E-selectin is a cytokine-inducible adhesion molecule that is expressed by activated endothelial cells at sites of inflammation. In addition to supporting rolling and stable arrest of leukocytes, there is increasing evidence that E-selectin functions in transmembrane signaling into endothelial cells during these adhesive interactions. We have previously shown that adhesion of HL-60 cells (which express ligands for E-selectin), or antibody-mediated cross-linking of E-selectin, results in formation of a Ras/Raf-1/phospho-MEK macrocomplex, extracellular signal-regulated protein kinase (ERK1/2) activation, and c-fos up-regulation. All of these downstream signaling events appear to require an intact cytoplasmic domain of E-selectin. Here we demonstrate that tyrosine 603 in the cytoplasmic domain of E-selectin is required for the E-selectin-dependent ERK1/2 activation. Tyrosine 603 plays an important role in mediating the association of E-selectin with SHP2, and the catalytic domain of SHP2 is, in turn, critical for E-selectin-dependent ERK1/2 activation. An adapter protein complex consisting of Shc·Grb2·Sos bridges between SHP2 and the Ras·Raf·phospho-MEK macrocomplex. These molecular events thus outline a mechanism by which cross-linking of E-selectin by engagement of ligands on adherent leukocytes can initiate a multifunctional signaling pathway in the activated endothelial cell at sites of inflammation. E-selectin is a cytokine-inducible adhesion molecule that is expressed by activated endothelial cells at sites of inflammation. In addition to supporting rolling and stable arrest of leukocytes, there is increasing evidence that E-selectin functions in transmembrane signaling into endothelial cells during these adhesive interactions. We have previously shown that adhesion of HL-60 cells (which express ligands for E-selectin), or antibody-mediated cross-linking of E-selectin, results in formation of a Ras/Raf-1/phospho-MEK macrocomplex, extracellular signal-regulated protein kinase (ERK1/2) activation, and c-fos up-regulation. All of these downstream signaling events appear to require an intact cytoplasmic domain of E-selectin. Here we demonstrate that tyrosine 603 in the cytoplasmic domain of E-selectin is required for the E-selectin-dependent ERK1/2 activation. Tyrosine 603 plays an important role in mediating the association of E-selectin with SHP2, and the catalytic domain of SHP2 is, in turn, critical for E-selectin-dependent ERK1/2 activation. An adapter protein complex consisting of Shc·Grb2·Sos bridges between SHP2 and the Ras·Raf·phospho-MEK macrocomplex. These molecular events thus outline a mechanism by which cross-linking of E-selectin by engagement of ligands on adherent leukocytes can initiate a multifunctional signaling pathway in the activated endothelial cell at sites of inflammation. interleukin 1b extracellular signal-regulated kinase rat sarcoma virus proto-oncogene mitogen-activated protein kinase/ERK1/2 Src homology domain 2 SH2-containing protein-tyrosinep hosphatase 2 SH2-domain-containing α2-c ollagen-related growth factorreceptor-bound protein 2 sonof sevenless human umbilical vein endothelial cells 4-morpholinepropanesulfonic acid fetal bovine serum monoclonal antibody E-selectin is an inducible adhesion molecule that is expressed at relatively high density on the surface of cultured endothelial cells that have been activated by proinflammatory cytokines, such as interleukin-1b (IL-1β),1 tumor necrosis factor-α, or bacterial endotoxin. It is detectable in vivo at the sites of inflammation (1Bevilacqua M.P. Stengelin S. Gimbrone M.A.J. Seed B. Science. 1989; 243: 1160-1165Crossref PubMed Scopus (1660) Google Scholar, 2Bevilacqua M.P. Nelson R.M. J. Clin. Invest. 1993; 91: 379-387Crossref PubMed Scopus (1055) Google Scholar, 3Milstone D.S. Fukumura D. Padgett R.C. O'Donnell P.E. Davis V.M. Benavidez O.J. Monsky W.L. Melder R.J. Jain R.K. Gimbrone M.A.J. Microcirculation. 1998; 5: 153-171Crossref PubMed Google Scholar). In addition to supporting the rolling and stable arrest of leukocytes on activated endothelium, there is increasing evidence that E-selectin can transduce outside-in signals (4Lorenzon P. Vecile E. Nardon E. Ferrero E. Harlan J.M. Tedesco F. Dobrina A. J. Cell Biol. 1998; 142: 1381-1391Crossref PubMed Scopus (205) Google Scholar, 5Yoshida M. Westlin W.F. Wang N. Ingber D.E. Rosenzweig A. Resnick N. Gimbrone M.A.J. J. Cell Biol. 1996; 133: 445-455Crossref PubMed Scopus (188) Google Scholar, 6Yoshida M. Szente B.E. Kiely J.M. Rosenzweig A. Gimbrone Jr., M.A. J. Immunol. 1998; 161: 933-941PubMed Google Scholar). Recently our laboratory demonstrated that leukocyte adhesion to cell surface E-selectin-activated extracellular signal-regulated protein kinase (ERK1/2), formed a macrocomplex containing Ras/Raf-1/phospho-MEK, and resulted in the up-regulation of c-fos expression (7Hu Y. Kiely J.M. Szente B.E. Rosenzweig A. Gimbrone Jr., M.A. J. Immunol. 2000; 165: 2142-2148Crossref PubMed Scopus (73) Google Scholar). In this study, we have investigated the molecular events occurring immediately upstream of Ras/Raf/phospho-MEK and downstream of cell surface E-selectin.The cytoplasmic domain of E-selectin has been implicated in transmembrane signaling (5Yoshida M. Westlin W.F. Wang N. Ingber D.E. Rosenzweig A. Resnick N. Gimbrone M.A.J. J. Cell Biol. 1996; 133: 445-455Crossref PubMed Scopus (188) Google Scholar, 7Hu Y. Kiely J.M. Szente B.E. Rosenzweig A. Gimbrone Jr., M.A. J. Immunol. 