Regulation of neuronal nitric-oxide synthase by calmodulin kinases

Phosphorylation of neuronal nitric-oxide synthase (nNOS) by Ca2+/calmodulin (CaM)-dependent protein kinases (CaM kinases) including CaM kinase I alpha (CaM-K I alpha), CaM kinase II alpha (CaM-K II alpha), and CaM kinase IV (CaM-K IV), was studied. It was found that purified recombinant nNOS was phosphorylated by CaM-K I alpha, CaM-K II alpha, and CaM-K IV at Ser(847) in vitro. Replacement of Ser(847) with Ala (S847A) prevented phosphorylation by CaM kinases, Phosphorylated recombinant wild-type nNOS at Ser(847) (approximate to 0.5 mol of phosphate incorporation into nNOS) exhibited a 30% decrease of V-max with little change of both the K-m for L-arginine and K-act for CaM relative to unphosphorylated enzyme. The activity of mutant S847D was decreased to a level 50-60% as much as the wild-type enzyme. The decreased NOS enzyme activity of phosphorylated nNOS at Ser(847) and mutant S847D was partially due to suppression of CaM binding, but not to impairment of dimer formation which is thought to be essential for enzyme activation. Inactive nNOS lacking CaM-binding ability was generated by mutation of Lys(732)-Lys-Leu to Asp(732)-Asp-Glu (Watanabe, Y., Hu, Y., and Hidaka, H. (1997) FEBS Lett. 403, 75-78). It was phosphorylated by CaM kinases, as was the wild-type enzyme, indicating that CaM-nNOS binding was not required for the phosphorylation reaction. We developed antibody NP847, which specifically recognize nNOS in its phosphorylated state at Ser(847). Using the antibody NP847, we obtained evidence that nNOS is phosphorylated at Ser(847) in rat brain. Thus, our results suggest that CaM kinase-induced phosphorylation of nNOS at Ser(847) alters the activity control of this enzyme.