title: The Chk1 protein kinase and the Cdc25C regulatory pathways are targets of the anticancer agent UCN-01.
abstract: 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.
authors.affiliation: Department of Cell Biology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
authors.forename: P R
authors.initials: PR
authors.lastname: Graves
authors.forename: L
authors.initials: L
authors.lastname: Yu
authors.forename: J K
authors.initials: JK
authors.lastname: Schwarz
authors.forename: J
authors.initials: J
authors.lastname: Gales
authors.forename: E A
authors.initials: EA
authors.lastname: Sausville
authors.forename: P M
authors.initials: PM
authors.lastname: O'Connor
authors.forename: H
authors.initials: H
authors.lastname: Piwnica-Worms
mesh_terms: D000470:Alkaloids / Q000494:pharmacology*; D000970:Antineoplastic Agents / Q000494:pharmacology*; D018797:Cell Cycle Proteins / Q000037:antagonists & inhibitors*; D000071877:Checkpoint Kinase 1; D064447:Checkpoint Kinase 2; D003001:Cloning, Molecular; D004249:DNA Damage; D004307:Dose-Response Relationship, Radiation; D016195:G2 Phase / Q000187:drug effects; D006367:HeLa Cells; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism; D008954:Models, Biological; D010766:Phosphorylation / Q000187:drug effects / Q000528:radiation effects; D010957:Plasmids; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D012694:Serine / Q000378:metabolism; D019311:Staurosporine / Q000031:analogs & derivatives; D013997:Time Factors; D020687:cdc25 Phosphatases / Q000037:antagonists & inhibitors*
chemical_list: D000470:Alkaloids; D000970:Antineoplastic Agents; D018797:Cell Cycle Proteins; D008565:Membrane Proteins; D047428:Protein Kinase Inhibitors; D012694:Serine; C054852:7-hydroxystaurosporine; D011494:Protein Kinases; D064447:Checkpoint Kinase 2; D011505:Protein-Tyrosine Kinases; C000605867:CHEK1 protein, human; C578634:CHEK2 protein, human; D000071877:Checkpoint Kinase 1; C490679:PKMYT1 protein, human; D017346:Protein Serine-Threonine Kinases; C517009:CDC25C protein, human; D020687:cdc25 Phosphatases; D019311:Staurosporine