2000; 165: 2142-2148Crossref PubMed Scopus (73) Google Scholar); however, the molecular mechanisms involved have not been well defined. The cytoplasmic domain of E-selectin consists of 32 amino acids (1Bevilacqua M.P. Stengelin S. Gimbrone M.A.J. Seed B. Science. 1989; 243: 1160-1165Crossref PubMed Scopus (1660) Google Scholar), including two tyrosine residues. It has been well documented that phosphorylation of tyrosine residues in the cytoplasmic domains of various types of receptors can play an important role in receptor-mediated transmembrane signal transduction, especially ERK1/2 activation. Therefore, it is reasonable to hypothesize that the two tyrosine residues on the cytoplasmic domain of E-selectin may be involved in the E-selectin-dependent ERK1/2 activation described by our laboratory (7Hu Y. Kiely J.M. Szente B.E. Rosenzweig A. Gimbrone Jr., M.A. J. Immunol. 2000; 165: 2142-2148Crossref PubMed Scopus (73) Google Scholar).The protein-tyrosine phosphatase SHP2 is a ubiquitously expressed cytosolic protein, which contains two amino-terminal tandem SH2 domains and a carboxyl-terminal catalytic domain (8Neel B.G. Semin. Cell Biol. 1993; 4: 419-432Crossref PubMed Scopus (106) Google Scholar). SHP2 associates with tyrosine-phosphorylated epidermal growth factor receptor, the platelet-derived growth factor receptor (9Case R.D. Piccione E. Wolf G. Benett A.M. Lechleider R.J. Neel B.G. Shoelson S.E. J. Biol. Chem. 1994; 269: 10467-10474Abstract Full Text PDF PubMed Google Scholar), insulin receptor (10Hausdorff S.F. Bennett A.M. Neel B.G. Birnbaum M.J. J. Biol. Chem. 1995; 270: 12965-12968Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar), and with the T and B cell receptors (11Siminovitch K.A. Neel B.G. Semin. Immunol. 1998; 10: 329-3247Crossref PubMed Scopus (61) Google Scholar). It becomes tyrosine phosphorylated upon cell stimulation and can become associated with other adapter proteins (12Bennett A.M. Tang T.L. Sugimoto S. Walsh C.T. Neel B.G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7335-7339Crossref PubMed Scopus (344) Google Scholar, 13Tang T.L. Freeman Jr., R.M. O'Reilly A.M. Neel B.G. Sokol S.Y. Cell. 1995; 80: 473-483Abstract Full Text PDF PubMed Scopus (307) Google Scholar, 14Neel B.G. Tonks N.K. Curr. Opin. Cell Biol. 1997; : 193-204Crossref PubMed Scopus (732) Google Scholar). Moreover, SHP2 has been show to have a positive effect on ERK1/2 signaling pathway (15Oh E.S. Gu H. Saxton T.M. Timms J.F. Hausdorff S. Frevert E.U. Kahn B.B. Pawson T. Neel B.G. Thomas S.M. Mol. Cell. Biol. 1999; 19: 3205-3215Crossref PubMed Scopus (194) Google Scholar), (16Yamauchi K. Milarski K.L. Saltiel A.R. Pessin J.E. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 664-668Crossref PubMed Scopus (268) Google Scholar).In this study, we describe the molecular mechanisms involved in the initiation of transmembrane signaling by cross-linking of cell surface E-selectin. These include tyrosine phosphorylation of E-selectin, association with SHP2, and assembly of the adapter proteins, Shc, Grb2, and Sos, to form a signaling complex, which then bridges SHP2 to the Ras·Raf-1·phospho-MEK macrocomplex, resulting in ERK1/2 activation.DISCUSSIONThere is ample evidence that transmembrane adhesion molecules can both physically bridge and biochemically transduce signals among interacting cells. The selectins, a family of adhesion molecules involved in leukocyte adhesion to activated vascular endothelium, are no exception. Binding to L-selectin has been shown to generate both inside-out and outside-in signals, such as the activation of the Ras pathway and potentiation of the oxidative burst of human neutrophils (20Brenner B. Gulbins E. Schlottmann K. Koppenhoefer U. Busch G.L. Walzog B. Steinhausen M. Coggeshall K.M. Linderkamp O. Lang F. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 15376-15381Crossref PubMed Scopus (142) Google Scholar, 26Crockett-Torabi E. Fantone J.C. Am. J. Physiol. 1997; 272: H1302-1308PubMed Google Scholar, 27Steeber D.A. Engel P. Miller A.S. Sheetz M.P. Tedder T.F. J. Immunol. 1997; 159: 952-963PubMed Google Scholar, 28Brenner B. Gulbins E. Busch G.L. Koppenhoefer U. Lang F. Linderkamp O. Biochem. Biophys. Res. Commun. 1997; 231: 802-807Crossref PubMed Scopus (61) Google Scholar, 29Brenner B. Grassme H.U. Muller C. Lang F. Speer C.P. Gulbins E. Exp. Cell Res. 1998; 243: 123-128Crossref PubMed Scopus (32) Google Scholar, 30Waddell T.K. Fialkow L. Chan C.K. Kishimoto T.K. Downey G.P. J. Biol. Chem. 1994; 269: 18485-18491Abstract Full Text PDF PubMed Google Scholar, 31Waddell T.K. Fialkow L. Chan C.K. Kishimoto T.K. Downey G.P. J. Biol. Chem. 1995; 270: 15403-15411Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar). P-selectin also has been shown to transduce signals across the plasma membrane, resulting in the induction of tyrosine phosphorylation of focal adhesion kinase and transient increases in intracellular Ca2+ (4Lorenzon P. Vecile E. Nardon E. Ferrero E. Harlan J.M. Tedesco F. Dobrina A. J. Cell Biol. 1998; 142: 1381-1391Crossref PubMed Scopus (205) Google Scholar, 32Haller H. Kunzendorf U. Sacherer K. Lindschau C. Walz G. Distler A. Luft F.C. J. Immunol. 