The Chk1 protein kinase and the Cdc25C regulatory pathways are targets of the anticancer agent UCN-01

J Biol Chem. 2000 Feb 25;275(8):5600-5. doi: 10.1074/jbc.275.8.5600.

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Alkaloids / pharmacology*
  • Antineoplastic Agents / pharmacology*
  • Cell Cycle Proteins / antagonists & inhibitors*
  • Checkpoint Kinase 1
  • Checkpoint Kinase 2
  • Cloning, Molecular
  • DNA Damage
  • Dose-Response Relationship, Radiation
  • G2 Phase / drug effects
  • HeLa Cells
  • Humans
  • Membrane Proteins / metabolism
  • Models, Biological
  • Phosphorylation / drug effects
  • Phosphorylation / radiation effects
  • Plasmids
  • Protein Kinase Inhibitors*
  • Protein Kinases / metabolism
  • Protein-Serine-Threonine Kinases / antagonists & inhibitors
  • Protein-Serine-Threonine Kinases / metabolism
  • Protein-Tyrosine Kinases / antagonists & inhibitors
  • Protein-Tyrosine Kinases / metabolism
  • Serine / metabolism
  • Staurosporine / analogs & derivatives
  • Time Factors
  • cdc25 Phosphatases / antagonists & inhibitors*

Substances

  • Alkaloids
  • Antineoplastic Agents
  • Cell Cycle Proteins
  • Membrane Proteins
  • Protein Kinase Inhibitors
  • Serine
  • 7-hydroxystaurosporine
  • Protein Kinases
  • Checkpoint Kinase 2
  • Protein-Tyrosine Kinases
  • CHEK1 protein, human
  • CHEK2 protein, human
  • Checkpoint Kinase 1
  • PKMYT1 protein, human
  • Protein-Serine-Threonine Kinases
  • CDC25C protein, human
  • cdc25 Phosphatases
  • Staurosporine