The Chk1 Protein Kinase and the Cdc25C Regulatory Pathways Are Targets of the Anticancer Agent UCN-01

doi:10.1074/jbc.275.8.5600

The Chk1 Protein Kinase and the Cdc25C Regulatory Pathways Are Targets of the Anticancer Agent UCN-01*

From the ^‡Department of Cell Biology and Physiology,^**Howard Hughes Medical Institute, Washington University School of Medicine, St. Louis, Missouri 63110, the ^§Laboratory of Molecular Pharmacology, Division of Basic Sciences, and the ^¶Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis, NCI, National Institutes of Health, Bethesda, Maryland 20892

Abstract

A checkpoint operating in the G₂ phase of the cell cycle prevents entry into mitosis in the presence of DNA damage. UCN-01, a protein kinase inhibitor currently undergoing clinical trials for cancer treatment, abrogates G₂ checkpoint function and sensitizes p53-defective cancer cells to DNA-damaging agents. In most species, the G₂ checkpoint prevents the Cdc25 phosphatase from removing inhibitory phosphate groups from the mitosis-promoting kinase Cdc2. This is accomplished by maintaining Cdc25 in a phosphorylated form that binds 14-3-3 proteins. The checkpoint kinases, Chk1 and Cds1, are proposed to regulate the interactions between human Cdc25C and 14-3-3 proteins by phosphorylating Cdc25C on serine 216. 14-3-3 proteins, in turn, function to keep Cdc25C out of the nucleus. Here we report that UCN-01 caused loss of both serine 216 phosphorylation and 14-3-3 binding to Cdc25C in DNA-damaged cells. In addition, UCN-01 potently inhibited the ability of Chk1 to phosphorylate Cdc25C in vitro. In contrast, Cds1 was refractory to inhibition by UCN-01in vitro, and Cds1 was still phosphorylated in irradiated cells treated with UCN-01. Thus, neither Cds1 nor kinases upstream of Cds1, such as ataxia telangiectasia-mutated, are targets of UCN-01 action in vivo. Taken together our results identify the Chk1 kinase and the Cdc25C pathway as potential targets of G₂ checkpoint abrogation by UCN-01.

Footnotes

↵* This work was supported in part by a grant from the American Heart Association, Heartland Affiliate (to P. R. G.), and the National Institutes of Health (to H. P. W.).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.
↵‖ Current address: Agouron Pharmaceuticals, Inc., 3565 General Atomics Ct., San Diego, CA 92121.
↵‡ Investigator of the Howard Hughes Medical Institute. To whom correspondence should be addressed: Dept. of Cell Biology and Physiology, Howard Hughes Medical Institute, Washington University School of Medicine, Box 8228, 660 South Euclid Ave., St. Louis, MO 63110.
↵2 P. R. Graves and H. Piwnica-Worms, unpublished observations.
Abbreviations:

NES

nuclear export signal

GST

glutathione S-transferase

PCR

polymerase chain reaction

ATM

ataxia telangiectasia-mutated

DTT

dithiothreitol

PBS

phosphate-buffered saline

RACE

rapid amplification of cDNA ends

GFP

green fluorescent protein

Gy

gray
- Received October 5, 1999.
The American Society for Biochemistry and Molecular Biology, Inc.