Bradykinin-stimulated protein tyrosine phosphorylation promotes endothelial nitric oxide synthase translocation to the cytoskeleton.
Stimulation of bovine aortic endothelial cells (BAEC) with bradykinin produces cycles of tyrosine phosphorylation/dephosphorylation of a 90 kDa endothelial nitric oxide synthase (eNOS)-associated protein which we have termed ENAP-1 (for endothelial nitric oxide synthase-associated protein 1). ENAP-1 interacts specifically and tightly with eNOS in BAEC and is co-immunoprecipitated from cell lysates with anti-eNOS antibodies. In addition, anti-phosphotyrosine antibodies co-precipitate eNOS. Bradykinin-stimulated tyrosine phosphorylation of ENAP-1 is blocked by the tyrosine kinase inhibitor, tyrphostin. Dephosphorylation is blocked by the tyrosine phosphatase inhibitor, orthovanadate. Treatment of BAEC with bradykinin or the tyrosine phosphatase inhibitor, phenylarsine oxide promotes tyrosine phosphorylation of detergent-insoluble, cytoskeletal proteins accompanied by translocation of eNOS to the cytoskeletal subcellular compartment. Translocation is blocked by the tyrosine kinase inhibitor, geldanamycin and does not appear to alter enzyme catalytic activity. Tyrosine phosphorylation-dependent association of eNOS with the cytoskeleton may have a role in targeting NO production to specific subcellular locations.
Venema
V J
VJ
Department of Pediatrics, Medical College of Georgia, Augusta 30912, USA.
Marrero
M B
MB
Venema
R C
RC
D000818:Animals; D000906:Antibodies / Q000494:pharmacology; D001011:Aorta; D001152:Arsenicals / Q000494:pharmacology; D016227:Benzoquinones; D001920:Bradykinin / Q000494:pharmacology*; D002352:Carrier Proteins / Q000378:metabolism*; D002417:Cattle; D002478:Cells, Cultured; D003599:Cytoskeleton / Q000201:enzymology*; D004730:Endothelium, Vascular / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007527:Isoenzymes / Q000378:metabolism; D007700:Kinetics; D047029:Lactams, Macrocyclic; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D019000:Phosphotyrosine / Q000276:immunology / Q000378:metabolism*; D017027:Protein Tyrosine Phosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011809:Quinones / Q000494:pharmacology
D000906:Antibodies; D001152:Arsenicals; D016227:Benzoquinones; D002352:Carrier Proteins; D004791:Enzyme Inhibitors; D007527:Isoenzymes; D047029:Lactams, Macrocyclic; D011809:Quinones; C029341:oxophenylarsine; D019000:Phosphotyrosine; D019001:Nitric Oxide Synthase; D011505:Protein-Tyrosine Kinases; D017027:Protein Tyrosine Phosphatases; D001920:Bradykinin; C001277:geldanamycin