Abstract 18468: ERK-Mediated Phosphorylation of GRK2 at Ser 670 is Essential for its Hsp90-dependent, Stress-Activated Mitochondrial Localization
Background: G protein-coupled receptor (GPCR) kinase-2 (GRK2) regulates intracellular signaling via phosphorylation of agonist-occupied GPCRs. Identification of novel binding partners for GRK2, including heat shock protein 90 (Hsp90), point towards additional cellular roles for GRK2. We have found that GRK2 associates with mitochondria (Mito) and tested the hypothesis that GRK2 binding to Hsp90 regulates its stress-induced Mito translocation.
Methods: Oxidative stress in myocytes was induced by treatment with chelerythrin (Chele) and ischemia/reperfusion (IR) was used in vivo in mice. Whole cell lysates and Mito fractions were prepared from cells or hearts. Immunoprecipitation (IP) and Western blotting was conducted to examine binding of GRK2 to Hsp90 and Mito translocation of these proteins. Direct in vitro binding of Hsp90 and GRK2 was studied using fusion proteins. To study the role of Ser670, a site for ERK phosphorylation, in stress-dependent GRK2 Mito translocation, a site-specific, phospho-antibody along with a GRK2-S670A mutant was used.
Results: GRK2 and Hsp90 protein were present in myocyte Mito fractions and oxidative stress increased the amount of these proteins in Mito. The Chele-induced translocation of both GRK2 and Hsp90 to Mito was inhibited by an inhibitor of Hsp90. Co-IP studies demonstrated a Hsp90-GRK2 association with a direct interaction confirmed by in vitro binding experiments. Further, Chele-induced apoptosis was accompanied by an increased amount of p-Ser670 of GRK2 that was associated with Mito. Similar results were found with a carboxyl-terminal peptide containing S670. Interestingly, ERK inhibition blocked GRK2 Mito translocation following stress and Chele failed to induce the Mito translocation of GRK2-S670A.
Conclusions: Mito translocation and accumulation of GRK2 after oxidative stress in myocytes is mediated by binding to Hsp90. The carboxyl-terminal domain of GRK2 consisting of AA 485-689 is at least sufficient for binding to Mito and HSP90. ERK-dependent phosphorylation of GRK2 at Ser670 appears to be essential for stress-induced translocation of GRK2 to cardiac Mito, perhaps through enhanced binding with Hsp90. Further details regarding this mechanism remain to be investigated.
- © 2010 by American Heart Association, Inc.