Src Kinase Mediates Phosphatidylinositol 3-Kinase/Akt-dependent Rapid Endothelial Nitric-oxide Synthase Activation by Estrogen*

17 (cid:1) -Estradiol activates endothelial nitric oxide synthase (eNOS), enhancing nitric oxide (NO) release from endothelial cells via the phosphatidylinositol 3-kinase (PI3-kinase)/Akt pathway. The upstream regulators of this pathway are unknown. We now demonstrate that 17 (cid:1) -estradiol rapidly activates eNOS through Src kinase in human endothelial cells. The Src family kinase spe-cific-inhibitor 4-amino-5-(4-chlorophenyl)-7-( t -bu-tyl)pyrazolo[3,4- d ]pyrimidine (PP2) abrogates 17 (cid:1) -es-tradiol- but not ionomycin-stimulated NO release. Consistent with these results, PP2 blocked 17 (cid:1) -estra-diol-induced Akt phosphorylation but did not inhibit NO release from cells transduced with a constitutively active Akt. PP2 abrogated 17 (cid:1) -estradiol-induced activation of PI3-kinase, indicating that the PP2-inhibitable kinase is upstream of PI3-kinase and Akt. A 17 (cid:1)

The cardioprotective effects of estrogen are diverse, including both rapid non-genomic and delayed genomic effects on the blood vessel wall (reviewed in Ref. 1). Specific, rapid vascular effects, such as moderation of vasomotor tone, have been linked to an estrogen-stimulated increase in bioavailable nitric oxide (NO) 1 (2)(3)(4). 17␤-estradiol (E2) treatment of human endothe-lial cells (EC) induces rapid release of NO by estrogen receptor (ER)-dependent activation of endothelial nitric oxide synthase (eNOS) (5). Many factors regulate eNOS enzyme activity, including fatty acid modification, subcellular localization, and binding to numerous proteins and cofactors, including calmodulin, caveolin-1, the 90-kDa heat shock protein (HSP90), and tetrahydrobiopterin (see Ref. 6 for review). eNOS is a Ca 2ϩ / calmodulin-dependent enzyme, the activity of which is also regulated by phosphorylation. Specific phosphorylation of eNOS by the serine/threonine kinase Akt renders the enzyme more active at much lower Ca 2ϩ concentrations (7,8). We demonstrated previously that the ER-dependent activation of eNOS occurs at resting Ca 2ϩ concentrations and requires activation of the phosphatidylinositol-3-OH kinase (PI3-kinase)/ Akt pathway (9). The regulatory subunit of PI3-kinase, P85, acts to stabilize and inhibit the catalytic activity of PI3-kinase. Recently, ER was shown to specifically bind to P85 in vitro (10). The E2-induced association correlated with increases in PI3kinase activity in EC. However, the specific mechanism for E2 activation of PI3-kinase is not known.
Evidence is emerging that membrane forms of steroid hormone receptors exist and participate in signaling pathways (11)(12)(13)(14). The activity of E2 at the cell membrane has been shown in EC, neurons, and breast cancer cell lines. We previously determined that rapid E2 activation of eNOS and MAP kinase occurs through a membrane-associated ER (9,12). The EC line EAhy.926 used in these experiments exhibits rapid E2-induced signaling but is unable to stimulate ER-dependent gene transactivation. Additionally, EAhy.926 cells do not express the traditional 66-kDa ER␣ or ER␤ but express a 46-kDa protein immunoreactive with C-terminal ER antibodies. Recently, a protein of similar size reactive with E2 and anti-ER antibodies was found to be associated with the plasma membrane in MCF-7 cells (13,14). Additionally, a 46-kDa putative ER, reactive with anti-ER antibodies, was found in wild-type and in the initial ER␣ knockout mice. This form of the receptor was thought to be responsible for E2 enhancement of basal NO production in the initial ER␣ knockout mice, because this E2 effect was lost in the complete ER␣ knockout mouse (15). In human ECs expressing both the 66-and the 46-kDa receptor, both rapid signaling to MAP kinase and gene transactivation of estrogen-responsive element-luciferase reporter was stimulated with E2 treatment (12). As previously indicated, the specific mechanism of membrane-associated ER coupling to P85 is unknown. E2-mediated actions are sensitive to serine/ threonine and tyrosine kinase inhibition. Previously, the activation of the tyrosine kinase c-Src was associated with rapid E2 effects in breast cancer cells (16,17). Src activation induces MAP kinase through a Shc/Grb2/Ras signaling cascade. In addition to MAP kinase, Ras-GTP has been shown to bind and activate PI3-kinase. Because E2 rapidly activates both EC MAP kinase and PI3-kinase, we investigated the ability of E2 to activate Src kinase in human EC and whether the consequences of this activation include activation of PI3-kinase, Akt, eNOS, and MAP kinase. Here, we present evidence that the non-receptor tyrosine kinase, c-Src, is rapidly activated in EC upon stimulation by E2. This activation leads to formation of a functional signaling complex composed of ER, c-Src, and P85.

