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ATRMec1 phosphorylation-independent activation of Chk1 in vivo

Yinhuai Chen1, Julie M Caldwell1, Elizabeth Pereira1

  • 1Department of Pharmacology and Toxicology, Norris Cotton Cancer Center, Dartmouth Medical School, Hanover, New Hampshire 03755 and Department of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267-0524.

The Journal of Biological Chemistry
|November 6, 2008
PubMed

Insights

The protein kinase Chk1 is vital for DNA damage response. A specific C-terminal mutation bypasses the need for ATR/Mec1 phosphorylation, enabling Chk1 checkpoint function.

Area of Science:

  • Molecular Biology
  • Cellular Biology
  • Biochemistry

Background:

  • The protein kinase Chk1 is crucial for cellular defense against DNA damage and replication stress.
  • Current models suggest ATR/Mec1 phosphorylates Chk1's C-terminal domain, but its activation mechanism and C-terminus function remain unclear.

Purpose of the Study:

  • To investigate the role of the Chk1 C-terminus in DNA damage and replication block response in vivo.
  • To elucidate the mechanism of Chk1 activation and the significance of ATR/Mec1 phosphorylation sites.

Main Methods:

  • Utilized an in vivo assay to study Chk1 C-terminus function.
  • Introduced mutations in ATR/Mec1 phosphorylation sites and specific C-terminal amino acids.
  • Assessed cell viability and Chk1 kinase activity in vitro.

Main Results:

  • ATR/Mec1 phosphorylation sites are essential for checkpoint response and cell viability during replication stress.
  • Loss of these phosphorylation sites did not affect Chk1 kinase activity in vitro.
  • A single amino acid substitution in the C-terminus restored viability and bypassed the need for ATR/Mec1 phosphorylation for Chk1 activation.

Conclusions:

  • The Chk1 C-terminus plays a critical role in checkpoint activation.
  • A specific C-terminal mutation can allosterically alter Chk1, bypassing the requirement for ATR/Mec1 phosphorylation sites for checkpoint function.

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