Regulation of endothelial nitric oxide synthase by protein kinase C.
Endothelial nitric oxide synthase (eNOS) is a key enzyme in nitric oxide-mediated signal transduction in mammalian cells. Its catalytic activity is regulated both by regulatory proteins, such as calmodulin and caveolin, and by a variety of post-translational modifications including phosphorylation and acylation. We have previously shown that the calmodulin-binding domain peptide is a good substrate for protein kinase C [Matsubara, M., Titani, K., and Taniguchi, H. (1996) Biochemistry 35, 14651-14658]. Here we report that bovine eNOS protein is phosphorylated at Thr497 in the calmodulin-binding domain by PKC both in vitro and in vivo, and that the phosphorylation negatively regulates eNOS activity. A specific antibody that recognizes only the phosphorylated form of the enzyme was raised against a synthetic phosphopeptide corresponding to the phosphorylated domain. The antibody recognized eNOS immunoprecipitated with anti-eNOS antibody from the soluble fraction of bovine aortic endothelial cells, and the immunoreactivity increased markedly when the cells were treated with phorbol 12-myristate 13-acetate. PKC phosphorylated eNOS specifically at Thr497 with a concomitant decrease in the NOS activity. Furthermore, the phosphorylated eNOS showed reduced affinity to calmodulin. Therefore, PKC regulates eNOS activity by changing the binding of calmodulin, an eNOS activator, to the enzyme.
Matsubara
Mamoru
M
Division of Biomedical Polymer Science, Institute for Comprehensive Medical Science, Fujita Health University, Toyoake, Aichi 470-1192. Mamoru.Matsubara@organon.jp
Hayashi
Nobuhiro
N
Jing
Tao
T
Titani
Koiti
K
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002147:Calmodulin / Q000378:metabolism; D002384:Catalysis; D002417:Cattle; D004730:Endothelium, Vascular / Q000201:enzymology*; D066298:In Vitro Techniques; D008565:Membrane Proteins / Q000378:metabolism; D008969:Molecular Sequence Data; D019001:Nitric Oxide Synthase / Q000235:genetics / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D011993:Recombinant Fusion Proteins; D013912:Threonine / Q000378:metabolism
D002147:Calmodulin; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D013912:Threonine; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III; D011493:Protein Kinase C