EXPERIMENTAL PROCEDURES
Materials-E2 and ionomycin were purchased from Sigma. Stock solutions were prepared in ethanol with final ethanol concentrations less than 0.1%. Stock solutions of LY294002 (Calbiochem) and ICI 182,780 (Zeneca Pharmaceuticals) were prepared in Me 2 SO, with final Me 2 SO concentrations less than 0.1%. Anti-phosphorylated Akt, anti-Akt, anti-phosphorylated p60, and anti-phosphorylated eNOS were purchased from Cell Signaling. Anti-P85 and anti-c-Src were purchased from Santa Cruz. Anti-eNOS antibody was purchased from BD Transduction Laboratories. All other reagents were purchased from Sigma unless otherwise noted.
Cell Culture-The EC line EAhy.926, described previously (9,18), was maintained in DMEM and 10% fetal bovine serum, supplemented with 5 mM hypoxanthine, 0.8 mM thymidine, and 20 M aminopterin. Human umbilical vein EC (HUVEC) were isolated and maintained as described previously (5). Murine embryonic fibroblasts derived from embryos deficient in c-Src, yes, and fyn (SYF Ϫ/Ϫ ) or fibroblasts derived from control animals lacking both yes and fyn but maintaining normal levels of c-Src (YF Ϫ/Ϫ S ϩ/ϩ ), described previously (19), were maintained in DMEM and 10% fetal bovine serum. Before E2 stimulation, cells were cultured in E2-free medium consisting of phenol red-free DMEM and 10% gelding horse serum and were subsequently serum-starved in phenol red-free DMEM containing 0.1% fatty acid-free bovine serum albumin.
NO Release-EC monolayer NO release was quantified by NO-specific chemiluminescence using potassium iodide and acetic acid reflux, as described previously (8,9). Cells were stimulated with E2 and ionomycin for 30 min at 37°C, and supernatants were collected for NO analysis. The Src family kinase inhibitor, PP2, or vehicle was added 30 min before agonist stimulation and NO collection.
Immunoprecipitation and Western Blotting-Cell monolayers were stimulated as described in the figure legends. Cells were either lysed directly in SDS-PAGE sample buffer or in 20 mM Tris-HCl, pH 7.4, 2.5 mM EDTA, 1% Triton X-100, 1% Nonidet P-40, 1 mM Na 3 VO 4 , 1 mM NaP i PO 4 , supplemented with a protease inhibitor mixture (Roche Molecular Biochemicals). Lysates were either directly subjected to SDS-PAGE or first incubated with the appropriate primary antibody, immunoprecipitated with protein A/G agarose (Santa Cruz) subjected to SDS-PAGE and immunoblotting. Immunoblots were probed with horseradish peroxidase-coupled species-specific secondary antibodies and visualized by enhanced chemiluminescence.
