Variant estrogen receptor-c-Src molecular interdependence and c-Src structural requirements for endothelial NO synthase activation

Abstract

Little is known about the tyrosine kinase c-Src's function in the systemic circulation, in particular its role in arterial responses to hormonal stimuli. In human aortic and venous endothelial cells, c-Src is indispensable for 17beta-estradiol (E2)-stimulated phosphatidylinositol 3-kinase/Akt/endothelial NO synthase (eNOS) pathway activation, a possible mechanism in E2-mediated vascular protection. Here we show that c-Src supports basal and E2-stimulated NO production and is required for E2-induced vasorelaxation in murine aortas. Only membrane c-Src is structurally and functionally involved in E2-induced eNOS activation. Independent of c-Src kinase activity, c-Src is associated with an N-terminally truncated estrogen receptor alpha variant (ER46) and eNOS in the plasma membrane through its "open" (substrate-accessible) conformation. In the presence of E2, c-Src kinase is activated by membrane ER46 and in turn phosphorylates ER46 for subsequent ER46 and c-Src membrane recruitment, the assembly of an eNOS-centered membrane macrocomplex, and membrane-initiated eNOS activation. Overall, these results provide insights into a critical role for the tyrosine kinase c-Src in estrogen-stimulated arterial responses, and in membrane-initiated rapid signal transduction, for which obligate complex assembly and localization require the c-Src substrate-accessible structure.

Fig. 1.

c-Src-dependent basal and E2-induced vasorelaxation. (A) Impaired E2 induced vasodilation in PE-preconstricted c-Src^−/− aortas. Control and Src^−/− thoracic aorta rings were isolated, preconstricted with 10⁻⁶ M PE, and treated with E2, and the vascular tone was recorded by vessel myograph system. (B) Partially NO- and receptor-dependent vasorelaxation (10⁻⁸ M E2, 10⁻⁵ M ICI 182,780, and 10⁻⁴ M L-NAME). *, P < 0.05 (two-tailed ANOVA).

Fig. 2.

Requirement of c-Src in ER46-mediated eNOS activation. (A) Activation of eNOS by E2 in reconstituted YF^−/−, Src^+/+ but not SYF^−/− cells. Cells were transfected with eNOS and ER46, challenged with 30 nM E2 for 10 min, and subjected to eNOS activity assay. *, P < 0.05, n = 4. (B) Phosphorylation of Y419 without dephosphorylation of Y530 of c-Src by E2 in ECs. Pretreated cells were treated with 30 nM E2 for the indicated time, followed by lysate immunoblot with pY419-Src, pY530-Src, npY530 (non-phospho-Y530)-Src, and c-Src antibodies. PDGF (5 ng/ml) treatment demonstrates a typical, parallel Y419 phosphorylation and Y530 dephosphorylation response using the same antibodies.

Fig. 3.

Assembly of a functional ER46–Src–eNOS macrocomplex in plasma membranes. (A) Complex formation in EC plasma membranes. Isolated plasma membranes and cytosol from E2-treated or E2BSA-treated (30 nM, 10 min) EA.hy926 cells were used for coimmunoprecipitation with ER antibody (F-10), followed by immunoblot with c-Src and eNOS antibodies. (B and C) Interdependent incorporation of c-Src and ER46 in plasma membranes. Electroporated EA.hy926 and Lipofectamine-transfected COS-7 cells were treated with 30 nM E2 or E2BSA for 10 min, fractionated, and immunoblotted. (D) Phosphorylation of ER46 by c-Src in vitro. ER46-centered complexes were immunoprecipitated with ER antibody (F-10) from EA.hy926 cell lysate and treated with 30 nM E2 and indicated inhibitors (10 μM LY 294002, 10 μM ICI 182,780, and 10 μM PP2) in the presence of [γ-³²P]ATP in vitro, followed by protein fractionation, autoradiography, and immunoblot.

Fig. 4.

Coimmunoprecipitation of ER with c-Src variants. COS-7 cells were transfected with ER46, eNOS, and the indicated avian c-Srcs, E2-deprived for 48 h, serum-starved for 12 h, and stimulated with 33 nM E2 for 10 min. Cell lysates were used for immunoprecipitation with F-10 antibody, and the coimmunoprecipitated avian c-Src was examined by immunoblotting. SrcW118K, c-Src SH3 mutant; SrcR175L, c-Src SH2 mutant.

Fig. 5.

Adaptation of membrane ER46 with open conformation of c-Src. (A) Requirement of open conformation of c-Src for membrane localization of ER46. (B–D) c-Src mediated membrane ER46 binding ligands at cell surface. Values are expressed as means ± SD (n = 4) of the indices [(sample readout/mg of protein)/(untransfected SYF^−/− readout)]. *, P < 0.05 (two-tailed ANOVA, sample vs. vector).

Fig. 6.

Activation of eNOS signaling by c-Src tyrosine kinase activity. (A and B) Requirement of c-Src kinase activity for eNOS activation. In the presence and absence of 10 μM PP2, transfected cells were treated with 30 nM E2 or E2BSA for 10 min, followed by eNOS activity assessment. Results are plotted as means ± SD (n = 3) of the indices [(sample readout/mg of protein)/(boiled lysate readout/mg of protein)]. *, P < 0.05 (two-tailed ANOVA, treated vs. vehicle). (C and D) Requirement of membrane c-Src kinase activity for eNOS activation in ECs. EA.hy926 cells electroporated with Src or SrcG2A were treated with 30 nM E2 or E2BSA for 10 min, fractionated, immunoblotted, and subjected to eNOS activity assay (n = 3 in D). Lysate immunoblotting (C) was conducted on two different membranes that were transblotted with identical samples.