Cyclin-dependent kinase 2 dependent phosphorylation of ATRIP regulates the G2-M checkpoint response to DNA damage.
The ATR-ATRIP kinase complex regulates cellular responses to DNA damage and replication stress. Mass spectrometry was used to identify phosphorylation sites on ATR and ATRIP to understand how the kinase complex is regulated by post-translational modifications. Two novel phosphorylation sites on ATRIP were identified, S224 and S239. Phosphopeptide-specific antibodies to S224 indicate that it is phosphorylated in a cell cycle-dependent manner. S224 matches a consensus site for cyclin-dependent kinase (CDK) phosphorylation and is phosphorylated by CDK2-cyclin A in vitro. S224 phosphorylation in cells is sensitive to CDK2 inhibitors. Mutation of S224 to alanine causes a defect in the ATR-ATRIP-dependent maintenance of the G(2)-M checkpoint to ionizing and UV radiation. Thus, ATRIP is a CDK2 substrate, and CDK2-dependent phosphorylation of S224 regulates the ability of ATR-ATRIP to promote cell cycle arrest in response to DNA damage.
Myers
Jeremy S
JS
Department of Biochemistry and Mass Spectrometry Research Center, Vanderbilt University, 23rd Pierce Avenue, Nashville, TN 37232, USA.
Zhao
Runxiang
R
Xu
Xin
X
Ham
Amy-Joan L
AJ
Cortez
David
D
D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D000818:Animals; D064007:Ataxia Telangiectasia Mutated Proteins; D002453:Cell Cycle; D018797:Cell Cycle Proteins / Q000096:biosynthesis*; D051357:Cyclin-Dependent Kinase 2 / Q000378:metabolism / Q000502:physiology*; D004249:DNA Damage; D004268:DNA-Binding Proteins; D005090:Exodeoxyribonucleases / Q000096:biosynthesis*; D015972:Gene Expression Regulation, Neoplastic; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D010748:Phosphopeptides / Q000737:chemistry; D010750:Phosphoproteins / Q000096:biosynthesis*; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D017346:Protein Serine-Threonine Kinases / Q000096:biosynthesis*; D017386:Sequence Homology, Amino Acid
C521194:ATRIP protein, human; D048868:Adaptor Proteins, Signal Transducing; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D010748:Phosphopeptides; D010750:Phosphoproteins; C576597:ATR protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; D017346:Protein Serine-Threonine Kinases; D051357:Cyclin-Dependent Kinase 2; D005090:Exodeoxyribonucleases; C429526:three prime repair exonuclease 1