Research article

Post-synaptic density-95 promotes calcium/calmodulin-dependent protein kinase II-mediated Ser847 phosphorylation of neuronal nitric oxide synthase

Yasuo WATANABE, Tao SONG, Katsuyoshi SUGIMOTO, Mariko HORII, Nobukazu ARAKI, Hiroshi TOKUMITSU, Tohru TEZUKA, Tadashi YAMAMOTO, Masaaki TOKUDA


Post-synaptic density-95 (PSD-95) is a neuronal scaffolding protein that associates with N-methyl-d-aspartate (NMDA) receptors and links them to intracellular signalling molecules. In neurons, neuronal nitric oxide synthase (nNOS) binds selectively to the second PDZ domain (PDZ2) of PSD-95, thereby exhibiting physiological activation triggered via NMDA receptors. We have demonstrated previously that Ca2+/calmodulin-dependent protein kinase IIα (CaM-K IIα) directly phosphorylates nNOS at residue Ser847, and can attenuate the catalytic activity of the enzyme in neuronal cells [Komeima, Hayashi, Naito and Watanabe (2000) J. Biol. Chem. 275, 28139–28143]. In the present study, we examined how CaM-K II participates in the phosphorylation by analysing the functional interaction between nNOS and PSD-95 in cells. The results showed that PSD-95 directly promotes the nNOS phosphorylation at Ser847 induced by endogenous CaM-K II. In transfected cells, this effect of PSD-95 required its dual palmitoylation and the PDZ2 domain, but did not rely on its guanylate kinase domain. CaM-K Iα and CaM-K IV failed to phosphorylate nNOS at Ser847 in transfected cells. Thus PSD-95 mediates cellular trafficking of nNOS, and may be required for the efficient phosphorylation of nNOS at Ser847 by CaM-K II in neuronal cells.

  • calmodulin-dependent protein kinase II
  • cellular trafficking
  • KN-93
  • neuronal nitric oxide synthase
  • PDZ domain
  • PSD-95


  • Abbreviations used: CaM, calmodulin; CaM-K Iα/IIα/IV, Ca2+/CaM-dependent protein kinases Iα, IIα and IV respectively; CaM-KK, Ca2+/CaM-dependent protein kinase kinase; CREB, cAMP-response-element-binding protein; GST, glutathione S-transferase; HA, haemagglutinin; mAb, monoclonal antibody; NMDA, N-methyl-d-aspartate; nNOS, neuronal nitric oxide synthase; pAb, polyclonal antibody; PSD-95, post-synaptic density-95; PDZ, PSD-95/Dlg/ZO-1 homology; T286A, etc., a mutation bearing a replacement of Thr286 with alanine, etc.