1997; 158: 1061-1067PubMed Google Scholar). Our laboratory has demonstrated that adhesion of HL-60 cells, which expresses ligands for E-selectin, or monoclonal antibody (mAb)-mediated cell surface E-selectin cross-linking, can induce cytoskeletal linkage of E-selectin (5Yoshida M. Westlin W.F. Wang N. Ingber D.E. Rosenzweig A. Resnick N. Gimbrone M.A.J. J. Cell Biol. 1996; 133: 445-455Crossref PubMed Scopus (188) Google Scholar), dephosphorylation of serine residues in the cytoplasmic domain of E-selectin (6Yoshida M. Szente B.E. Kiely J.M. Rosenzweig A. Gimbrone Jr., M.A. J. Immunol. 1998; 161: 933-941PubMed Google Scholar), and activation of ERK1/2, as well as the formation of a Ras·Raf·MEK macrocomplex (7Hu Y. Kiely J.M. Szente B.E. Rosenzweig A. Gimbrone Jr., M.A. J. Immunol. 2000; 165: 2142-2148Crossref PubMed Scopus (73) Google Scholar).In this study, we used E-selectin single-point mutants to demonstrate that one tyrosine residue, Tyr603, in the cytoplasmic domain of E-selectin plays an essential role in the association between E-selectin and SHP2, and in mediating the ERK1/2 activation induced by cross-linking cell surface E-selectin. A consensus sequence for SHP2 binding, Y(I/V)X (V/I/L/P), has been defined in several proteins, such as platelet-derived growth factor β-receptor and platelet endothelial cell adhesion molecule)-1 (33Ronnstrand L. Arvidsson A.K. Kallin A. Rorsman C. Hellman U. Engstrom U. Wernstedt C. Heldin C.H. Oncogene. 1999; 18: 3696-3702Crossref PubMed Scopus (62) Google Scholar, 34Masuda M. Osawa M. Shigematsu H. Harada N. Fujiwara K. FEBS Lett. 1997; 408: 331-336Crossref PubMed Scopus (91) Google Scholar). The cytoplasmic domain of E-selectin does contain similar motifs around the Tyr603 (YQKP) and Tyr608 (YIL) residues. Upon cross-linking of cell surface E-selectin, SHP2 becomes associated with E-selectin in a time-dependent (Fig. 1 B) and dose-dependent (data not shown) manner; however, only Tyr603 appeared to play an essential role in this association. Furthermore, Tyr603 plays an important role in E-selectin-dependent ERK1/2 activation (Fig. 2). Although the basal level of the association between SHP2 and E-selectin mutated at Tyr608 appears to be slightly higher than the wild-type E-selectin (Fig. 1 C), this increased association does not result in any E-selectin-dependent ERK1/2 activation (Fig.2), suggesting a nonspecific effect. Cross-linking another endothelial cell surface heterodimer, the HLA class I molecule, which is present at comparable density on the surface of IL-1β-activated HUVEC, did not generate any changes in tyrosine phosphorylation on E-selectin (Fig.1 A). Our previously studies have indicated that antibody-mediated cross-linking can be utilized to mimic the specific clustering of cell surface E-selectin that presumably occurs during leukocyte adhesion, and results in activation of ERK1/2 (5Yoshida M. Westlin W.F. Wang N. Ingber D.E. Rosenzweig A. Resnick N. Gimbrone M.A.J. J. Cell Biol. 1996; 133: 445-455Crossref PubMed Scopus (188) Google Scholar, 7Hu Y. Kiely J.M. Szente B.E. Rosenzweig A. Gimbrone Jr., M.A. J. Immunol. 2000; 165: 2142-2148Crossref PubMed Scopus (73) Google Scholar). Taken together, these data strongly suggest the existence of an E-selectin-specific signaling pathway leading to the activation of ERK1/2 that includes Tyr603 in the cytoplasmic domain of E-selectin. Previously, our laboratory had shown that serine residues on the cytoplasmic domain of E-selectin became de-phosphorylated upon HL-60 adhesion (6Yoshida M. Szente B.E. Kiely J.M. Rosenzweig A. Gimbrone Jr., M.A. J. Immunol. 1998; 161: 933-941PubMed Google Scholar). There have been reports that serine/threonine dephosphorylation may be involved in the subsequent tyrosine phosphorylation (35Artcanuthurry V. Grelac F. Maclouf J. Martin-Cramer E. Levy-Toledano S. Semin. Thromb. Hemostasis. 1996; 22: 317-326Crossref PubMed Scopus (13) Google Scholar). We are in the process of determining whether cross-linking-induced dephosphorylation on serine residues is required for the observed phosphorylation on tyrosine residues in the cytoplasmic domain of E-selectin.It has been shown that overexpression of a catalytically inactive SHP2 can block ERK1/2 activation in response to insulin, platelet-derived growth factor (36Milarski K.L. Saltiel A.R. J. Biol. Chem. 1994; 269: 21239-21243Abstract Full Text PDF PubMed Google Scholar, 37Rivard N. McKenzie F.R. Brondello J.M. Pouyssegur J. J. Biol. Chem. 1995; 270: 11017-11024Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar), epidermal growth factor (38Zhao Z. Tan Z. Wright J.H. Diltz C.D. Shen S.H. Krebs E.G. Fischer E.H. J. Biol. Chem. 1995; 270: 11765-11769Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar), and fibroblast growth factor (13Tang T.L. Freeman Jr., R.M. O'Reilly A.M. Neel B.G. Sokol S.Y. Cell. 1995; 80: 473-483Abstract Full Text PDF PubMed Scopus (307) Google Scholar), via a dominant-negative effect. In our system, COS-7 cells co-transfected with WT-E-selectin and catalytically inert SHP2 (dominant negative SHP2), showed significantly blunted E-selectin-dependent ERK1/2 activation (Fig. 3). This suggests that SHP2 is a positive effector upstream of E-selectin-dependent ERK1/2 activation. We have further observed that E-selectin-associated SHP2 can de-phosphorylate tyrosine-phosphorylated E-selectin in vitro (Fig. 4), however; the exact substrate(s) of SHP2 in the context of E-selectin-dependent signaling in vivo are yet to be determined.In other systems, tyrosine-phosphorylated SHP2 functions as an adapter protein with positive effects for downstream signaling (12Bennett A.M. Tang T.L. Sugimoto S. Walsh C.T. Neel B.G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7335-7339Crossref PubMed Scopus (344) Google Scholar, 16Yamauchi K. Milarski K.L. Saltiel A.R. Pessin J.E. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 664-668Crossref PubMed Scopus (268) Google Scholar, 22Pazdrak K. Adachi T. Alam R. J. Exp. Med. 1997; 186: 561-568Crossref PubMed Scopus (78) Google Scholar,39David M. Zhou G. Pine R. Dixon J.E. Larner A.C. J. Biol. Chem. 1996; 271: 15862-15865Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). In our system, we demonstrate by co-immunoprecipitation that SHP2 itself becomes tyrosine phosphorylated and associates with Shc upon cross-linking of cell surface E-selectin (Figs. 5 and 6 A). This further results in the formation of an E-selectin-dependent signaling complex, which includes Shc, Grb2, and Sos (Fig. 6, B and C). These adapter proteins form a macromolecular complex to bridge SHP2 to downstream signals. Previously we had described, that upon cross-linking cell surface E-selectin, Ras was activated and became associated with Raf-1 and phospho-MEK (7Hu Y. Kiely J.M. Szente B.E. Rosenzweig A. Gimbrone Jr., M.A. J. Immunol. 2000; 165: 2142-2148Crossref PubMed Scopus (73) Google Scholar). Here, we show that Sos becomes associated with Ras in an E-selectin-dependent manner (Fig. 6 D). This suggests that the E-selectin-dependent signaling complex, which includes SHP2, Shc, Grb2, and Sos, is physically related to the Ras/Raf-1/MEK/ERK1/2 pathway.In summary, we have shown that E-selectin can transduce signals across the endothelial cell cytoplasmic membrane, via the phosphorylation of a specific tyrosine residue in its cytoplasmic domain. This results in the association of the cytoplasmic domain of E-selectin with SHP2, a protein-tyrosine phosphatase. SHP2, in turn, acts as both a positive effector to the downstream E-selectin-dependent ERK1/2 activation and an adapter protein to bridge between E-selectin and a downstream adapter complex, comprised of Shc, Grb2, and Sos. These events thus outline a molecular mechanism by which cross-linking of E-selectin during leukocyte adhesive interactions on the surface of an activated endothelial cell, can initiate a transmembrane signaling cascade within that endothelial cell. The downstream consequences of these signaling events, including the modulation of endothelial gene expression, may have multifunctional implications for the pathology of inflammation. E-selectin is an inducible adhesion molecule that is expressed at relatively high density on the surface of cultured endothelial cells that have been activated by proinflammatory cytokines, such as interleukin-1b (IL-1β),1 tumor necrosis factor-α, or bacterial endotoxin. It is detectable in vivo at the sites of inflammation (1Bevilacqua M.P. Stengelin S. Gimbrone M.A.J. Seed B. Science. 1989; 243: 1160-1165Crossref PubMed Scopus (1660) Google Scholar, 2Bevilacqua M.P. Nelson R.M. J. Clin. Invest. 1993; 91: 379-387Crossref PubMed Scopus (1055) Google Scholar, 3Milstone D.S. Fukumura D. Padgett R.C. O'Donnell P.E. Davis V.M. Benavidez O.J. Monsky W.L. Melder R.J. Jain R.K. Gimbrone M.A.J. Microcirculation. 1998; 5: 153-171Crossref PubMed Google Scholar). In addition to supporting the rolling and stable arrest of leukocytes on activated endothelium, there is increasing evidence that E-selectin can transduce outside-in signals (4Lorenzon P. Vecile E. Nardon E. Ferrero E. Harlan J.M. Tedesco F. Dobrina A. J. Cell Biol. 1998; 142: 1381-1391Crossref PubMed Scopus (205) Google Scholar, 5Yoshida M. Westlin W.F. Wang N. Ingber D.E. Rosenzweig A. Resnick N. Gimbrone M.A.J. J. Cell Biol. 1996; 133: 445-455Crossref PubMed Scopus (188) Google Scholar, 6Yoshida M. Szente B.E. Kiely J.M. Rosenzweig A. Gimbrone Jr., M.A. J. Immunol. 1998; 161: 933-941PubMed Google Scholar). Recently our laboratory demonstrated that leukocyte adhesion to cell surface E-selectin-activated extracellular signal-regulated protein kinase (ERK1/2), formed a macrocomplex containing Ras/Raf-1/phospho-MEK, and resulted in the up-regulation of c-fos expression (7Hu Y. Kiely J.M. Szente B.E. Rosenzweig A. Gimbrone Jr., M.A. J. Immunol. 2000; 165: 2142-2148Crossref PubMed Scopus (73) Google Scholar). In this study, we have investigated the molecular events occurring immediately upstream of Ras/Raf/phospho-MEK and downstream of cell surface E-selectin. The cytoplasmic domain of E-selectin has been implicated in transmembrane signaling (5Yoshida M. Westlin W.F. Wang N. Ingber D.E. Rosenzweig A. Resnick N. Gimbrone M.A.J. J. Cell Biol. 1996; 133: 445-455Crossref PubMed Scopus (188) Google Scholar, 7Hu Y. Kiely J.M. Szente B.E. Rosenzweig A. Gimbrone Jr., M.A. J. Immunol. 2000; 165: 2142-2148Crossref PubMed Scopus (73) Google Scholar); however, the molecular mechanisms involved have not been well defined. The cytoplasmic domain of E-selectin consists of 32 amino acids (1Bevilacqua M.P. Stengelin S. Gimbrone M.A.J. Seed B. Science. 