AGONIST-MODULATED PALMITOYLATION OF ENDOTHELIAL NITRIC-OXIDE SYNTHASE

The nitric oxide synthases (NOS) comprise a family of enzymes which di ffer in primary structure, biological roles, subcellular distribution, and post-translational modifications. The endothelial nitric oxide sy nthase (ecNOS) is unique among the NOS isoforms in being modified by N -terminal myristoylation, which is necessary for its targeting to the endothelial cell membrane. The subcellular localization of the ecNOS, but not enzyme myristoylation, is dynamically regulated by agonists su ch as bradykinin, which promote ecNOS translocation from membrane to c ytosol, as well as enhancing enzyme phosphorylation. Using transiently transfected endothelial cells, we now show that a myristoylation-defi cient mutant ecNOS undergoes phosphorylation despite restriction to th e cytosol, suggesting that phosphorylation may be a consequence rather than a cause of ecNOS translocation. We therefore explored whether ot her post-translational modifications might regulate ecNOS targeting an d now report that ecNOS is reversibly palmitoylated. Biosynthetic labe ling of endothelial cells with [H-3]palmitic acid followed by immunopr ecipitation of ecNOS revealed that the enzyme is palmitoylated; the la bel is released by hydroxylamine, consistent with formation of a fatty acyl thioester, and authentic palmitate can be recovered from labeled ecNOS following acid hydrolysis. Importantly, pulse-chase experiments in endothelial cells biosynthetically labeled with [H-3]palmitate sho w that bradykinin treatment promotes ecNOS depalmitoylation. We conclu de that ecNOS palmitoylation is dynamically regulated by bradykinin an d propose that depalmitoylation of the enzyme may result in its cytoso lic translocation and subsequent phosphorylation.