1 acted upstream of p53. In vitro, Chk2/hCds1 phosphorylated p53 on Ser-20 and dissociated preformed complexes of p53 with Mdm2, a protein that targets p53 for degradation. In vivo, ectopic expression of wild-type Chk2/hCds1 led to increased p53 stabilization after DNA damage, whereas expression of a dominant-negative Chk2/hCds1 mutant abrogated both phosphorylation of p53 on Ser-20 and p53 stabilization. Thus, in response to DNA damage, Chk2/hCds1 stabilizes the p53 tumor suppressor protein leading to cell cycle arrest in G1.

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Footnotes

  • 4 Corresponding author.

  • E-MAIL halazonetis{at}wistar.upenn.edu; FAX (215) 898-3929.

    • Received November 16, 1999.
    • Accepted December 22, 1999.
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