1989; 243: 1160-1165Crossref PubMed Scopus (1660) Google Scholar), including two tyrosine residues. It has been well documented that phosphorylation of tyrosine residues in the cytoplasmic domains of various types of receptors can play an important role in receptor-mediated transmembrane signal transduction, especially ERK1/2 activation. Therefore, it is reasonable to hypothesize that the two tyrosine residues on the cytoplasmic domain of E-selectin may be involved in the E-selectin-dependent ERK1/2 activation described by our laboratory (7Hu Y. Kiely J.M. Szente B.E. Rosenzweig A. Gimbrone Jr., M.A. J. Immunol. 2000; 165: 2142-2148Crossref PubMed Scopus (73) Google Scholar). The protein-tyrosine phosphatase SHP2 is a ubiquitously expressed cytosolic protein, which contains two amino-terminal tandem SH2 domains and a carboxyl-terminal catalytic domain (8Neel B.G. Semin. Cell Biol. 1993; 4: 419-432Crossref PubMed Scopus (106) Google Scholar). SHP2 associates with tyrosine-phosphorylated epidermal growth factor receptor, the platelet-derived growth factor receptor (9Case R.D. Piccione E. Wolf G. Benett A.M. Lechleider R.J. Neel B.G. Shoelson S.E. J. Biol. Chem. 1994; 269: 10467-10474Abstract Full Text PDF PubMed Google Scholar), insulin receptor (10Hausdorff S.F. Bennett A.M. Neel B.G. Birnbaum M.J. J. Biol. Chem. 1995; 270: 12965-12968Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar), and with the T and B cell receptors (11Siminovitch K.A. Neel B.G. Semin. Immunol. 1998; 10: 329-3247Crossref PubMed Scopus (61) Google Scholar). It becomes tyrosine phosphorylated upon cell stimulation and can become associated with other adapter proteins (12Bennett A.M. Tang T.L. Sugimoto S. Walsh C.T. Neel B.G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7335-7339Crossref PubMed Scopus (344) Google Scholar, 13Tang T.L. Freeman Jr., R.M. O'Reilly A.M. Neel B.G. Sokol S.Y. Cell. 1995; 80: 473-483Abstract Full Text PDF PubMed Scopus (307) Google Scholar, 14Neel B.G. Tonks N.K. Curr. Opin. Cell Biol. 1997; : 193-204Crossref PubMed Scopus (732) Google Scholar). Moreover, SHP2 has been show to have a positive effect on ERK1/2 signaling pathway (15Oh E.S. Gu H. Saxton T.M. Timms J.F. Hausdorff S. Frevert E.U. Kahn B.B. Pawson T. Neel B.G. Thomas S.M. Mol. Cell. Biol. 1999; 19: 3205-3215Crossref PubMed Scopus (194) Google Scholar), (16Yamauchi K. Milarski K.L. Saltiel A.R. Pessin J.E. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 664-668Crossref PubMed Scopus (268) Google Scholar). In this study, we describe the molecular mechanisms involved in the initiation of transmembrane signaling by cross-linking of cell surface E-selectin. These include tyrosine phosphorylation of E-selectin, association with SHP2, and assembly of the adapter proteins, Shc, Grb2, and Sos, to form a signaling complex, which then bridges SHP2 to the Ras·Raf-1·phospho-MEK macrocomplex, resulting in ERK1/2 activation. DISCUSSIONThere is ample evidence that transmembrane adhesion molecules can both physically bridge and biochemically transduce signals among interacting cells. The selectins, a family of adhesion molecules involved in leukocyte adhesion to activated vascular endothelium, are no exception. Binding to L-selectin has been shown to generate both inside-out and outside-in signals, such as the activation of the Ras pathway and potentiation of the oxidative burst of human neutrophils (20Brenner B. Gulbins E. Schlottmann K. Koppenhoefer U. Busch G.L. Walzog B. Steinhausen M. Coggeshall K.M. Linderkamp O. Lang F. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 15376-15381Crossref PubMed Scopus (142) Google Scholar, 26Crockett-Torabi E. Fantone J.C. Am. J. Physiol. 1997; 272: H1302-1308PubMed Google Scholar, 27Steeber D.A. Engel P. Miller A.S. Sheetz M.P. Tedder T.F. J. Immunol. 1997; 159: 952-963PubMed Google Scholar, 28Brenner B. Gulbins E. Busch G.L. Koppenhoefer U. Lang F. Linderkamp O. Biochem. Biophys. Res. Commun. 1997; 231: 802-807Crossref PubMed Scopus (61) Google Scholar, 29Brenner B. Grassme H.U. Muller C. Lang F. Speer C.P. Gulbins E. Exp. Cell Res. 1998; 243: 123-128Crossref PubMed Scopus (32) Google Scholar, 30Waddell T.K. Fialkow L. Chan C.K. Kishimoto T.K. Downey G.P. J. Biol. Chem. 1994; 269: 18485-18491Abstract Full Text PDF PubMed Google Scholar, 31Waddell T.K. Fialkow L. Chan C.K. Kishimoto T.K. Downey G.P. J. Biol. Chem. 1995; 270: 15403-15411Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar). P-selectin also has been shown to transduce signals across the plasma membrane, resulting in the induction of tyrosine phosphorylation of focal adhesion kinase and transient increases in intracellular Ca2+ (4Lorenzon P. Vecile E. Nardon E. Ferrero E. Harlan J.M. Tedesco F. Dobrina A. J. Cell Biol. 1998; 142: 1381-1391Crossref PubMed Scopus (205) Google Scholar, 32Haller H. Kunzendorf U. Sacherer K. Lindschau C. Walz G. Distler A. Luft F.C. J. Immunol. 