Phosphorylation of nitric oxide synthase by protein kinase A.
Nitric oxide synthase was purified to apparent homogeneity from the cytosolic fractions obtained from rat and porcine cerebellum. Enzyme activity--measured as [3H]citrulline formation after incubation with [3H]arginine--was dependent on Ca2+/calmodulin, NADPH, and tetrahydro-L-biopterin. Specific activity varied between 450 to 780 nmol/min/mg protein. Purified nitric oxide synthases showed a single band on 8% SDS/PAGE gels and had an apparent molecular mass of 150,000 Da. The purified proteins were used as substrate for phosphorylation with different protein kinases. In the assays using two Ca2+/calmodulin-dependent protein kinases, CaM kinase II and CaM kinase-Gr, protein kinase C, and the catalytic subunit of protein kinase A, nitric oxide synthase was exclusively phosphorylated by protein kinase A. Such phosphorylation was linear over time for at least 60 min and resulted in nearly stoichiometric phosphate/protein incorporation. The serine in the protein kinase A-consensus sequence KRFGS is probably the site of phosphorylation in nitric oxide synthase. Kemptide, a known protein kinase A substrate, inhibited phosphorylation of nitric oxide synthase in a dose-dependent manner. No changes in nitric oxide synthase activity were observed upon phosphorylation by protein kinase A.
Brüne
B
B
Division of Cell Biology, Burroughs Wellcome Co., Research Triangle Park, N.C. 27709.
Lapetina
E G
EG
D000255:Adenosine Triphosphate / Q000378:metabolism; D000594:Amino Acid Oxidoreductases / Q000378:metabolism*; D000818:Animals; D002531:Cerebellum / Q000201:enzymology*; D003600:Cytosol / Q000201:enzymology; D007700:Kinetics; D008970:Molecular Weight; D019001:Nitric Oxide Synthase; D009842:Oligopeptides / Q000494:pharmacology; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D051381:Rats; D013552:Swine
D009842:Oligopeptides; C037642:kemptide; D000255:Adenosine Triphosphate; D019001:Nitric Oxide Synthase; D000594:Amino Acid Oxidoreductases; D011494:Protein Kinases