Abstract 3530: Hsp90-Tom70 interaction facilitates ATM localization to mitochondria and induces mitochondrial autophagy

Abstract

Abstract The serine/threonine protein kinase Ataxia telangiectasia mutated (ATM), plays a critical role in DNA damage response specifically to agents that induce DNA double strand breaks. Recently, mitochondrial autophagy (mitophagy)-associated non-nuclear functions have been attributed to ATM in AT fibroblast (Valentin-Vega YA et al., Blood 119: 1490; 2012). Our investigations suggested similar role of ATM in mantle cell lymphoma (MCL) cell lines (Sarkar, A et al., Proc. AACR, 74: Abstract # 313; 2014). This is clinically important, since ATM is lost in >50% of primary malignant B-cells in MCL patients. To identify the role of ATM in mitophagy, using MCL cell lines, we established that a fraction of ATM protein is located within mitochondria. The proportion of mitochondrial ATM increases following mitophagy induced by mitochondrial uncoupler CCCP or FCCP in ATM proficient cell lines. This process is compromised in MCL cells lacking ATM. However how ATM enters mitochondria is not known. Using quantitative liquid chromatography mass spectrometry analysis (LC/MS), we identified new ATM interacting proteins in HEK293 cells that facilitate mitochondrial transport of ATM. These new ATM interacting proteins (Hsp90, Hsp70 and the outer mitochondrial translocation (TOM) complex, including Tom70) are unrelated to DNA damage response. The physical association of ATM with Hsp90 and Tom70 was confirmed by co-immunoprecipitation experiments while ATM-Hsp70 interaction was mostly nonspecific. Additionally, using kinase dead FLAG-ATM construct in HEK293 cells or pharmacologic (KU55933) inhibition of ATM kinase activity in ATM proficient Jeko-1 and Mino MCL cell lines, we demonstrate that ATM phosphorylation is dispensable to enforce mitophagic exclusion of nonfunctional mitochondria since non-phosphorylated ATM can still bound to Hsp90 and induce mitophagy. These MCL cell lines when treated with an Hsp90 inhibitor; 17-AAG either alone or in combination with CCCP; abolished mitochondrial ATM localization and are defective in CCCP-induced mitochondrial clearance suggesting extra-mitochondrial ATM enters mitochondria via Hsp90 interaction. Further analysis revealed ATM-Hsp90 loosely associate with outer mitochondrial import receptor Tom70 via Hsp90 interaction and this targeting preference between Hsp90-Tom70 is abolished following deletion of the cytosolic tricopeptide repeat (TPR) clamp domain of Tom70 entailing that ATM-Hsp90-Tom70 complex may be required for ATM entry inside mitochondria. Moreover, trypsin digested purified mitochondrial fraction from HeLa cells revealed the entry of C terminal ATM protein within mitochondria while N terminal part remained in the cytosolic fraction. The molecular model of ATM's role in mitophagy and its biological consequence in ATM deficient MCL will be discussed. Citation Format: Aloke K. Sarkar, Tej K. Pandita, Varsha Gandhi. Hsp90-Tom70 interaction facilitates ATM localization to mitochondria and induces mitochondrial autophagy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3530.