1997; 158: 1061-1067PubMed Google Scholar). Our laboratory has demonstrated that adhesion of HL-60 cells, which expresses ligands for E-selectin, or monoclonal antibody (mAb)-mediated cell surface E-selectin cross-linking, can induce cytoskeletal linkage of E-selectin (5Yoshida M. Westlin W.F. Wang N. Ingber D.E. Rosenzweig A. Resnick N. Gimbrone M.A.J. J. Cell Biol. 1996; 133: 445-455Crossref PubMed Scopus (188) Google Scholar), dephosphorylation of serine residues in the cytoplasmic domain of E-selectin (6Yoshida M. Szente B.E. Kiely J.M. Rosenzweig A. Gimbrone Jr., M.A. J. Immunol. 1998; 161: 933-941PubMed Google Scholar), and activation of ERK1/2, as well as the formation of a Ras·Raf·MEK macrocomplex (7Hu Y. Kiely J.M. Szente B.E. Rosenzweig A. Gimbrone Jr., M.A. J. Immunol. 2000; 165: 2142-2148Crossref PubMed Scopus (73) Google Scholar).In this study, we used E-selectin single-point mutants to demonstrate that one tyrosine residue, Tyr603, in the cytoplasmic domain of E-selectin plays an essential role in the association between E-selectin and SHP2, and in mediating the ERK1/2 activation induced by cross-linking cell surface E-selectin. A consensus sequence for SHP2 binding, Y(I/V)X (V/I/L/P), has been defined in several proteins, such as platelet-derived growth factor β-receptor and platelet endothelial cell adhesion molecule)-1 (33Ronnstrand L. Arvidsson A.K. Kallin A. Rorsman C. Hellman U. Engstrom U. Wernstedt C. Heldin C.H. Oncogene. 1999; 18: 3696-3702Crossref PubMed Scopus (62) Google Scholar, 34Masuda M. Osawa M. Shigematsu H. Harada N. Fujiwara K. FEBS Lett. 1997; 408: 331-336Crossref PubMed Scopus (91) Google Scholar). The cytoplasmic domain of E-selectin does contain similar motifs around the Tyr603 (YQKP) and Tyr608 (YIL) residues. Upon cross-linking of cell surface E-selectin, SHP2 becomes associated with E-selectin in a time-dependent (Fig. 1 B) and dose-dependent (data not shown) manner; however, only Tyr603 appeared to play an essential role in this association. Furthermore, Tyr603 plays an important role in E-selectin-dependent ERK1/2 activation (Fig. 2). Although the basal level of the association between SHP2 and E-selectin mutated at Tyr608 appears to be slightly higher than the wild-type E-selectin (Fig. 1 C), this increased association does not result in any E-selectin-dependent ERK1/2 activation (Fig.2), suggesting a nonspecific effect. Cross-linking another endothelial cell surface heterodimer, the HLA class I molecule, which is present at comparable density on the surface of IL-1β-activated HUVEC, did not generate any changes in tyrosine phosphorylation on E-selectin (Fig.1 A). Our previously studies have indicated that antibody-mediated cross-linking can be utilized to mimic the specific clustering of cell surface E-selectin that presumably occurs during leukocyte adhesion, and results in activation of ERK1/2 (5Yoshida M. Westlin W.F. Wang N. Ingber D.E. Rosenzweig A. Resnick N. Gimbrone M.A.J. J. Cell Biol. 1996; 133: 445-455Crossref PubMed Scopus (188) Google Scholar, 7Hu Y. Kiely J.M. Szente B.E. Rosenzweig A. Gimbrone Jr., M.A. J. Immunol. 2000; 165: 2142-2148Crossref PubMed Scopus (73) Google Scholar). Taken together, these data strongly suggest the existence of an E-selectin-specific signaling pathway leading to the activation of ERK1/2 that includes Tyr603 in the cytoplasmic domain of E-selectin. Previously, our laboratory had shown that serine residues on the cytoplasmic domain of E-selectin became de-phosphorylated upon HL-60 adhesion (6Yoshida M. Szente B.E. Kiely J.M. Rosenzweig A. Gimbrone Jr., M.A. J. Immunol. 1998; 161: 933-941PubMed Google Scholar). There have been reports that serine/threonine dephosphorylation may be involved in the subsequent tyrosine phosphorylation (35Artcanuthurry V. Grelac F. Maclouf J. Martin-Cramer E. Levy-Toledano S. Semin. Thromb. Hemostasis. 1996; 22: 317-326Crossref PubMed Scopus (13) Google Scholar). We are in the process of determining whether cross-linking-induced dephosphorylation on serine residues is required for the observed phosphorylation on tyrosine residues in the cytoplasmic domain of E-selectin.It has been shown that overexpression of a catalytically inactive SHP2 can block ERK1/2 activation in response to insulin, platelet-derived growth factor (36Milarski K.L. Saltiel A.R. J. Biol. Chem. 1994; 269: 21239-21243Abstract Full Text PDF PubMed Google Scholar, 37Rivard N. McKenzie F.R. Brondello J.M. Pouyssegur J. J. Biol. Chem. 1995; 270: 11017-11024Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar), epidermal growth factor (38Zhao Z. Tan Z. Wright J.H. Diltz C.D. Shen S.H. Krebs E.G. Fischer E.H. J. Biol. Chem. 1995; 270: 11765-11769Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar), and fibroblast growth factor (13Tang T.L. Freeman Jr., R.M. O'Reilly A.M. Neel B.G. Sokol S.Y. Cell. 1995; 80: 473-483Abstract Full Text PDF PubMed Scopus (307) Google Scholar), via a dominant-negative effect. In our system, COS-7 cells co-transfected with WT-E-selectin and catalytically inert SHP2 (dominant negative SHP2), showed significantly blunted E-selectin-dependent ERK1/2 activation (Fig. 3). This suggests that SHP2 is a positive effector upstream of E-selectin-dependent ERK1/2 activation. We have further observed that E-selectin-associated SHP2 can de-phosphorylate tyrosine-phosphorylated E-selectin in vitro (Fig. 4), however; the exact substrate(s) of SHP2 in the context of E-selectin-dependent signaling in vivo are yet to be determined.In other systems, tyrosine-phosphorylated SHP2 functions as an adapter protein with positive effects for downstream signaling (12Bennett A.M. Tang T.L. Sugimoto S. Walsh C.T. Neel B.G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7335-7339Crossref PubMed Scopus (344) Google Scholar, 16Yamauchi K. Milarski K.L. Saltiel A.R. Pessin J.E. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 664-668Crossref PubMed Scopus (268) Google Scholar, 22Pazdrak K. Adachi T. Alam R. J. Exp. Med. 1997; 186: 561-568Crossref PubMed Scopus (78) Google Scholar,39David M. Zhou G. Pine R. Dixon J.E. Larner A.C. J. Biol. Chem. 1996; 271: 15862-15865Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). In our system, we demonstrate by co-immunoprecipitation that SHP2 itself becomes tyrosine phosphorylated and associates with Shc upon cross-linking of cell surface E-selectin (Figs. 5 and 6 A). This further results in the formation of an E-selectin-dependent signaling complex, which includes Shc, Grb2, and Sos (Fig. 6, B and C). These adapter proteins form a macromolecular complex to bridge SHP2 to downstream signals. Previously we had described, that upon cross-linking cell surface E-selectin, Ras was activated and became associated with Raf-1 and phospho-MEK (7Hu Y. Kiely J.M. Szente B.E. Rosenzweig A. Gimbrone Jr., M.A. J. Immunol. 2000; 165: 2142-2148Crossref PubMed Scopus (73) Google Scholar). Here, we show that Sos becomes associated with Ras in an E-selectin-dependent manner (Fig. 6 D). This suggests that the E-selectin-dependent signaling complex, which includes SHP2, Shc, Grb2, and Sos, is physically related to the Ras/Raf-1/MEK/ERK1/2 pathway.In summary, we have shown that E-selectin can transduce signals across the endothelial cell cytoplasmic membrane, via the phosphorylation of a specific tyrosine residue in its cytoplasmic domain. This results in the association of the cytoplasmic domain of E-selectin with SHP2, a protein-tyrosine phosphatase. SHP2, in turn, acts as both a positive effector to the downstream E-selectin-dependent ERK1/2 activation and an adapter protein to bridge between E-selectin and a downstream adapter complex, comprised of Shc, Grb2, and Sos. These events thus outline a molecular mechanism by which cross-linking of E-selectin during leukocyte adhesive interactions on the surface of an activated endothelial cell, can initiate a transmembrane signaling cascade within that endothelial cell. The downstream consequences of these signaling events, including the modulation of endothelial gene expression, may have multifunctional implications for the pathology of inflammation. There is ample evidence that transmembrane adhesion molecules can both physically bridge and biochemically transduce signals among interacting cells. The selectins, a family of adhesion molecules involved in leukocyte adhesion to activated vascular endothelium, are no exception. Binding to L-selectin has been shown to generate both inside-out and outside-in signals, such as the activation of the Ras pathway and potentiation of the oxidative burst of human neutrophils (20Brenner B. Gulbins E. Schlottmann K. Koppenhoefer U. Busch G.L. Walzog B. Steinhausen M. Coggeshall K.M. Linderkamp O. Lang F. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 15376-15381Crossref PubMed Scopus (142) Google Scholar, 26Crockett-Torabi E. Fantone J.C. Am. J. Physiol. 1997; 272: H1302-1308PubMed Google Scholar, 27Steeber D.A. Engel P. Miller A.S. Sheetz M.P. Tedder T.F. J. Immunol. 1997; 159: 952-963PubMed Google Scholar, 28Brenner B. Gulbins E. Busch G.L. Koppenhoefer U. Lang F. Linderkamp O. Biochem. Biophys. Res. Commun. 1997; 231: 802-807Crossref PubMed Scopus (61) Google Scholar, 29Brenner B. Grassme H.U. Muller C. Lang F. Speer C.P. Gulbins E. Exp. Cell Res. 1998; 243: 123-128Crossref PubMed Scopus (32) Google Scholar, 30Waddell T.K. Fialkow L. Chan C.K. Kishimoto T.K. Downey G.P. J. Biol. Chem. 1994; 269: 18485-18491Abstract Full Text PDF PubMed Google Scholar, 31Waddell T.K. Fialkow L. Chan C.K. Kishimoto T.K. Downey G.P. J. Biol. Chem. 1995; 270: 15403-15411Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar). P-selectin also has been shown to transduce signals across the plasma membrane, resulting in the induction of tyrosine phosphorylation of focal adhesion kinase and transient increases in intracellular Ca2+ (4Lorenzon P. Vecile E. Nardon E. Ferrero E. Harlan J.M. Tedesco F. Dobrina A. J. Cell Biol. 1998; 142: 1381-1391Crossref PubMed Scopus (205) Google Scholar, 32Haller H. Kunzendorf U. Sacherer K. Lindschau C. Walz G. Distler A. Luft F.C. J. Immunol. 1997; 158: 1061-1067PubMed Google Scholar). Our laboratory has demonstrated that adhesion of HL-60 cells, which expresses ligands for E-selectin, or monoclonal antibody (mAb)-mediated cell surface E-selectin cross-linking, can induce cytoskeletal linkage of E-selectin (5Yoshida M. Westlin W.F. Wang N. Ingber D.E. Rosenzweig A. Resnick N. Gimbrone M.A.J. J. Cell Biol. 1996; 133: 445-455Crossref PubMed Scopus (188) Google Scholar), dephosphorylation of serine residues in the cytoplasmic domain of E-selectin (6Yoshida M. Szente B.E. Kiely J.M. Rosenzweig A. Gimbrone Jr., M.A. J. Immunol. 1998; 161: 933-941PubMed Google Scholar), and activation of ERK1/2, as well as the formation of a Ras·Raf·MEK macrocomplex (7Hu Y. Kiely J.M. Szente B.E. Rosenzweig A. Gimbrone Jr., M.A. J. Immunol. 