GRP94 is a Novel ATM Interactor and Putative Substrate which Regulates Oncogenic Receptor Expression and Microglial Activation

Abstract

Background: Ataxia-telangiectasia (A-T) is a pleiotropic genetic disorder characterized by symptoms including cerebellar neurodegeneration, cancer predisposition, immunological abnormalities, pulmonary dysfunction, and telangiectasias. A-T is caused by bi-allelic mutations in the gene Ataxia-telangiectasia mutated (ATM). The gene product, ATM, is a 350 kDa protein kinase canonically involved in orchestrating the cellular response to double-stranded breaks in DNA. However, the ATM interactome is not fully characterized. Additionally, the symptomology of A-T is not fully attributable to dysfunction of the DNA damage response (DDR). Methods: We first utilized a proximity-based biotin-labeling (Bio-ID) interactor screen to label and isolate potential interactors of ATM and the autophagy regulator protein Beclin-1. From this screen, we identified the ER chaperone protein GRP94 as a protein of interest. We then characterized how CRISPR-Cas9 deletion and specific pharmacological inhibition of ATM, as well as specific pharmacological inhibition of GRP94, impacted 1) receptor tyrosine kinase (RTK) protein expression, 2) substrate phosphorylation 3) microglial activation (cytokine production and phagocytosis). Furthermore, we employed the use GRP94 wild-type and S64-mutant transgene constructs to study the functional impact of S64-modulation on these phenotypes. Results: GRP94 is a novel interactor and apparent substrate of ATM kinase. ATM-mediated modulation of the putative phosphorylation site Ser 64 impacts N-glycosylation of GRP94. Loss of ATM function leads to aberrant localization of GRP94 to the cell surface, where it enhances expression and pathway activation of the RTKs EGFR and IGF1-R; these phenotypes are reversed with specific pharmacological inhibition of cell surface GRP94. MycBP2 is a putative ATM substrate and novel interactor of both ATM and GRP94. ATM activity may also regulate MycBP2 intracellular localization and interaction with GRP94. The ATM-GRP94 signaling axis also regulates microglial activation, with ATM loss being associated with increased inflammatory cytokine (CXCL-8, IL1-, CXCL-10) expression and increased phagocytic activity, both of which are rescued by inhibition of GRP94. Conclusions: Modulation of GRP94 by ATM kinase regulates important cellular functions which are implicated in oncogenesis and neurodegeneration. These findings provide a potential molecular mechanism for the cancer development and neurodegenerative phenotypes observed in A-T, but may extend to other A-T symptomology and broadly into cancer biology and other neurodegenerative disorders.