Phosphoinositide 3-Kinase Assay-The PI3-kinase assay was performed essentially according to the manufacturer's instructions (Upstate Biotechnology). Briefly, monolayers of EAhy.926 cells were E2deprived for 48 h and serum-starved in 0.25% bovine serum albumin overnight before E2-stimulation. Some plates were additionally preincubated with inhibitors or vehicle control before stimulation. Cells were washed and lysed in the presence of a protease inhibitor mixture (Roche Molecular Biochemicals), 1 mM Na 3 VO 4 , and 1 mM NaF. The supernatant was collected and precleared by irrelevant mouse IgG. Approximately 500 g of soluble proteins were subjected to immunoprecipitation with anti-P85 or anti-ER␣ (Santa Cruz Biotechnology, Santa Cruz, CA) antibodies for 2 h at 4°C. The immunocomplexes were harvested by protein A/G agarose, washed, and incubated with 15 g of phosphatidylinositol (Avanti Polar Lipids, Inc) for 10 min at room temperature. PI3-kinase activity as monitored at 37°C for 15 min after addition of 20 Ci of [␥-32 P]ATP (3000 Ci/nmol; 0.88 mM ATP) and 20 mM MgCl 2 to the reaction. The reaction was terminated by 6N HCl, after which the lipids were extracted by chloroform/methanol (1:1) and fractionated by thin layer chromatography in chloroform/methanol/water/ammonium hy-droxide (129:114:21:5). The thin layer chromatography plate was then air-dried and subjected to autoradiography.
Adenoviral Infection-Recombinant adenoviruses expressing ␤-galactosidase (␤-gal), or the membrane-targeted, myristoylated Akt (myr-Akt), described previously (8), were obtained from K. Walsh (St. Elizabeth's Medical Center, Boston, MA). Monolayers were incubated with the recombinant adenoviruses at a multiplicity of infection of 100 for ␤-gal and myr-Akt. After infection, E2-free medium was added for the cell recovery period followed by serum starvation in phenol red-free DMEM plus 0.1% bovine serum albumin. Adenovirally infected cells were stimulated as described in the figure legends.
Transient Transfection-Cell monolayers were incubated with empty vector (pcDNA3), kinase dead Src kinase (Src K295M), or constitutively active Src kinase (Src Y527F) and Fugene (Roche Molecular Biochemicals) at a 1:6 DNA-to-lipid ratio according to the manufacturer's directions in E2-free medium. After transfection, cells were serum-starved in phenol red-free DMEM and 0.1% bovine serum albumin before stimulation with E2.

Effect of Src Family Kinase Inhibition on NO Release-We
demonstrated previously that induced NO release occurs through activation of E2-stimulated PI3-kinase and Akt activation, resulting in phosphorylation and enhanced activation of eNOS (9). Once Akt is targeted to the membrane via a PI3kinase-dependent mechanism, it can be phosphorylated on at least two residues, Ser 473 and Thr 308 . Phosphorylation of Thr 308 is thought to be largely constitutive, whereas Ser 473 phosphorylation is highly inducible. Thus "activation" of Akt is almost exclusively measured by Ser 473 phosphorylation (20).
Recently, in addition to phosphorylation of Ser 473 and Thr 308 , Akt has been shown to be phosphorylated on Tyr 315 and Tyr 326 by Src kinase (21). This tyrosine phosphorylation is thought to be important for full activation of Akt but is independent of serine/threonine phosphorylation. These authors demonstrated that the activity of a constitutively active Akt (myr-Akt) was further augmented by transfection of a constitutively active Src kinase (Src527F). Therefore, we attempted to determine whether Src kinase was a primary upstream mediator of the signal transduction pathway leading to E2-mediated NO release. EC were pretreated with PP2, a pharmacological inhibitor specific for Src family tyrosine kinases (22,23), or vehicle for 30 min before agonist stimulation and NO collection. As in our prior work, the EAhy.926 EC line was used, largely because the cells are phenotypically homogeneous, contain good levels of eNOS, and display rapid signaling responses to estrogen (9,12). PP2 completely abrogated E2-induced NO release but had no effect on ionomycin-stimulated NO release (Fig. 1). This demonstrates that a member of the Src kinase family is involved in E2-but not calcium ionophore-enhanced eNOS activation.
