Abstract 1175: The Proline-Rich Tyrosine Kinase Attenuates eNOS Activity in Shear Stress-Stimulated Endothelial Cells by the Phosphorylation of Tyr657
In native and primary cultures of human and porcine endothelial cells, fluid shear stress (12 dynes cm−2, 30 min) elicits the tyrosine phosphorylation of the endothelial NO synthase (eNOS), however, the consequences of this modification on enzyme activity and localisation are unclear. We found that fluid shear stress induces the physical association of eNOS with the proline-rich tyrosine kinase 2 (PYK2) and that the eNOS immunoprecipited from HEK293 cells co-transfected with eNOS and PYK2 was tyrosine-phosphorylated on Tyr657 (as determined using MALDI-TOF spectroscopy). In lung endothelial cells isolated from eNOS−/− mice and transduced with an eNOS adenovirus, the overexpression of PYK2 decreased eNOS activity (by approximately 50%), whereas the downregulation of PYK2 using a siRNA approach increased ionomycin-induced NO production. These data indicate that the tyrosine phosphorylation of eNOS by PYK2 negatively regulates enzyme activity. Point mutation of Tyr657 to the phosphomimetic residues aspartate (D) or glutamate (E) abolished enzyme activity, while a non-phosphorylatable mutant (phenylalanine; F) showed comparable activity to that of the wild-type enzyme. Immunohistochemistry revealed that the Y657E eNOS mutant largely co-localised with the cytoskeleton and was less soluble in Triton X-100 than the wild-type enzyme. A normal flow-induced vasodilatation was apparent in carotid arteries from eNOS−/−mice overexpressing either the wild-type eNOS or the Y657F mutant. However, no flow-induced vasodilatation was apparent in arteries expressing the Y657E eNOS mutant. These data indicate that PYK2 mediates the tyrosine phosphorylation of eNOS on Tyr657 in endothelial cells in response to fluid shear stress and that this modification attenuates the activity of the enzyme. The PYK2-dependent inhibition of NO production may at least partially account for the observation that shear stress elicits only a modest (2– 4 fold) increase in endothelial cell NO output and thus limits the detrimental consequences of maintained high NO output e.g. the generation of peroxynitrite.