カルモデュリンキナーゼI及びIIによるリン酸化を介した神経型NO合成酵素の活性制御

抄録

Neuronal nitric-oxide synthase (nNOS) is regulated by Ca²⁺-dependent binding of calmodulin (CaM) and kinase-dependent phosphorylation. We demonstrate here that nNOS is phosphorylated and inhibited by a constitutively active form of Ca²⁺/CaM-dependent protein kinase I (CaM-K I_1-293) in vitro. Substitution of Ser⁷⁴¹ to Ala blocked the phosphorylation and the inhibitory effect. Mimicking phosphorylation at Ser⁷⁴¹ by Ser to Asp mutation resulted in decreased binding of and activation by CaM since the mutation was within the CaM-binding domain. Phosphorylation of wild-type but not Ser⁷⁴¹Ala mutant nNOS by CaM-K I_1-293 decreased CaM binding to the enzyme. CaM-K I_1-293 also gave phosphorylation of nNOS at Ser⁷⁴¹ in transfected cells, which was determined using phosphorylation site-specific antibody against phospho-Ser⁷⁴¹ nNOS, resulting in 60-70% inhibition of nNOS activity. Furthermore, we obtained evidence that wild-type CaM-K I but neither CaM-K II nor CaM-K IV phosphorylated nNOS at Ser⁷⁴¹ in transfected cells. We have previously demonstrated that CaM-K II directly phosphorylates nNOS at Ser⁸⁴⁷ and can attenuate the catalytic activity of the enzyme in neuronal cells. These results raise the possibility of a novel cross-talk between nNOS and CaM-K I through the phosphorylation of Ser⁷⁴¹ on nNOS in neuronal cells. [Jpn J Physiol 54 Suppl:S66 (2004)]