Effect of PP2 on Constitutively Active Akt-enhanced NO Release-To begin dissecting the level at which Src kinase transduces the aforementioned E2-stimulated responses, EAhy.926 cells were infected with recombinant adenovirus encoding either ␤-gal as a control or a membrane targeted and thus constitutively active myr-Akt. After infection, the cells were pretreated with PP2, followed by E2 or vehicle stimulation for 30 min, and the NO release was quantified. PP2 has no effect on NO release induced by constitutively active Akt (Fig. 2), indicating that Akt itself is not a critical substrate for Src kinase. That is, Src kinase-mediated Akt tyrosine phosphorylation is not a required step in Akt-dependent eNOS activation. This demonstrates that a Src kinase is playing a role upstream of Akt-mediated eNOS phosphorylation. Because PP2 was unable to prevent NO release induced by constitutively active Akt, it was important to determine where in the pathway Src kinase was involved. PP2 abrogates E2-induced phosphorylation of Akt on Ser 473 in immortalized EAhy.926 EC (Fig. 3A) and in c-Src Mediates Estrogen Receptor-dependent eNOS Activation HUVEC (Fig. 3B), indicating Src kinase involvement in the primary activation of Akt (Fig. 3). Phosphorylation of a downstream target of E2-activated Akt, eNOS Ser 1177 , was inhibited by pretreatment of HUVEC with PP2 (Fig. 3C), correlating with PP2 inhibition of E2-stimulated NO release (Fig. 1). These data also indicate that the E2-induced signaling responses seen in the immortalized EAhy.926 cells are functionally identical to that seen in primary HUVEC.
Effect of E2 Stimulation on EC c-Src-Although the PP2 data do not define the precise Src kinase involved, c-Src is rapidly phosphorylated in response to E2 in mammary tumor cell lines and osteoblasts (17, 24 -26). In these cells, Src kinase activation results in the induction of the Shc/Ras/Erk signal transduction pathway. We have previously demonstrated rapid EC Erk 1/2 activation in response to E2 (12). Human EC, including EAhy.926 cells, contain easily detectable levels of c-Src. Therefore, E2-induced c-Src activation was evaluated. c-Src activation kinase requires carboxy-terminal Tyr 530 dephosphorylation and subsequent kinase domain Tyr 416 autophosphorylation (27). Fig. 4A demonstrates induced c-Src Tyr 416 phospho-rylation within 2 min of E2 stimulation. As with all other E2-stimulated rapid signaling responses we have observed in human EC (5,9,12,28), this activation is completely inhibited by the conventional ER antagonist ICI 182,780, indicating that this is an ER-mediated event (see below). PP2 also abrogates E2-induced c-Src phosphorylation, consistent with the requirement for autophosphorylation. However, E2-induced c-Src phosphorylation was not inhibited by the specific PI3-kinase inhibitor LY294002, indicating E2 activation of Src kinase occurs before activation of the PI3-kinase/Akt pathway.
Although c-Src phosphorylation/activation is clearly E2-induced in EC (Fig. 4A) and in osteoclasts (16,25,29), the precise mechanism has not been defined. In osteoclasts, an interaction between ligand-activated steroid receptors and c-Src seems required for kinase activity (16,25,29). Western blots performed on coimmunoprecipitates from E2-stimulated EAhy.926 cells demonstrated a rapidly induced c-Src/ER association (Fig. 4B). The anti-ER antibody used is a monoclonal antibody directed at the carboxyl terminus of ER␣. In human EC, including EAhy.926, the antibody blots/immunoprecipitates a 46-kDa protein that we believe to be the predominate membrane-associated ER in these cells (Ref. 12, and below). Thus, E2 stimulation promotes the formation of a putative signaling complex between the 46-kDa, signal-transducing ER and c-Src. As might be expected from our previous results, PP2 also inhibited E2-but not ionomycin-stimulated Erk 1/2 activation (data not shown). These data support the idea that E2induced c-Src activation results in parallel activation of the MAP kinase (Erk 1/2 ) and Akt pathways, the latter of which results in eNOS phosphorylation and augmented NO release.
The Role of Src Kinase in E2-induced PI3-kinase Activation-We have previously demonstrated that E2-activated NO FIG. 2. Effect of PP2 on constitutive Akt activity. EC were virally transduced with either control ␤-gal or myr-Akt. After E2 deprivation and serum starvation, the indicated EC were preincubated with PP2 (10 M) and stimulated with E2 (10 ng/ml) for 30 min, after which the medium was collected and NO was measured by NO-specific chemiluminescence. *, p Յ 0.01 difference compared with ␤-gal control cells (C, ␤-gal).

