Quantitative proteomics reveal ATM kinase-dependent exchange in DNA damage response complexes.
ATM is a protein kinase that initiates a well-characterized signaling cascade in cells exposed to ionizing radiation (IR). However, the role for ATM in coordinating critical protein interactions and subsequent exchanges within DNA damage response (DDR) complexes is unknown. We combined SILAC-based tandem mass spectrometry and a subcellular fractionation protocol to interrogate the proteome of irradiated cells treated with or without the ATM kinase inhibitor KU55933. We developed an integrative network analysis to identify and prioritize proteins that were responsive to KU55933, specifically in chromatin, and that were also enriched for physical interactions with known DNA repair proteins. This analysis identified 53BP1 and annexin A1 (ANXA1) as strong candidates. Using fluorescence recovery after photobleaching, we found that the exchange of GFP-53BP1 in DDR complexes decreased with KU55933. Further, we found that ANXA1 knockdown sensitized cells to IR via a mechanism that was not potentiated by KU55933. Our study reveals a role for ATM kinase activity in the dynamic exchange of proteins in DDR complexes and identifies a role for ANXA1 in cellular radioprotection.
Medical Scientist Training Program, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, United States.
Serah
S
Choi
Rohith
R
Srivas
Katherine Y
KY
Fu
Brian L
BL
Hood
Banu
B
Dost
Gregory A
GA
Gibson
Simon C
SC
Watkins
Bennett
B
Van Houten
Nuno
N
Bandeira
Thomas P
TP
Conrads
Trey
T
Ideker
Christopher J
CJ
Bakkenist
D017305:Annexin A1 / Q000235:genetics / Q000378:metabolism*; D064007:Ataxia Telangiectasia Mutated Proteins; D018797:Cell Cycle Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002460:Cell Line; D049109:Cell Proliferation; D002470:Cell Survival / Q000528:radiation effects; D002843:Chromatin / Q000378:metabolism; D004249:DNA Damage; D045643:DNA Repair Enzymes / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D055785:Gene Knockdown Techniques; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism; D009025:Morpholines / Q000494:pharmacology; D046912:Multiprotein Complexes / Q000378:metabolism*; D011485:Protein Binding; D060066:Protein Interaction Maps; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D040901:Proteomics; D011753:Pyrones / Q000494:pharmacology; D034622:RNA Interference; D025521:Tumor Suppressor Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000071857:Tumor Suppressor p53-Binding Protein 1
C495818:2-morpholin-4-yl-6-thianthren-1-yl-pyran-4-one; D017305:Annexin A1; D018797:Cell Cycle Proteins; D002843:Chromatin; D004268:DNA-Binding Proteins; D047908:Intracellular Signaling Peptides and Proteins; D009025:Morpholines; D046912:Multiprotein Complexes; D011753:Pyrones; C498301:TP53BP1 protein, human; D025521:Tumor Suppressor Proteins; D000071857:Tumor Suppressor p53-Binding Protein 1; C576324:ATM protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; D017346:Protein Serine-Threonine Kinases; D045643:DNA Repair Enzymes