Src Kinase Activates Endothelial Nitric-oxide Synthase by Phosphorylating Tyr-83

Abstract

The endothelial nitric-oxide synthase (eNOS) is regulated in part by serine/threonine phosphorylation, but eNOS tyrosine phosphorylation is less well understood. In the present study we have examined the tyrosine phosphorylation of eNOS in bovine aortic endothelial cells (BAECs) exposed to oxidant stress. Hydrogen peroxide and pervanadate (PV) treatment stimulates eNOS tyrosine phosphorylation in BAECs. Phosphorylation is blocked by the Src kinase family inhibitor, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2). Moreover, eNOS and c-Src can be coimmunoprecipitated from BAEC lysates by antibodies directed against either protein. Domain mapping and site-directed mutagenesis studies in COS-7 cells transfected with either eNOS alone and then treated with PV or cotransfected with eNOS and constitutively active v-Src identified Tyr-83 (bovine sequence) as the major eNOS tyrosine phosphorylation site. Tyr-83 phosphorylation is associated with a 3-fold increase in basal NO release from cotransfected cells. Furthermore, the Y83F eNOS mutation attenuated thapsigargin-stimulated NO production. Taken together, these data indicate that Src-mediated tyrosine phosphorylation of eNOS at Tyr-83 modulates eNOS activity in endothelial cells.The endothelial nitric-oxide synthase (eNOS), 4 which catalyzes the conversion of L-arginine to L-citrulline and nitric oxide (NO), is posttranslationally regulated by diverse protein-protein interactions and by covalent modification with fatty acylation and phosphorylation. eNOS, like the other two NOS isoforms termed neuronal NOS and inducible NOS, is a homodimer, with each of the two subunits having a bidomain structure consisting of an N-terminal oxygenase domain containing a heme moiety and binding sites for zinc, arginine, and the cofactor, tetrahydrobiopterin, and a C-terminal reductase domain that contains binding sites for FAD, FMN, and NADPH (1). Located between the oxygenase and reductase domains is a Ca 2ϩ calmodulin (CaM) binding sequence (2). Five serine/threonine sites of phosphorylation of bovine eNOS have been identified at Ser-116, Thr-497, Ser-617, Ser-635, and Ser-1179 (3-10). eNOS phosphorylation at Ser-1179 increases eNOS activity by increasing the rate of electron flux through the reductase domain (11). Based on mutagenesis experiments, phosphorylation at Ser-635 increases the maximal activity of eNOS by an as yet unknown mechanism, whereas phosphorylation at Ser-617 does not alter maximal activity but significantly increases the Ca 2ϩ -CaM sensitivity of the enzyme (8). In contrast, eNOS phosphorylation at Thr-497 within the CaM binding sequence reduces eNOS catalytic activity by decreasing the binding affinity for Ca 2ϩ -CaM (7). The function of eNOS phosphorylation at Ser-116 is currently not well understood (12).Evidence exists that eNOS may also be regulated by tyrosine phosphorylation. have reported that treatment of endothelial cells with the tyrosine phosphatase inhibitor, sodium orthovanadate, or the oxidant, hydrogen peroxide (H 2 O 2 )...

Src-mediated Phosphorylation of Hsp90 in Response to Vascular Endothelial Growth Factor (VEGF) Is Required for VEGF Receptor-2 Signaling to Endothelial NO Synthase

“…This phosphorylation of eNOS on Y83 increases the activity of the enzyme. However, it seems specific for calcium mobilizing agonists because VEGF stimulation did not increase the tyrosine phosphorylation levels of eNOS (Fulton et al, 2005). We now provide evidence that c-Src catalyzes the tyrosine phosphorylation of Hsp90 on Y300 in response to VEGF stimulation.…”

“…In addition, Src kinase activity is necessary for VEGF-mediated vascular permeability involved in angiogenesis, tumor cell extravasation, and metastasis (Eliceiri et al, 1999;Weis et al, 2004). Recently, eNOS as been shown to be tyrosine-phosphorylated by c-Src in response to increases in intracellular calcium concentration (Fulton et al, 2005). This phosphorylation of eNOS on Y83 increases the activity of the enzyme.…”

The Roles of Proteinase-Activated Receptors in the Vascular Physiology and Pathophysiology

“…64,66 The phosphorylation of eNOS at Ser1177 (in human) has been shown to activate NO production in a Ca 2ϩ -independent manner. 67,68 In addition, phosphorylation at Ser615, Ser633, 69 and Tyr81 70 has been reported to be associated with an increase in NO production, whereas phosphorylation at Ser495 was associated with a decrease in NO production. 71 The changes in the state of eNOS phosphorylation could thus contribute to the Ca 2ϩ -independent component of thrombin-induced NO production.…”

Integrin α9β1 mediates enhanced cell migration through nitric oxide synthase activity regulated by Src tyrosine kinase

“…The enzyme iNOS is known to interact with Rho GTPases, including Rac-1 (Kuncewicz et al, 2001), and NO itself can act as a heme cofactor for guanylyl cyclase (GC) generating cGMP, a second messenger acting coordinately to facilitate cell migration. The biological effects of NO are contextand cell-type-specific, resulting from either increased or decreased NOS activity (Pan et al, 1996;Fulton et al, 2005;Hausel et al, 2006;Milward et al, 2006). The enzymatic activity of NOS is often modulated by phosphorylation and/or its direct interactions with other intracellular proteins such as Src tyrosine kinases and Erk mitogen-activated protein (MAP) kinase (Kone et al, 2003;Korhonen et al, 2005;Zhang et al, 2007).…”