The activation of neuronal NO synthase is mediated by G-protein betagamma subunit and the tyrosine phosphatase SHP-2.
In CHO cells we had found that CCK positively regulated cell proliferation via the activation of a soluble guanylate cyclase. Here we demonstrate that CCK stimulated a nitric oxide synthase (NOS) activity. The production of NO was involved in the proliferative response elicited by CCK regarding the inhibitory effect of NOS inhibitors L-NAME and alpha-guanidinoglutaric acid. We identified the NOS activated by the peptide as the neuronal isoform: the expression of the C415A neuronal NOS mutant inhibited both CCK-induced stimulation of NOS activity and cell proliferation. These two effects were also inhibited after expression of the C459S tyrosine phosphatase SHP-2 mutant and the betaARK1 (495-689) sequestrant peptide, indicating the requirement of activated SHP-2 and G-betagamma subunit. Kinetic analysis (Western blot after coimmunoprecipitation and specific SHP-2 activity) revealed that in response to CCK-treatment, SHP-2 associated to G-beta1 subunit, became activated, and then dephosphorylated the neuronal NOS through a direct association. These data demonstrate that the neuronal NOS is implicated in proliferative effect evoked by CCK. A novel growth signaling pathway is described, involving the activation of neuronal NOS by dephosphorylation of tyrosyl residues.
Cordelier
P
P
INSERM U151, IFR 31, CHU Rangueil, 31403 Toulouse, France.
Estève
J P
JP
Rivard
N
N
Marletta
M
M
Vaysse
N
N
Susini
C
C
Buscail
L
L
D000818:Animals; D016466:CHO Cells; D002455:Cell Division / Q000187:drug effects; D006224:Cricetinae; D019281:Dimerization; D004789:Enzyme Activation; D019204:GTP-Binding Proteins / Q000502:physiology*; D047908:Intracellular Signaling Peptides and Proteins; D009569:Nitric Oxide / Q000502:physiology; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D052248:Nitric Oxide Synthase Type I; D009599:Nitroprusside / Q000494:pharmacology; D010766:Phosphorylation; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11; D053824:Protein Tyrosine Phosphatase, Non-Receptor Type 6; D017027:Protein Tyrosine Phosphatases / Q000502:physiology*; D011949:Receptors, Cholecystokinin / Q000502:physiology; D012844:Sincalide / Q000494:pharmacology; D014443:Tyrosine / Q000378:metabolism
D047908:Intracellular Signaling Peptides and Proteins; D011949:Receptors, Cholecystokinin; D009599:Nitroprusside; D009569:Nitric Oxide; D014443:Tyrosine; D019001:Nitric Oxide Synthase; D052248:Nitric Oxide Synthase Type I; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11; D053824:Protein Tyrosine Phosphatase, Non-Receptor Type 6; D017027:Protein Tyrosine Phosphatases; D019204:GTP-Binding Proteins; D012844:Sincalide