Depalmitoylation of endothelial nitric-oxide synthase by acyl-protein thioesterase 1 is potentiated by Ca2+-calmodulin

Protein palmitoylation represents an important mechanism governing the dyna mic subcellular localization of many signaling proteins, Palmitoylation of endothelial nitric-oxide synthase (eNOS) promotes its targeting to plasmale mmal caveolae; agonist-promoted depalmitoylation leads to eNOS translocatio n, Depalmitoylation and translocation of eNOS modulate the agonist response , but the pathways that regulate eNOS palmitoylation and depalmitoylation a re poorly understood. We now show that the newly characterized acylprotein thioesterase 1 (APT1) regulates eNOS depalmitoylation. Immunoblot analyses indicate that APT1 is expressed in bovine aortic endothelial cells, which e xpress eNOS. APT1 overexpression appears to accelerate the depalmitoylation of eNOS in COS-7 cells cotransfected with eNOS and APT1 cDNAs. Additionall y, purified recombinant APT1 depalmitoylates eNOS assayed in biological mem branes isolated from endothelial cells biosynthetically labeled with [H-3]p almitate or COS-7 cells transfected with eNOS cDNA. More important, the APT 1-catalyzed depalmitoylation of palmitoyl-eNOS is potentiated by Ca2+-calmo dulin (CaM), a key allosteric activator of eNOS. In contrast, APT1-catalyze d depalmitoylation of the G protein G alpha(s) is unaffected by Ca2+-CaM. F urthermore, caveolin, a palmitoylated membrane protein, does not appear to be a substrate for APT1. Taken together, these results support a role for A PT1 in the regulation of eNOS depalmitoylation and suggest that Ca2+-CaM ac tivation of eNOS renders the enzyme more susceptible to APT1-catalyzed depa lmitoylation.