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

2000; Elsevier BV; Volume: 275; Issue: 8 Linguagem: Inglês

10.1074/jbc.275.8.5600

1083-351X

Paul R. Graves, Lijia Yu, Julie K. Schwarz, Janis Gales, Edward A. Sausville, Patrick M. O’Connor, Helen Piwnica‐Worms,

Ubiquitin and proteasome pathways

A checkpoint operating in the G 2 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 G 2 checkpoint function and sensitizes p53-defective cancer cells to DNA-damaging agents. In most species, the G 2 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-01 in 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 G 2 checkpoint abrogation by UCN-01.