FIG. 3. Effect of PP2 on induced Akt and eNOS phosphorylation. EC (EAhy.926 (A) and HUVEC (B)
) were pretreated with PP2 (10 M) or vehicle for 20 min, followed by E2 (10 ng/ml) activation for 15 min. EC were washed, lysed, subjected to SDS-PAGE, transferred to nitrocellulose, and immunoblotted with phosphorylation-specific Akt antibody (pAKT) and reprobed with total Akt antibody (AKT). C, HU-VEC lysates were additionally subjected to SDS-PAGE, transferred to nitrocellulose, immunoblotted with phosphorylation-specific eNOS antibody (peNOS) and reprobed with total eNOS antibody (eNOS). release can be completely inhibited by the PI3-kinase inhibitor LY294002 (9), indicating an absolute requirement for PI3-kinase. It was recently demonstrated that ER␣ can associate with the regulatory subunit of PI3-kinase, P85, and that this association correlates with increased production of phosphatidylinositol 3,4,5-phosphates (10). The mechanism by which ligand-induced ER/P85 association activates PI3-kinase remains to be determined. In EAhy.926 cells, which do not express the full-length ER, we thus evaluated whether this alternative form of ER could associate with P85. E2-treated cell lysates were immunoprecipitated with anti-ER␣ antibodies and Western blotted for the presence of P85. E2 rapidly stimulated ER/P85 association in EAhy.926 cells (Fig. 5A). This association was blocked by the ER antagonist, ICI 182,780 and PP2, indicating a role for Src kinase in this complex formation (Fig. 5B). Furthermore, c-Src was inducibly associated with P85 in response to E2 (Fig. 5C), an effect that was also inhibited by ICI 182,780 and PP2. Because PP2 treatment inhibited the apparent upstream ER/P85 association, as well as downstream Akt and eNOS activation, it was important to determine the effect of Src kinase inhibition on E2-induced PI3kinase activity. EAhy.926 cells were pretreated with ICI 182,780, LY294002, PP2, or vehicle control for 30 min before E2 stimulation, after which PI3-kinase was immunoprecipitated from stimulated cells with anti-P85 (Fig. 6) or anti-ER␣ antibodies. The production of PI3-kinase generated D3-phosphoinositides (PIP) was determined by an in vitro kinase assay. Fig. 6 demonstrates that in P85 immunoprecipitates, E2 stimulation rapidly increased the production of PIP, with maximum levels achieved in 10 min. These increases in PIP were completely abrogated by ER antagonist ICI 182,780, LY294002, and PP2. Identical results were obtained when E2-induced PI3-kinase activity was immunoprecipitated with anti-ER␣ antibodies. E2-stimulated increased production of PIP in ER im-munoprecipitates was also inhibited by pretreatment with ICI 182,780, LY294002 and PP2 (data not shown). This indicates that Src kinase is required for the E2-mediated increase in PI3-kinase activity and that Src is a component of the activated ER/PI3-kinase signaling complex. Notably, PI3-kinase inhibition with LY294002 did not inhibit E2 activation of Src kinase (Fig. 4A), suggesting a sequential activation cascade in which ER/c-Src association induces Src kinase activity and the activated ER/c-Src complex consequently associates with P85, effecting increased PI3-kinase activity.
The Role of c-Src in E2-stimulated Akt Activation-E2-induced phosphorylation of Akt was absent in fibroblasts devoid of Src family kinases c-Src, fyn, and yes (SYF Ϫ/Ϫ ). However, in cells lacking fyn and yes, but maintaining normal expression of c-Src (YF Ϫ/Ϫ S ϩ/ϩ ), E2 markedly stimulated Akt phosphorylation (Fig. 7A)  E2 was added to the monolayer in serum-free medium for the indicated times. A, cells were washed, lysed and immunoblotted with phosphorylation-specific c-Src antibody (pSrc). Membranes were reprobed with total c-Src antibody (c-Src). B, E2 (10 ng/ml) was added to the monolayer in serum-free medium for the indicated times. Cells were washed, lysed, and immunoprecipitated with anti-c-Src antibody-agarose conjugate. Immunoprecipitates (IP) were immunoblotted with anti-ER antibody and reprobed with anti-c-Src antibody. The ratio of ER that was co-immunoprecipitated with c-Src was determined by densitometry based upon total c-Src detected in the immunoblots. WB, Western blot.

FIG. 5. Effect of E2 on ER/P85/c-Src interaction.
