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-associate d protein 1). ENAP-1 interacts specifically and tightly with eNOS in B AEC and is co-immunoprecipitated from cell lysates with anti-eNOS anti bodies. In addition, anti-phosphotyrosine antibodies co-precipitate eN OS. Bradykinin-stimulated tyrosine phosphorylation of ENAP-1 is blocke d by the tyrosine kinase inhibitor, tyrphostin. Dephosphorylation is b locked by the tyrosine phosphatase inhibitor, orthovanadate. Treatment of BAEC with bradykinin or the tyrosine phosphatase inhibitor, phenyl arsine oxide promotes tyrosine phosphorylation of detergent- insoluble , cytoskeletal proteins accompanied by translocation of eNOS to the cy toskeletal subcellular compartment. Translocation is blocked by the ty rosine kinase inhibitor, geldanamycin and does not appear to alter enz yme catalytic activity. Tyrosine phosphorylation-dependent association of eNOS with the cytoskeleton may have a role in targeting NO product ion to specific subcellular locations. (C) 1996 Academic Press, Inc.