Advertisement

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

Open AccessPublished:November 12, 2002DOI:https://doi.org/10.1074/jbc.M210828200
      17β-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 17β-estradiol rapidly activates eNOS through Src kinase in human endothelial cells. The Src family kinase specific-inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) abrogates 17β-estradiol- but not ionomycin-stimulated NO release. Consistent with these results, PP2 blocked 17β-estradiol-induced Akt phosphorylation but did not inhibit NO release from cells transduced with a constitutively active Akt. PP2 abrogated 17β-estradiol-induced activation of PI3-kinase, indicating that the PP2-inhibitable kinase is upstream of PI3-kinase and Akt. A 17β-estradiol-induced estrogen receptor/c-Src association correlated with rapid c-Src phosphorylation. Moreover, transfection of kinase-dead c-Src inhibited 17β-estradiol-induced Akt phosphorylation, whereas constitutively active c-Src increased basal Akt phosphorylation. Estrogen stimulation of murine embryonic fibroblasts with homozygous deletions of the c-src, fyn, and yes genes failed to induce Akt phosphorylation, whereas cells maintaining c-Src expression demonstrated estrogen-induced Akt activation. Estrogen rapidly activated c-Src inducing an estrogen receptor, c-Src, and P85 (regulatory subunit of PI3-kinase) complex formation. This complex formation results in the successive activation of PI3-kinase, Akt, and eNOS with consequent enhanced NO release, implicating c-Src as a critical upstream regulator of the estrogen-stimulated PI3-kinase/Akt/eNOS pathway.
      NO
      nitric oxide
      E2
      17β-estradiol
      EC
      endothelial cells
      ER
      estrogen receptor
      eNOS
      endothelial nitric-oxide synthase
      PI3-kinase
      phosphatidylinositol 3-kinase
      MAP
      mitogen-activated protein
      DMEM
      Dulbecco's modified Eagle's medium
      PP2
      4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine
      β-gal
      β-galactosidase
      myr-Akt
      membrane-targeted, myristoylated Akt
      HUVEC
      human umbilical vein endothelial cell
      PIP
      phosphatidylinositol phosphate
      The cardioprotective effects of estrogen are diverse, including both rapid non-genomic and delayed genomic effects on the blood vessel wall (reviewed in Ref.
      • Mendelsohn M.E.
      • Karas R.H.
      ). Specific, rapid vascular effects, such as moderation of vasomotor tone, have been linked to an estrogen-stimulated increase in bioavailable nitric oxide (NO)1 (
      • Rubanyi G.M.
      • Freay A.D.
      • Kauser K.
      • Sukovich D.
      • Burton G.
      • Lubahn D.B.
      • Couse J.F.
      • Curtis S.W.
      • Korach K.S.
      ,
      • Guetta V.
      • Quyyumi A.A.
      • Prasad A.
      • Panza J.A.
      • Waclawiw M.
      • Cannon 3rd, R.O.
      ,
      • Best P.J.
      • Berger P.B.
      • Miller V.M.
      • Lerman A.
      ). 17β-estradiol (E2) treatment of human endothelial cells (EC) induces rapid release of NO by estrogen receptor (ER)-dependent activation of endothelial nitric oxide synthase (eNOS) (
      • Caulin-Glaser T.
      • Garcia-Cardena G.
      • Sarrel P.
      • Sessa W.C.
      • Bender J.R.
      ). 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.
      • Fulton D.
      • Gratton J.P.
      • Sessa W.C.
      for review). eNOS is a Ca2+/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 Ca2+ concentrations (
      • McCabe T.J.
      • Fulton D.
      • Roman L.J.
      • Sessa W.C.
      ,
      • Fulton D.
      • Gratton J.P.
      • McCabe T.J.
      • Fontana J.
      • Fujio Y.
      • Walsh K.
      • Franke T.F.
      • Papapetropoulos A.
      • Sessa W.C.
      ). We demonstrated previously that the ER-dependent activation of eNOS occurs at resting Ca2+ concentrations and requires activation of the phosphatidylinositol-3-OH kinase (PI3-kinase)/Akt pathway (
      • Haynes M.P.
      • Sinha D.
      • Russell K.S.
      • Collinge M.
      • Fulton D.
      • Morales-Ruiz M.
