p90 RSK-1 associates with and inhibits neuronal nitric oxide synthase.
Evidence is presented that RSK1 (ribosomal S6 kinase 1), a downstream target of MAPK (mitogen-activated protein kinase), directly phosphorylates nNOS (neuronal nitric oxide synthase) on Ser847 in response to mitogens. The phosphorylation thus increases greatly following EGF (epidermal growth factor) treatment of rat pituitary tumour GH3 cells and is reduced by exposure to the MEK (MAPK/extracellular-signal-regulated kinase kinase) inhibitor PD98059. Furthermore, it is significantly enhanced by expression of wild-type RSK1 and antagonized by kinase-inactive RSK1 or specific reduction of endogenous RSK1. EGF treatment of HEK-293 (human embryonic kidney) cells, expressing RSK1 and nNOS, led to inhibition of NOS enzyme activity, associated with an increase in phosphorylation of nNOS at Ser847, as is also the case in an in vitro assay. In addition, these phenomena were significantly blocked by treatment with the RSK inhibitor Ro31-8220. Cells expressing mutant nNOS (S847A) proved resistant to phosphorylation and decrease of NOS activity. Within minutes of adding EGF to transfected cells, RSK1 associated with nNOS and subsequently dissociated following more prolonged agonist stimulation. EGF-induced formation of the nNOS-RSK1 complex was significantly decreased by PD98059 treatment. Treatment with EGF further revealed phosphorylation of nNOS on Ser847 in rat hippocampal neurons and cerebellar granule cells. This EGF-induced phosphorylation was partially blocked by PD98059 and Ro31-8220. Together, these data provide substantial evidence that RSK1 associates with and phosphorylates nNOS on Ser847 following mitogen stimulation and suggest a novel role for RSK1 in the regulation of nitric oxide function in brain.
Song
Tao
T
Department of Cell Physiology, Kagawa University, Faculty of Medicine, Kagawa 761-0793, Japan.
Sugimoto
Katsuyoshi
K
Ihara
Hideshi
H
Mizutani
Akihiro
A
Hatano
Naoya
N
Kume
Kodai
K
Kambe
Toshie
T
Yamaguchi
Fuminori
F
Tokuda
Masaaki
M
Watanabe
Yasuo
Y
D000595:Amino Acid Sequence; D000818:Animals; D002531:Cerebellum / Q000166:cytology / Q000378:metabolism; D004815:Epidermal Growth Factor / Q000494:pharmacology / Q000502:physiology; D006624:Hippocampus / Q000378:metabolism; D009474:Neurons / Q000378:metabolism; D052248:Nitric Oxide Synthase Type I / Q000037:antagonists & inhibitors* / Q000378:metabolism; D010766:Phosphorylation; D051381:Rats; D038744:Ribosomal Protein S6 Kinases, 90-kDa / Q000378:metabolism*; D012694:Serine / Q000378:metabolism
D012694:Serine; D004815:Epidermal Growth Factor; D052248:Nitric Oxide Synthase Type I; D038744:Ribosomal Protein S6 Kinases, 90-kDa