Quantitative Proteomics
Reveal ATM Kinase-dependent Exchange in DNA Damage Response Complexes
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Abstract
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