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Cyclin-dependent kinase 2 dependent phosphorylation of ATRIP regulates the G2-M checkpoint response to DNA damage

Jeremy S Myers1, Runxiang Zhao, Xin Xu

  • 1Department of Biochemistry and Mass Spectrometry Research Center, Vanderbilt University, 23rd Pierce Avenue, Nashville, TN 37232, USA.

Cancer Research
|July 20, 2007
PubMed

Insights

The ATR-ATRIP complex

Area of Science:

  • Cellular biology
  • Molecular biology
  • Biochemistry

Background:

  • The ATR-ATRIP kinase complex is crucial for cellular responses to DNA damage and replication stress.
  • Understanding the regulation of this complex via post-translational modifications is key to cellular health.

Purpose of the Study:

  • To identify novel phosphorylation sites on ATR and ATRIP to elucidate the regulatory mechanisms of the ATR-ATRIP kinase complex.
  • To investigate the role of specific phosphorylation sites in ATRIP's function.

Main Methods:

  • Mass spectrometry was employed to identify phosphorylation sites on ATR and ATRIP.
  • Phosphopeptide-specific antibodies were generated to study phosphorylation at S224.
  • In vitro kinase assays and cell-based experiments using CDK2 inhibitors and S224 alanine mutants were performed.

Main Results:

  • Two novel phosphorylation sites, S224 and S239, were identified on ATRIP.
  • Phosphorylation at S224 occurs in a cell cycle-dependent manner and is mediated by CDK2-cyclin A.
  • Mutation of S224 to alanine impaired the ATR-ATRIP complex's ability to maintain the G(2)-M checkpoint following DNA damage.

Conclusions:

  • ATRIP is a substrate for CDK2, with S224 phosphorylation being a critical regulatory event.
  • CDK2-dependent phosphorylation of ATRIP at S224 is essential for the ATR-ATRIP complex's role in promoting cell cycle arrest after DNA damage.

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