Inhibition of endothelial nitric oxide synthase activity by proline-rich tyrosine kinase 2 in response to fluid shear stress and insulin.
In native and primary cultures of endothelial cells, fluid shear stress elicits the tyrosine phosphorylation of the endothelial NO synthase (eNOS), however, the consequences of this modification on enzyme activity are unclear. We found that fluid shear stress induces the association of eNOS with the proline-rich tyrosine kinase 2 (PYK2) in endothelial cells and that the eNOS immunoprecipitated from eNOS- and PYK2-overexpressing HEK293 cells was tyrosine-phosphorylated on Tyr657. In mouse carotid arteries, the overexpression of wild-type PYK2, but not a dominant-negative PYK2, decreased eNOS activity (approximately 50%), whereas in murine lung endothelial cells, the downregulation of PYK2 (small interfering RNA) increased ionomycin-induced NO production. Mutation of Tyr657 to the phosphomimetic residues aspartate (D) or glutamate (E) abolished enzyme activity, whereas a nonphosphorylatable mutant (phenylalanine [F]) showed activity comparable to the wild-type enzyme. Moreover, normal flow-induced vasodilatation was apparent in carotid arteries from eNOS(-/-) mice overexpressing either the wild-type eNOS or the Y657F mutant, whereas no flow-induced vasodilatation was apparent in arteries expressing the Y657E eNOS mutant. Insulin also activated PYK2 and stimulated eNOS in endothelial cells expressing the Y657F mutant but not wild-type eNOS. These data indicate that PYK2 mediates the tyrosine phosphorylation of eNOS on Tyr657 in response to fluid shear stress and insulin stimulation and that this modification attenuates the activity of the enzyme. The PYK2-dependent inhibition of NO production may serve to keep eNOS activity low and limit the detrimental consequences of maintained high NO output, ie, the generation of peroxynitrite.
Fisslthaler
Beate
B
Vascular Signalling Group, Institut für Kardiovaskuläre Physiologie, Goethe-Universität, Theodor-Stern-Kai 7, D-60590 Frankfurt am Main, Germany.
Loot
Annemarieke E
AE
Mohamed
Annisuddin
A
Busse
Rudi
R
Fleming
Ingrid
I
D000818:Animals; D001011:Aorta / Q000166:cytology; D002478:Cells, Cultured / Q000187:drug effects / Q000201:enzymology; D042783:Endothelial Cells / Q000187:drug effects / Q000201:enzymology*; D004789:Enzyme Activation / Q000187:drug effects; D004847:Epithelial Cells / Q000187:drug effects / Q000201:enzymology; D051418:Focal Adhesion Kinase 2 / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D007328:Insulin / Q000494:pharmacology*; D015759:Ionomycin / Q000494:pharmacology; D008168:Lung / Q000166:cytology; D051379:Mice; D020125:Mutation, Missense; D009569:Nitric Oxide / Q000096:biosynthesis; D052247:Nitric Oxide Synthase Type II / Q000037:antagonists & inhibitors* / Q000172:deficiency / Q000235:genetics; D052250:Nitric Oxide Synthase Type III / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics / Q000502:physiology; D017354:Point Mutation; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000502:physiology; D012212:Rheology; D013314:Stress, Mechanical; D034421:Sus scrofa
D007328:Insulin; D034741:RNA, Small Interfering; D011993:Recombinant Fusion Proteins; D009569:Nitric Oxide; D015759:Ionomycin; C496314:NOS3 protein, human; D052247:Nitric Oxide Synthase Type II; D052250:Nitric Oxide Synthase Type III; C496315:Nos3 protein, mouse; D051418:Focal Adhesion Kinase 2; C496053:Ptk2b protein, mouse