A, E2-deprived and serum-starved EC monolayers were stimulated with E2 (10 ng/ml) for 15 min. Cells were washed, lysed, and immunoprecipitated with anti-P85 antibody. Immunoprecipitates (IP) were immunoblotted with anti-ER antibody and reprobed with anti-P85 antibody. WB, Western blot. B and C, E2-deprived and serum-starved EC monolayers were pretreated with ICI 182,780 (ICI, 10 M), PP2 (10 M), or vehicle for 30 min and then stimulated with E2 for 10 min. Cells were washed, lysed, and immunoprecipitated with anti-ER or anti-c-Src antibodies. Immunoprecipitates were immunoblotted with anti-P85 antibody. Membranes were reprobed with anti-ER antibody or anti-c-Src antibody.

c-Src Mediates Estrogen Receptor-dependent eNOS Activation
c-Src in E2 activation of the PI3-kinase/Akt pathway, EC were transiently transfected with either control vector (Fig. 7B,  lanes 1-3), a kinase-dead c-Src (SrcK295M) (Fig. 7B, lanes 4  and 5), or a constitutively active c-Src (Src527F) (Fig. 7B, lanes  6 and 7). Cells were then stimulated with E2, and the activation of Akt was determined by presence of Ser 473 phosphorylation. E2 treatment of endothelial cells transfected with control vector resulted in increased phosphorylation of Akt (Fig. 7B,  compare lane 1 with lanes 2 and 3). In cells expressing the kinase-dead Src, E2 was unable to stimulate Akt phosphorylation (Fig. 7B, compare lanes 4 and 5). Moreover, cells transfected with the constitutively active c-Src in the absence of additional stimulation demonstrated increased basal Akt phosphorylation (Fig. 7B, compare lane 1 with lanes 6 and 7). These data demonstrate that c-Src can mediate the E2-induced Akt/ eNOS activation response and that activated c-Src, by itself, can effect Akt activation. DISCUSSION There are now numerous reports of estrogen-induced endothelial NO release in vitro and vasodilation in vivo (5, 9, 12, 28, 30 -32). Although we have learned a great deal about the downstream effectors in this important signaling pathway, the paradox that a steroid hormone receptor can, upon engagement, be responsible for triggering rapid "transmembrane" signal transduction remains. In particular, the most proximal molecular components of this pathway remain obscure. We previously demonstrated that estrogen, similar to shear stress and insulin, can stimulate the enhancement of eNOS activity in an ER-dependent fashion that does not require an intracellular Ca 2ϩ flux (5). Since that initial observation, we and others have demonstrated that E2 treatment of EC results in rapid phosphorylation and activation of Akt with consequent phosphorylation of eNOS on Ser 1177 . This phosphorylation enhances electron flux through the eNOS reductase domain with a reduced rate of calmodulin dissociation at low (resting) Ca 2ϩ levels (7,9). This provided the first mechanistic explanation for E2-stimulated NO release in the absence of a Ca 2ϩ flux. However, the mechanism of E2-induced Akt phosphorylation remains unknown.
Our data, and those of others, have defined PI3-kinase as a critical upstream activator in the E2-stimulated Akt/eNOS activation pathway (9,10). In fact, a direct interaction between ER and P85, the regulatory subunit of PI3-kinase, has been demonstrated, correlating with increased PI3-kinase activity (10). However, the E2-stimulated molecular switches responsible for the ER/PI3-kinase association are not defined. There are several reasons to suspect that Src family kinases could be the link between ER and PI3-kinase. First, we and others have demonstrated, in parallel to Akt activation, that E2 stimulation of EC results in rapid ERK 1/2 activation (12,30). This response resembles that mediated by receptor tyrosine kinases, which, in some cases, recruit Src family kinases as a part of a MAP kinase cascade. Second, P85 has been shown to be a Src kinase (lck and abl) substrate (33)(34)(35), and fyn, lyn, and lck can, through their SH3 domains, interact with P85 (36 -41). Third, estrogen-induced c-Src phosphorylation has been demonstrated in osteoclasts and breast cancer cell lines (24,29).