      • Sessa W.C.
      • Bender J.R.
      ). 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 (
      • Simoncini T.
      • Hafezi-Moghadam A.
      • Brazil D.P.
      • Ley K.
      • Chin W.W.
      • Liao J.K.
      ). The E2-induced association correlated with increases in PI3-kinase 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 (
      • Pappas T.C.
      • Gametchu B.
      • Watson C.S.
      ,
      • Russell K.
      • Haynes M.
      • Sinha D.
      • Clerisme E.
      • Bender J.
      ,
      • Marquez D.C.
      • Pietras R.J.
      ,
      • Chambliss K.L.
      • Yuhanna I.S.
      • Mineo C.
      • Liu P.
      • German Z.
      • Sherman T.S.
      • Mendelsohn M.E.
      • Anderson R.G.
      • Shaul P.W.
      ). 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 (
      • Haynes M.P.
      • Sinha D.
      • Russell K.S.
      • Collinge M.
      • Fulton D.
      • Morales-Ruiz M.
      • Sessa W.C.
      • Bender J.R.
      ,
      • Russell K.
      • Haynes M.
      • Sinha D.
      • Clerisme E.
      • Bender J.
      ). 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 ERα or ERβ 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 (
      • Marquez D.C.
      • Pietras R.J.
      ,
      • Chambliss K.L.
      • Yuhanna I.S.
      • Mineo C.
      • Liu P.
      • German Z.
      • Sherman T.S.
      • Mendelsohn M.E.
      • Anderson R.G.
      • Shaul P.W.
      ). Additionally, a 46-kDa putative ER, reactive with anti-ER antibodies, was found in wild-type and in the initial ERα knockout mice. This form of the receptor was thought to be responsible for E2 enhancement of basal NO production in the initial ERα knockout mice, because this E2 effect was lost in the complete ERα knockout mouse (
      • Pendaries C.
      • Darblade B.
      • Rochaix P.
      • Krust A.
      • Chambon P.
      • Korach K.S.
      • Bayard F.
      • Arnal J.F.
      ). 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 (
      • Russell K.
      • Haynes M.
      • Sinha D.
      • Clerisme E.
      • Bender J.
      ). 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 (
      • Migliaccio A., Di
      • Domenico M.
      • Castoria G.
      • de Falco A.
      • Bontempo P.
      • Nola E.
      • Auricchio F.
      ,
      • Migliaccio A.
      • Pagano M.
      • Auricchio F.
      ). 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.

      DISCUSSION

      There are now numerous reports of estrogen-induced endothelial NO release in vitro and vasodilation in vivo (
      • Caulin-Glaser T.
      • Garcia-Cardena G.
      • Sarrel P.
      • Sessa W.C.
      • Bender J.R.
      ,
      • Haynes M.P.
      • Sinha D.
      • Russell K.S.
      • Collinge M.
      • Fulton D.
      • Morales-Ruiz M.
      • Sessa W.C.
      • Bender J.R.
      ,
      • Russell K.
      • Haynes M.
      • Sinha D.
      • Clerisme E.
      • Bender J.
      ,
      • Russell K.S.
      • Haynes M.P.
      • Caulin-Glaser T.
      • Rosneck J.
      • Sessa W.C.
      • Bender J.
      ,
      • Chen Z.
      • Yuhanna I.S.
      • Galcheva-Gargova Z.
      • Karas R.H.
      • Mendelsohn M.E.
      • Shaul P.W.
      ,
      • Lantin-Hermoso R.L.
      • Rosenfeld C.R.
      • Yuhanna I.S.
      • German Z.
      • Chen Z.
      • Shaul P.W.
      ,
      • Kirsch E.A.
      • Yuhanna I.S.
      • Chen Z.
      • German Z.
      • Sherman T.S.
      • Shaul P.W.
      ). 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 Ca2+ flux (
      • Caulin-Glaser T.
      • Garcia-Cardena G.
      • Sarrel P.
      • Sessa W.C.
      • Bender J.R.
      ). 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 Ser1177. This phosphorylation enhances electron flux through the eNOS reductase domain with a reduced rate of calmodulin dissociation at low (resting) Ca2+levels (
      • McCabe T.J.
      • Fulton D.
      • Roman L.J.
