Ischemia induces ligand-independent activation of EGFR in model intestinal epithelia: Role of SHC and GRB2 in stress-induced MAPK response

Abstract

Abstract Introduction: Ischemic stress activates mitogen-activated protein kinases (MAPKs) in intestinal epithelia, a response that directs cell fate after injury. We reported that ischemic stress triggers ligand-independent activation of the epidermal growth factor receptor (EGFR), an event associated with downstream activation of MAPK. This study examines the role of EGFR-associated adaptor proteins SHC and GRB2 in ischemic activation of MAPK. Methods: Human intestinal T84 cells underwent 60min of ischemic stress (ATP depletion) by substrate withdrawal and chemical inhibition of mitochondrial respiration and glycolysis (glucose-free buffer containing 1mM oligomycin-A plus 10mM 2-deoxyglucose). Ischemic stress was terminated by return to glucose-containing buffer (ATP repletion). SHC activation was assessed by immunoprecipitation of SHC and blotting with phosphotyrosine specific Ab. Blots were probed for total SHC to ensure equal loading. SHC interactions with GRB2 were assessed by immunoprecipitation of SHC followed by blotting for GRB2. Results: ERK 1/2 phosphorylation is maximal and EGFR was activated within 20min of ATP repletion. At this time point, levels of SHC tyrosine phosphorylation (indicative of SHC activation) were increased (131% +/− 23%, n=3) without change in total SHC levels. Additionally, the interaction of SHC with GRB2 increased by nearly 3-fold (291 +/− 63.7%, n=3, p Conclusions: Following ischemic stress in model intestinal epithelia, SHC phosphorylation/activation is increased in parallel with increased association with GRB2. We conclude that ischemic stress induces the recruitment of EGFR adaptor proteins SHC and GRB2. These data further support a role for ligand-independent activation of EGFR in post-ischemic activation of MAPK signaling.