Here, we provide the first demonstration of ER-dependent c-Src activation in EC, and that this activation provides a functional link between ER engagement and the PI3-kinase/ Akt/eNOS pathway. A pharmacological inhibitor of the Src family tyrosine kinases inhibited not only Akt activation and NO release but also PI3-kinase dependent generation of phosphatidylinositol phosphates, indicating that Src activation is upstream of PI3-kinase. The c-Src specificity was documented by inhibiting E2-induced Akt phosphorylation with a kinasedead c-Src. We now demonstrate an estrogen-stimulated molecular complex formation, between ER, P85, and c-Src, that includes activated c-Src, phosphorylated within 2 min of E2 treatment. The basis and direct consequence of a P85/c-Src interaction remain to be determined, although several possibilities exist. As noted above, P85 was shown to be specifically phosphorylated on Tyr 688 by the Src kinases lck and abl (33)(34)(35), and PI3-kinase has been shown to be a preferential substrate for c-Src (42,43). It is also possible that estrogen-activated c-Src could tyrosine phosphorylate docking proteins containing binding sites for the SH2 domain of P85, thus alleviating, upon interaction, the inhibitory constraint on the PI3kinase P110 catalytic subunit (44,45). Alternatively, the SH3 domains of several Src kinases have been shown to bind directly to P85 and regulate its activity (36 -41). This includes c-Src that, in osteoclasts, interacts directly through its SH3 domain with P85, in response to colony stimulating factor-1 (46).
Although we believe that these rapid signaling responses to estrogen have important implications in vascular tissue, other ligand-activated steroid hormone receptors may have similar effects. Engagement of the androgen receptor, but not the progesterone receptor, can result in phosphatidylinositol 3,4,5phosphate generation (10). As might be expected, ER and androgen receptor have been shown to directly couple with c-Src, whereas the progesterone receptor has not (25,29), consistent with the notion that steroid hormone receptor-induced PI3kinase activation is c-Src-dependent. In contrast, if steroid hormone receptors heteromultimerize, responses can be diversified. For example, PR and ER can associate in the absence of ligand; in this setting, either progestins or estrogens can rapidly trigger c-Src activation (25). Also, a ternary androgen receptor/ER/c-Src complex has been demonstrated, through an ER-pTyr 537 /c-Src-SH2 and androgen receptor/c-Src-SH3 interaction (29). Whether ER-pTyr 537 is constitutively or inducibly (by estrogen) phosphorylated remains unclear. FIG. 7. Effect of c-Src mutant expression on E2-induced Akt activation. A, either YF Ϫ/Ϫ S ϩ/ϩ or SYF Ϫ/Ϫ murine embryonic fibroblast monolayers were E2-deprived, serum-starved, and stimulated with E2 for 15 min. Monolayers were washed, lysed, subjected to SDS-PAGE, transferred to nitrocellulose, and immunoblotted with phosphorylationspecific Akt antibody (pAKT) and reprobed with anti-Akt antibody (AKT). B, EC monolayers were transiently transfected with either control vector (pcDNA3) (lanes 1-3), kinase-dead c-Src (Src-K295M) (lanes 4 and 5) or constitutively active c-Src (Src 527F) (lanes 6 and 7). Cells were E2-deprived, serum-starved, and stimulated with E2 for 15 min. Cells were then washed, lysed, and immunoblotted with phosphorylation-specific Akt antibody (pAKT) and reprobed with anti-Akt antibody (AKT). Lanes 2 and 3 represent duplicate E2 stimulation of control cells.

c-Src Mediates Estrogen Receptor-dependent eNOS Activation
The expectation is that those ER-dependent sequential c-Src/ PI3-kinase/Akt activation events are rapidly catalyzed at the plasma membrane. This brings the focus back to that of a non-conventional, membrane-localized steroid hormone receptor-signaling pathway. There is an impressive and growing list of membrane steroid hormone-mediated responses in a variety of cells (9,(11)(12)(13)(14)(47)(48)(49)(50)(51)(52)(53)(54)(55). We have recently taken advantage of the EAhy.926 EC line, which, under the described culture conditions, does not express the 66-kDa, estrogen-responsive element-enhancing ER but does express a 46-kDa ER that is capable of transducing the signals we have described previously (9,12). We are currently identifying the requirements for membrane localization and preferentially expressed forms of ER in vascular tissue, which are responsible for ligand-induced c-Src activation and consequent NO release. As we come closer to identifying the most proximal components of this signal transduction cascade, the feasibility of targeting reagents to positively modulate cardiovascular responses expands.