      • Sessa W.C.
      ,
      • Haynes M.P.
      • Sinha D.
      • Russell K.S.
      • Collinge M.
      • Fulton D.
      • Morales-Ruiz M.
      • Sessa W.C.
      • Bender J.R.
      ). This provided the first mechanistic explanation for E2-stimulated NO release in the absence of a Ca2+ 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 (
      • Haynes M.P.
      • Sinha D.
      • Russell K.S.
      • Collinge M.
      • Fulton D.
      • Morales-Ruiz M.
      • Sessa W.C.
      • Bender J.R.
      ,
      • Simoncini T.
      • Hafezi-Moghadam A.
      • Brazil D.P.
      • Ley K.
      • Chin W.W.
      • Liao J.K.
      ). In fact, a direct interaction between ER and P85, the regulatory subunit of PI3-kinase, has been demonstrated, correlating with increased PI3-kinase activity (
      • Simoncini T.
      • Hafezi-Moghadam A.
      • Brazil D.P.
      • Ley K.
      • Chin W.W.
      • Liao J.K.
      ). 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 ERK1/2 activation (
      • Russell K.
      • Haynes M.
      • Sinha D.
      • Clerisme E.
      • Bender J.
      ,
      • Chen Z.
      • Yuhanna I.S.
      • Galcheva-Gargova Z.
      • Karas R.H.
      • Mendelsohn M.E.
      • Shaul P.W.
      ). 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 (
      • von Willebrand M.
      • Williams S.
      • Saxena M.
      • Gilman J.
      • Tailor P.
      • Jascur T.
      • Amarante-Mendes G.P.
      • Green D.R.
      • Mustelin T.
      ,
      • Cuevas B., Lu, Y.
      • Watt S.
      • Kumar R.
      • Zhang J.
      • Siminovitch K.A.
      • Mills G.B.
      ,
      • Cuevas B.D., Lu, Y.
      • Mao M.
      • Zhang J.
      • LaPushin R.
      • Siminovitch K.
      • Mills G.B.
      ), and fyn, lyn, and lck can, through their SH3 domains, interact with P85 (
      • Herrera-Velit P.
      • Reiner N.E.
      ,
      • Kapeller R.
      • Prasad K.V.
      • Janssen O.
      • Hou W.
      • Schaffhausen B.S.
      • Rudd C.E.
      • Cantley L.C.
      ,
      • Mak P., He, Z.
      • Kurosaki T.
      ,
      • Prasad K.V.
      • Janssen O.
      • Kapeller R.
      • Raab M.
      • Cantley L.C.
      • Rudd C.E.
      ,
      • Prasad K.V.
      • Kapeller R.
      • Janssen O.
      • Duke-Cohan J.S.
      • Repke H.
      • Cantley L.C.
      • Rudd C.E.
      ,
      • Susa M.
      • Rohner D.
      • Bichsel S.
      ). Third, estrogen-induced c-Src phosphorylation has been demonstrated in osteoclasts and breast cancer cell lines (
      • Kousteni S.
      • Bellido T.
      • Plotkin L.I.
      • O'Brien C.A.
      • Bodenner D.L.
      • Han L.
      • Han K.
      • DiGregorio G.B.
      • Katzenellenbogen J.A.
      • Katzenellenbogen B.S.
      • Roberson P.K.
      • Weinstein R.S.
      • Jilka R.L.
      • Manolagas S.C.
      ,
      • Migliaccio A.
      • Castoria G., Di
      • Domenico M.
      • de Falco A.
      • Bilancio A.
      • Lombardi M.
      • Barone M.V.
      • Ametrano D.
      • Zannini M.S.
      • Abbondanza C.
      • Auricchio F.
      ).
      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 kinase-dead 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 Tyr688 by the Src kinases lck and abl (
      • von Willebrand M.
      • Williams S.
      • Saxena M.
      • Gilman J.
      • Tailor P.
      • Jascur T.
      • Amarante-Mendes G.P.
      • Green D.R.
      • Mustelin T.
      ,
      • Cuevas B., Lu, Y.
      • Watt S.
      • Kumar R.
      • Zhang J.
      • Siminovitch K.A.
      • Mills G.B.
      ,
      • Cuevas B.D., Lu, Y.
      • Mao M.
      • Zhang J.
