Hsp90α regulates ATM and NBN functions in sensing and repair of DNA double-strand breaks

The molecular chaperone heat shock protein 90 (Hsp90 alpha) regulates cell proteostasis and mitigates the harmful effects of endogenous and exogenous stressors on the proteome. Indeed, the inhibition of Hsp90 alpha ATPase activity affects the cellular response to ionizing radiation (IR). Although the interplay between Hsp90 alpha and several DNA damage response (DDR) proteins has been reported, its role in the DDR is still unclear. Here, we show that ataxia-telangiectasia-mutated kinase (ATM) and nibrin (NBN), but not 53BP1, RAD50, and MRE11, are Hsp90 alpha clients as the Hsp90 alpha inhibitor 17-(allylamino)-17-demethoxygeldanamycin (17-AAG) induces ATM and NBN polyubiquitination and proteosomal degradation in normal fibroblasts and lymphoblastoid cell lines. Hsp90 alpha-ATM and Hsp90 alpha-NBN complexes are present in unstressed and irradiated cells, allowing the maintenance of ATM and NBN stability that is required for the MRE11/RAD50/NBN complex-dependent ATM activation and the ATM dependent phosphorylation of both NBN and Hsp90 alpha in response to IR-induced DNA double-strand breaks (DSBs). Hsp90 alpha forms a complex also with ph-Ser1981-ATM following IR. Upon phosphorylation, NBN dissociates from Hsp90 alpha and translocates at the DSBs, while phThr5/7Hsp90 alpha is not recruited at the damaged sites. The inhibition of Hsp90 alpha affects nuclear localization of MRE11 and RAD50, impairs DDR signaling (e.g., BRCA1 and CHK2 phosphorylation), and slows down DSBs repair. Hsp90 alpha inhibition does not affect DNA-dependent protein kinase (DNA-PK) activity, which possibly phosphorylates Hsp90 alpha and H2AX after IR. Notably, Hsp90 alpha inhibition causes H2AX phosphorylation in proliferating cells, this possibly indicating replication stress events. Overall, present data shed light on the regulatory role of Hsp90 alpha on the DDR, controlling ATM and NBN stability and influencing the DSBs signaling and repair.