Abstract 11455: Mechanism Underlying Increased eNOS Expression and Neuroprotection in Mice With Targeted Deletion of ROCK2

Background: The Rho kinases (ROCK1 and ROCK2) are important regulators of the actin cytoskeleton. We have recently shown that ROCK activity is increased during the onset of ischemic stroke and that the ROCK inhibitor, fasudil, decreased the severity of focal cerebral ischemia, in part, through the upregulation of endothelial nitric oxide synthase (eNOS). However, the precise mechanism by which ROCK regulates eNOS expression is unknown. Methods and Results: Using RNA affinity chromatography with 3’UTR of eNOS mRNA followed by MALDI-TOF-MS analysis, we have identified eukaryotic elongation factor 1 alpha 1 (eEF1A1) as an actin-binding protein, which directly binds to eNOS mRNA and regulates eNOS mRNA stability. Overexpression of eEF1A1 decreased, and knock-down of eEF1A1 by siRNA increased, eNOS expression. Protein kinase assays using eEF1A1 with purified ROCK1 and ROCK2 showed that eEF1A1 is strongly phosphorylated by ROCK2, but not ROCK1, and that eEF1A1 phosphorylation by ROCK2 is required for binding to...