      • LaPushin R.
      • Siminovitch K.
      • Mills G.B.
      ), and PI3-kinase has been shown to be a preferential substrate for c-Src (
      • Cantley L.C.
      • Auger K.R.
      • Carpenter C.
      • Duckworth B.
      • Graziani A.
      • Kapeller R.
      • Soltoff S.
      ,
      • Carpenter C.L.
      • Duckworth B.C.
      • Auger K.R.
      • Cohen B.
      • Schaffhausen B.S.
      • Cantley L.C.
      ). 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 PI3-kinase P110 catalytic subunit (
      • Lee A.W.
      • States D.J.
      ,
      • Shinohara M.
      • Kodama A.
      • Matozaki T.
      • Fukuhara A.
      • Tachibana K.
      • Nakanishi H.
      • Takai Y.
      ). Alternatively, the SH3 domains of several Src kinases have been shown to bind directly to P85 and regulate its activity (
      • Herrera-Velit P.
      • Reiner N.E.
      ,
      • Kapeller R.
      • Prasad K.V.
      • Janssen O.
      • Hou W.
      • Schaffhausen B.S.
      • Rudd C.E.
      • Cantley L.C.
      ,
      • Mak P., He, Z.
      • Kurosaki T.
      ,
      • Prasad K.V.
      • Janssen O.
      • Kapeller R.
      • Raab M.
      • Cantley L.C.
      • Rudd C.E.
      ,
      • Prasad K.V.
      • Kapeller R.
      • Janssen O.
      • Duke-Cohan J.S.
      • Repke H.
      • Cantley L.C.
      • Rudd C.E.
      ,
      • Susa M.
      • Rohner D.
      • Bichsel S.
      ). This includes c-Src that, in osteoclasts, interacts directly through its SH3 domain with P85, in response to colony stimulating factor-1 (
      • Grey A.
      • Chen Y.
      • Paliwal I.
      • Carlberg K.
      • Insogna K.
      ).
      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 3,4,5-phosphate generation (
      • Simoncini T.
      • Hafezi-Moghadam A.
      • Brazil D.P.
      • Ley K.
      • Chin W.W.
      • Liao J.K.
      ). As might be expected, ER and androgen receptor have been shown to directly couple with c-Src, whereas the progesterone receptor has not (
      • Migliaccio A.
      • Piccolo D.
      • Castoria G., Di
      • Domenico M.
      • Bilancio A.
      • Lombardi M.
      • Gong W.
      • Beato M.
      • Auricchio F.
      ,
      • Migliaccio A.
      • Castoria G., Di
      • Domenico M.
      • de Falco A.
      • Bilancio A.
      • Lombardi M.
      • Barone M.V.
      • Ametrano D.
      • Zannini M.S.
      • Abbondanza C.
      • Auricchio F.
      ), consistent with the notion that steroid hormone receptor-induced PI3-kinase 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 (
      • Migliaccio A.
      • Piccolo D.
      • Castoria G., Di
      • Domenico M.
      • Bilancio A.
      • Lombardi M.
      • Gong W.
      • Beato M.
      • Auricchio F.
      ). Also, a ternary androgen receptor/ER/c-Src complex has been demonstrated, through an ER-pTyr537/c-Src-SH2 and androgen receptor/c-Src-SH3 interaction (
      • Migliaccio A.
      • Castoria G., Di
      • Domenico M.
      • de Falco A.
      • Bilancio A.
      • Lombardi M.
      • Barone M.V.
      • Ametrano D.
      • Zannini M.S.
      • Abbondanza C.
      • Auricchio F.
      ). Whether ER-pTyr537 is constitutively or inducibly (by estrogen) phosphorylated remains unclear.
      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 (
      • Haynes M.P.
      • Sinha D.
      • Russell K.S.
      • Collinge M.
      • Fulton D.
      • Morales-Ruiz M.
      • Sessa W.C.
      • Bender J.R.
      ,
      • Pappas T.C.
      • Gametchu B.
      • Watson C.S.
      ,
      • Russell K.
      • Haynes M.
      • Sinha D.
      • Clerisme E.
      • Bender J.
      ,
      • Marquez D.C.
      • Pietras R.J.
      ,
      • Chambliss K.L.
      • Yuhanna I.S.
