Regulation of neuronal nitric-oxide synthase activity by somatostatin analogs following SST5 somatostatin receptor activation.
Somatostatin receptor SST5 is an inhibitory G protein-coupled receptor that exerts a strong cytostatic effect on various cell types. We reported previously that the SST5 anti-proliferative effect results in the inhibition of mitogen-induced increases in intracellular cGMP levels and MAPK activity. This study was conducted to define the early molecular events accountable for the SST5-mediated anti-proliferative effect. Here, we demonstrate that, in Chinese hamster ovary cells expressing SST5 (CHO/SST5 cells), somatostatin inhibited cell proliferation induced by nitric oxide donors and overexpression of the neuronal nitric-oxide synthase (nNOS) protein isoform. Accordingly, nNOS activity and dimerization were strongly inhibited following SST5 activation by the somatostatin analog RC-160. In CHO/SST5 cells, nNOS was dynamically recruited by the SST5 receptor and phosphorylated at tyrosyl residues following RC-160 treatment. RC-160 induced SST5-p60(src) kinase complex formation and subsequent p60(src) kinase activation. Coexpression of an inactive p60(src) kinase mutant with SST5 blocked RC-160-induced nNOS phosphorylation and inactivation and prevented the SST5-mediated anti-proliferative effect. In CHO/SST5 cells, p60(src) kinase associated with nNOS to induce its inactivation by phosphorylation at tyrosyl residues following RC-160 treatment. Using recombinant proteins, we demonstrated that such phosphorylation prevented nNOS homodimerization. Next, surface plasmon resonance and mutation analysis revealed that p60(src) directly associated with nNOS phosphorylated Tyr604. SST5-mediated inhibition of nNOS activity was demonstrated to be essential to the RC-160 anti-proliferative effect on pancreatic endocrine tumor-derived cells. We therefore identified nNOS as a new p60(src) kinase substrate essential for SST5-mediated anti-proliferative action.
Cordelier
Pierre
P
INSERM U531, IFR31, Centre Hospitalier Universitaire Rangueil, 31432 Toulouse Cedex 4, France. pierre.cordelier@toulouse.inserm.fr
Estève
Jean-Pierre
JP
Najib
Souad
S
Moroder
Luis
L
Vaysse
Nicole
N
Pradayrol
Lucien
L
Susini
Christiane
C
Buscail
Louis
L
D000818:Animals; D016466:CHO Cells; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D006224:Cricetinae; D019281:Dimerization; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D052248:Nitric Oxide Synthase Type I / Q000378:metabolism*; D010766:Phosphorylation; D020033:Protein Isoforms; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000737:chemistry; D051381:Rats; D017481:Receptors, Somatostatin / Q000378:metabolism*; D011994:Recombinant Proteins; D013004:Somatostatin / Q000031:analogs & derivatives*
D020033:Protein Isoforms; D017481:Receptors, Somatostatin; D011994:Recombinant Proteins; D013004:Somatostatin; C094573:somatostatin receptor 5; D052248:Nitric Oxide Synthase Type I; D016392:Proto-Oncogene Proteins pp60(c-src)