Endothelial nitric-oxide synthase (eNOS) is activated through G-protein-coupled receptor kinase-interacting protein 1 (GIT1) tyrosine phosphorylation and Src protein.
Nitric oxide (NO) is a critical regulator of vascular tone and plays an especially prominent role in liver by controlling portal blood flow and pressure within liver sinusoids. Synthesis of NO in sinusoidal endothelial cells by endothelial nitric-oxide synthase (eNOS) is regulated in response to activation of endothelial cells by vasoactive signals such as endothelins. The endothelin B (ETB) receptor is a G-protein-coupled receptor, but the mechanisms by which it regulates eNOS activity in sinusoidal endothelial cells are not well understood. In this study, we built on two previous strands of work, the first showing that G-protein βγ subunits mediated activation of phosphatidylinositol 3-kinase and Akt to regulate eNOS and the second showing that eNOS directly bound to the G-protein-coupled receptor kinase-interacting protein 1 (GIT1) scaffold protein, and this association stimulated NO production. Here we investigated the mechanisms by which the GIT1-eNOS complex is formed and regulated. GIT1 was phosphorylated on tyrosine by Src, and Y293F and Y554F mutations reduced GIT1 phosphorylation as well as the ability of GIT1 to bind to and activate eNOS. Akt phosphorylation activated eNOS (at Ser(1177)), and Akt also regulated the ability of Src to phosphorylate GIT1 as well as GIT1-eNOS association. These pathways were activated by endothelin-1 through the ETB receptor; inhibiting receptor-activated G-protein βγ subunits blocked activation of Akt, GIT1 tyrosine phosphorylation, and ET-1-stimulated GIT1-eNOS association but did not affect Src activation. These data suggest a model in which Src and Akt cooperate to regulate association of eNOS with the GIT1 scaffold to facilitate NO production.
From the Department of Medicine, Medical University of South Carolina, Charleston, South Carolina 29425 and.
Songling
S
Liu
Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710.
Richard T
RT
Premont
From the Department of Medicine, Medical University of South Carolina, Charleston, South Carolina 29425 and rockey@musc.edu.
Don C
DC
Rockey
D020816:Amino Acid Motifs; D000818:Animals; D018797:Cell Cycle Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D042783:Endothelial Cells / Q000201:enzymology* / Q000378:metabolism; D004789:Enzyme Activation; D006801:Humans; D008297:Male; D009569:Nitric Oxide / Q000378:metabolism; D052250:Nitric Oxide Synthase Type III / Q000235:genetics / Q000378:metabolism*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010750:Phosphoproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D014443:Tyrosine / Q000378:metabolism*
D018797:Cell Cycle Proteins; C485648:Git1 protein, rat; D010750:Phosphoproteins; D009569:Nitric Oxide; D014443:Tyrosine; D052250:Nitric Oxide Synthase Type III; D019869:Phosphatidylinositol 3-Kinases; D016392:Proto-Oncogene Proteins pp60(c-src); D051057:Proto-Oncogene Proteins c-akt
Endothelial Cell; Endothelial Dysfunction; Endothelin; G-protein; Nitric Oxide; Nitric-oxide Synthase