      • Mineo C.
      • Liu P.
      • German Z.
      • Sherman T.S.
      • Mendelsohn M.E.
      • Anderson R.G.
      • Shaul P.W.
      ,
      • Brubaker K.D.
      • Gay C.V.
      ,
      • Falkenstein E.
      • Heck M.
      • Gerdes D.
      • Grube D.
      • Christ M.
      • Weigel M.
      • Buddhikot M.
      • Meizel S.
      • Wehling M.
      ,
      • Germain P.S.
      • Metezeau P.
      • Tiefenauer L.X.
      • Kiefer H.
      • Ratinaud M.H.
      • Habrioux G.
      ,
      • Kelly M.J.
      • Levin E.R.
      ,
      • Pietras R.J.
      • Szego C.M.
      ,
      • Razandi M.
      • Pedram A.
      • Greene G.L.
      • Levin E.R.
      ,
      • Razandi M.
      • Pedram A.
      • Levin E.R.
      ,
      • Watson C.S.
      • Gametchu B.
      ,
      • Watters J.J.
      • Campbell J.S.
      • Cunningham M.J.
      • Krebs E.G.
      • Dorsa D.M.
      ). 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 (
      • Haynes M.P.
      • Sinha D.
      • Russell K.S.
      • Collinge M.
      • Fulton D.
      • Morales-Ruiz M.
      • Sessa W.C.
      • Bender J.R.
      ,
      • Russell K.
      • Haynes M.
      • Sinha D.
      • Clerisme E.
      • Bender J.
      ). 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.

      Acknowledgments

      We gratefully acknowledge Lynn O'Donnell for technical assistance and all those providing valuable reagents, including K. Walsh for the recombinant adenoviruses used.

      REFERENCES

        • Mendelsohn M.E.
        • Karas R.H.
        N. Engl. J. Med. 1999; 340: 1801-1811
        • Rubanyi G.M.
        • Freay A.D.
        • Kauser K.
        • Sukovich D.
        • Burton G.
        • Lubahn D.B.
        • Couse J.F.
        • Curtis S.W.
        • Korach K.S.
        J. Clin. Invest. 1997; 99: 2429-2437
        • Guetta V.
        • Quyyumi A.A.
        • Prasad A.
        • Panza J.A.
        • Waclawiw M.
        • Cannon 3rd, R.O.
        Circulation. 1997; 96: 2795-2801
        • Best P.J.
        • Berger P.B.
        • Miller V.M.
        • Lerman A.
        Ann. Intern. Med. 1998; 128: 285-288
        • Caulin-Glaser T.
        • Garcia-Cardena G.
        • Sarrel P.
        • Sessa W.C.
        • Bender J.R.
        Circ. Res. 1997; 81: 885-892
        • Fulton D.
        • Gratton J.P.
        • Sessa W.C.
        J. Pharmacol. Exp. Ther. 2001; 299: 818-824
        • McCabe T.J.
        • Fulton D.
        • Roman L.J.
        • Sessa W.C.
        J. Biol. Chem. 2000; 275: 6123-6128
        • Fulton D.
        • Gratton J.P.
        • McCabe T.J.
        • Fontana J.
        • Fujio Y.
        • Walsh K.
        • Franke T.F.
        • Papapetropoulos A.
        • Sessa W.C.
        Nature. 1999; 399: 597-601
        • Haynes M.P.
        • Sinha D.
        • Russell K.S.
        • Collinge M.
        • Fulton D.
        • Morales-Ruiz M.
        • Sessa W.C.
        • Bender J.R.
        Circ. Res. 2000; 87: 677-682
        • Simoncini T.
        • Hafezi-Moghadam A.
        • Brazil D.P.
        • Ley K.
        • Chin W.W.
        • Liao J.K.
        Nature. 2000; 407: 538-541
        • Pappas T.C.
        • Gametchu B.
        • Watson C.S.
        FASEB J. 1995; 9: 404-410
        • Russell K.
        • Haynes M.
        • Sinha D.
        • Clerisme E.
        • Bender J.
        Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5930-5935
        • Marquez D.C.
        • Pietras R.J.
        Oncogene. 2001; 20: 5420-5430
        • Chambliss K.L.
        • Yuhanna I.S.
        • Mineo C.
