Radiation-induced phosphorylation of Chk1 at S345 is associated with p53-dependent cell cycle arrest pathways

Neoplasia. Mar-Apr 2002;4(2):171-80. doi: 10.1038/sj.neo.7900219.

Abstract

Because DNA damage-inducible cell cycle checkpoints are thought to protect cells from the lethal effects of ionizing radiation, a better understanding of the mechanistic functions of cell cycle regulatory proteins may reveal new molecular targets for cancer therapy. The two major regulatory proteins of G2 arrest are Chk1 and p53. Yet, it is unclear how these two proteins interact and coordinate their functional roles during radiation-induced G2 arrest. To determine Chk1's role in p53-dependent G2 arrest, we used p53 proficient cells and examined expression of G2 arrest proteins under conditions in which G2 arrest was inhibited by the staurosporine analog, UCN-01. We found that UCN-01 inhibited both G1 and G2 arrest in irradiated p53 proficient cells. The arrest inhibition was associated with suppression of radiation-induced expression of both p21 and 14-3-3 sigma -- two known p53-dependent G2 arrest proteins. The suppression occurred despite normal induction of p53 and normal phosphorylation of p53 at S20 and Cdc25C at S216 -- the two known substrates of Chk1 kinase activity. In contrast, we showed that radiation-induced phosphorylation of Chk1 at S345 was associated with binding of Chk1 to p53, p21, and 14-3-3 sigma, and that UCN-01 inhibited S345 phosphorylation. We suggest that DNA damage-induced phosphorylation of Chk1 at S345, and subsequent p53 binding, links Chk1 with p53 downstream responses and may provide a coordinated interaction between DNA damage responses and cell cycle arrest functions.

Publication types

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

MeSH terms

  • 14-3-3 Proteins
  • Alkaloids / pharmacology
  • Antineoplastic Agents / pharmacology
  • Blotting, Western
  • Cell Cycle / drug effects
  • Cell Cycle / radiation effects
  • Checkpoint Kinase 1
  • Colonic Neoplasms / drug therapy
  • Colony-Forming Units Assay
  • Cyclin-Dependent Kinase Inhibitor p21
  • Cyclins / metabolism
  • DNA / radiation effects
  • DNA Damage
  • Drug Resistance
  • Enzyme Inhibitors / pharmacology
  • Humans
  • Mutation / genetics
  • Phosphorylation
  • Protein Kinases / metabolism*
  • Signal Transduction
  • Staurosporine / analogs & derivatives
  • Tumor Cells, Cultured / drug effects
  • Tumor Cells, Cultured / metabolism
  • Tumor Cells, Cultured / radiation effects
  • Tumor Suppressor Protein p53 / metabolism*
  • Tyrosine 3-Monooxygenase / metabolism

Substances

  • 14-3-3 Proteins
  • Alkaloids
  • Antineoplastic Agents
  • CDKN1A protein, human
  • Cyclin-Dependent Kinase Inhibitor p21
  • Cyclins
  • Enzyme Inhibitors
  • Tumor Suppressor Protein p53
  • 7-hydroxystaurosporine
  • DNA
  • Tyrosine 3-Monooxygenase
  • Protein Kinases
  • CHEK1 protein, human
  • Checkpoint Kinase 1
  • Staurosporine