        • Liu P.
        • German Z.
        • Sherman T.S.
        • Mendelsohn M.E.
        • Anderson R.G.
        • Shaul P.W.
        Circ. Res. 2000; 87: E44-E52
        • Pendaries C.
        • Darblade B.
        • Rochaix P.
        • Krust A.
        • Chambon P.
        • Korach K.S.
        • Bayard F.
        • Arnal J.F.
        Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 2205-2210
        • Migliaccio A., Di
        • Domenico M.
        • Castoria G.
        • de Falco A.
        • Bontempo P.
        • Nola E.
        • Auricchio F.
        EMBO J. 1996; 15: 1292-1300
        • Migliaccio A.
        • Pagano M.
        • Auricchio F.
        Oncogene. 1993; 8: 2183-2191
        • Edgell C.J.
        • McDonald C.C.
        • Graham J.B.
        Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 3734-3737
        • Klinghoffer R.A.
        • Sachsenmaier C.
        • Cooper J.A.
        • Soriano P.
        EMBO J. 1999; 18: 2459-2471
        • Persad S.
        • Attwell S.
        • Gray V.
        • Mawji N.
        • Deng J.T.
        • Leung D.
        • Yan J.
        • Sanghera J.
        • Walsh M.P.
        • Dedhar S.
        J. Biol. Chem. 2001; 276: 27462-27469
        • Chen R.
        • Kim O.
        • Yang J.
        • Sato K.
        • Eisenmann K.M.
        • McCarthy J.
        • Chen H.
        • Qiu Y.
        J. Biol. Chem. 2001; 276: 31858-31862
        • Hanke J.H.
        • Gardner J.P.
        • Dow R.L.
        • Changelian P.S.
        • Brissette W.H.
        • Weringer E.J.
        • Pollok B.A.
        • Connelly P.A.
        J. Biol. Chem. 1996; 271: 695-701
        • Salazar E.P.
        • Rozengurt E.
        J. Biol. Chem. 1999; 274: 28371-28378
        • Kousteni S.
        • Bellido T.
        • Plotkin L.I.
        • O'Brien C.A.
        • Bodenner D.L.
        • Han L.
        • Han K.
        • DiGregorio G.B.
        • Katzenellenbogen J.A.
        • Katzenellenbogen B.S.
        • Roberson P.K.
        • Weinstein R.S.
        • Jilka R.L.
        • Manolagas S.C.
        Cell. 2001; 104: 719-730
        • Migliaccio A.
        • Piccolo D.
        • Castoria G., Di
        • Domenico M.
        • Bilancio A.
        • Lombardi M.
        • Gong W.
        • Beato M.
        • Auricchio F.
        EMBO J. 1998; 17: 2008-2018
        • Brubaker K.D.
        • Gay C.V.
        J. Cell. Biochem. 1999; 76: 206-216
        • Bjorge J.D.
        • Jakymiw A.
        • Fujita D.J.
        Oncogene. 2000; 19: 5620-5635
        • Russell K.S.
        • Haynes M.P.
        • Caulin-Glaser T.
        • Rosneck J.
        • Sessa W.C.
        • Bender J.
        J. Biol. Chem. 2000; 275: 5026-5030
        • Migliaccio A.
        • Castoria G., Di
        • Domenico M.
        • de Falco A.
        • Bilancio A.
        • Lombardi M.
        • Barone M.V.
        • Ametrano D.
        • Zannini M.S.
        • Abbondanza C.
        • Auricchio F.
        EMBO J. 2000; 19: 5406-5417
        • Chen Z.
        • Yuhanna I.S.
        • Galcheva-Gargova Z.
        • Karas R.H.
        • Mendelsohn M.E.
        • Shaul P.W.
        J. Clin. Invest. 1999; 103: 401-406
        • Lantin-Hermoso R.L.
        • Rosenfeld C.R.
        • Yuhanna I.S.
        • German Z.
        • Chen Z.
        • Shaul P.W.
        Am. J. Physiol. 1997; 273: L119-L126
        • Kirsch E.A.
        • Yuhanna I.S.
        • Chen Z.
        • German Z.
        • Sherman T.S.
        • Shaul P.W.
        Am. J. Respir. Cell Mol. Biol. 1999; 20: 658-666
        • von Willebrand M.
        • Williams S.
        • Saxena M.
        • Gilman J.
        • Tailor P.
        • Jascur T.
        • Amarante-Mendes G.P.
        • Green D.R.
        • Mustelin T.
        J. Biol. Chem. 1998; 273: 3994-4000
        • Cuevas B., Lu, Y.
        • Watt S.
        • Kumar R.
        • Zhang J.
        • Siminovitch K.A.
        • Mills G.B.
        J. Biol. Chem. 1999; 274: 27583-27589
        • Cuevas B.D., Lu, Y.
        • Mao M.
        • Zhang J.
        • LaPushin R.
        • Siminovitch K.
        • Mills G.B.
        J. Biol. Chem. 2001; 276: 27455-27461
        • Herrera-Velit P.
        • Reiner N.E.
        J. Immunol. 1996; 156: 1157-1165
        • Kapeller R.
        • Prasad K.V.
        • Janssen O.
        • Hou W.
        • Schaffhausen B.S.
        • Rudd C.E.
        • Cantley L.C.
        J. Biol. Chem. 1994; 269: 1927-1933
        • Mak P., He, Z.
        • Kurosaki T.
        FEBS Lett. 1996; 397 (2–3): 183-185
        • Prasad K.V.
        • Janssen O.
        • Kapeller R.
        • Raab M.
        • Cantley L.C.
        • Rudd C.E.
        Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7366-7370
        • Prasad K.V.
        • Kapeller R.
        • Janssen O.
        • Duke-Cohan J.S.
        • Repke H.
        • Cantley L.C.
        • Rudd C.E.
        Philos. Trans. R. Soc. Lond-Biol. Sci. 1993; 342: 35-42
        • Susa M.
        • Rohner D.
        • Bichsel S.
        Biochem. Biophys. Res. Commun. 1996; 220: 729-734
        • Cantley L.C.
        • Auger K.R.
        • Carpenter C.
        • Duckworth B.
        • Graziani A.
        • Kapeller R.
        • Soltoff S.
        Cell. 1991; 64: 281-302
        • Carpenter C.L.
        • Duckworth B.C.
        • Auger K.R.
        • Cohen B.
        • Schaffhausen B.S.
        • Cantley L.C.
        J. Biol. Chem. 1990; 265: 19704-19711
        • Lee A.W.
        • States D.J.
        Mol. Cell. Biol. 2000; 20: 6779-6798
        • Shinohara M.
        • Kodama A.
        • Matozaki T.
        • Fukuhara A.
        • Tachibana K.
        • Nakanishi H.
        • Takai Y.
        J. Biol. Chem. 2001; 276: 18941-18946
        • Grey A.
        • Chen Y.
        • Paliwal I.
        • Carlberg K.
        • Insogna K.
        Endocrinology. 2000; 141: 2129-2138
        • Brubaker K.D.
        • Gay C.V.
        Calcif. Tissue Int. 1999; 64: 459-462
        • Falkenstein E.
        • Heck M.
        • Gerdes D.
        • Grube D.
        • Christ M.
        • Weigel M.
        • Buddhikot M.
        • Meizel S.
        • Wehling M.
        Endocrinology. 1999; 140: 5999-6002
        • Germain P.S.
        • Metezeau P.
        • Tiefenauer L.X.
        • Kiefer H.
        • Ratinaud M.H.
        • Habrioux G.
        Anticancer Res. 1993; 13: 2347-2353
        • Kelly M.J.
        • Levin E.R.
        Trends Endocrinol. Metab. 2001; 12: 152-156
        • Pietras R.J.
        • Szego C.M.
        Nature. 1977; 265: 69-72
        • Razandi M.
        • Pedram A.
        • Greene G.L.
        • Levin E.R.
        Mol. Endocrinol. 1999; 13: 307-319
        • Razandi M.
        • Pedram A.
        • Levin E.R.
        J. Biol. Chem. 2000; 275: 38540-38546
        • Watson C.S.
        • Gametchu B.
        Proc. Soc. Exp. Biol. Med. 1999; 220: 9-19
        • Watters J.J.
        • Campbell J.S.
        • Cunningham M.J.
        • Krebs E.G.
        • Dorsa D.M.
        Endocrinology. 1997; 138: 4030-4033