2000; 165: 2142-2148Crossref PubMed Scopus (73) Google Scholar). In this study, we used E-selectin single-point mutants to demonstrate that one tyrosine residue, Tyr603, in the cytoplasmic domain of E-selectin plays an essential role in the association between E-selectin and SHP2, and in mediating the ERK1/2 activation induced by cross-linking cell surface E-selectin. A consensus sequence for SHP2 binding, Y(I/V)X (V/I/L/P), has been defined in several proteins, such as platelet-derived growth factor β-receptor and platelet endothelial cell adhesion molecule)-1 (33Ronnstrand L. Arvidsson A.K. Kallin A. Rorsman C. Hellman U. Engstrom U. Wernstedt C. Heldin C.H. Oncogene. 1999; 18: 3696-3702Crossref PubMed Scopus (62) Google Scholar, 34Masuda M. Osawa M. Shigematsu H. Harada N. Fujiwara K. FEBS Lett. 1997; 408: 331-336Crossref PubMed Scopus (91) Google Scholar). The cytoplasmic domain of E-selectin does contain similar motifs around the Tyr603 (YQKP) and Tyr608 (YIL) residues. Upon cross-linking of cell surface E-selectin, SHP2 becomes associated with E-selectin in a time-dependent (Fig. 1 B) and dose-dependent (data not shown) manner; however, only Tyr603 appeared to play an essential role in this association. Furthermore, Tyr603 plays an important role in E-selectin-dependent ERK1/2 activation (Fig. 2). Although the basal level of the association between SHP2 and E-selectin mutated at Tyr608 appears to be slightly higher than the wild-type E-selectin (Fig. 1 C), this increased association does not result in any E-selectin-dependent ERK1/2 activation (Fig.2), suggesting a nonspecific effect. Cross-linking another endothelial cell surface heterodimer, the HLA class I molecule, which is present at comparable density on the surface of IL-1β-activated HUVEC, did not generate any changes in tyrosine phosphorylation on E-selectin (Fig.1 A). Our previously studies have indicated that antibody-mediated cross-linking can be utilized to mimic the specific clustering of cell surface E-selectin that presumably occurs during leukocyte adhesion, and results in activation of ERK1/2 (5Yoshida M. Westlin W.F. Wang N. Ingber D.E. Rosenzweig A. Resnick N. Gimbrone M.A.J. J. Cell Biol. 1996; 133: 445-455Crossref PubMed Scopus (188) Google Scholar, 7Hu Y. Kiely J.M. Szente B.E. Rosenzweig A. Gimbrone Jr., M.A. J. Immunol. 2000; 165: 2142-2148Crossref PubMed Scopus (73) Google Scholar). Taken together, these data strongly suggest the existence of an E-selectin-specific signaling pathway leading to the activation of ERK1/2 that includes Tyr603 in the cytoplasmic domain of E-selectin. Previously, our laboratory had shown that serine residues on the cytoplasmic domain of E-selectin became de-phosphorylated upon HL-60 adhesion (6Yoshida M. Szente B.E. Kiely J.M. Rosenzweig A. Gimbrone Jr., M.A. J. Immunol. 1998; 161: 933-941PubMed Google Scholar). There have been reports that serine/threonine dephosphorylation may be involved in the subsequent tyrosine phosphorylation (35Artcanuthurry V. Grelac F. Maclouf J. Martin-Cramer E. Levy-Toledano S. Semin. Thromb. Hemostasis. 1996; 22: 317-326Crossref PubMed Scopus (13) Google Scholar). We are in the process of determining whether cross-linking-induced dephosphorylation on serine residues is required for the observed phosphorylation on tyrosine residues in the cytoplasmic domain of E-selectin. It has been shown that overexpression of a catalytically inactive SHP2 can block ERK1/2 activation in response to insulin, platelet-derived growth factor (36Milarski K.L. Saltiel A.R. J. Biol. Chem. 1994; 269: 21239-21243Abstract Full Text PDF PubMed Google Scholar, 37Rivard N. McKenzie F.R. Brondello J.M. Pouyssegur J. J. Biol. Chem. 1995; 270: 11017-11024Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar), epidermal growth factor (38Zhao Z. Tan Z. Wright J.H. Diltz C.D. Shen S.H. Krebs E.G. Fischer E.H. J. Biol. Chem. 1995; 270: 11765-11769Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar), and fibroblast growth factor (13Tang T.L. Freeman Jr., R.M. O'Reilly A.M. Neel B.G. Sokol S.Y. Cell. 1995; 80: 473-483Abstract Full Text PDF PubMed Scopus (307) Google Scholar), via a dominant-negative effect. In our system, COS-7 cells co-transfected with WT-E-selectin and catalytically inert SHP2 (dominant negative SHP2), showed significantly blunted E-selectin-dependent ERK1/2 activation (Fig. 3). This suggests that SHP2 is a positive effector upstream of E-selectin-dependent ERK1/2 activation. We have further observed that E-selectin-associated SHP2 can de-phosphorylate tyrosine-phosphorylated E-selectin in vitro (Fig. 4), however; the exact substrate(s) of SHP2 in the context of E-selectin-dependent signaling in vivo are yet to be determined. In other systems, tyrosine-phosphorylated SHP2 functions as an adapter protein with positive effects for downstream signaling (12Bennett A.M. Tang T.L. Sugimoto S. Walsh C.T. Neel B.G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7335-7339Crossref PubMed Scopus (344) Google Scholar, 16Yamauchi K. Milarski K.L. Saltiel A.R. Pessin J.E. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 664-668Crossref PubMed Scopus (268) Google Scholar, 22Pazdrak K. Adachi T. Alam R. J. Exp. Med. 1997; 186: 561-568Crossref PubMed Scopus (78) Google Scholar,39David M. Zhou G. Pine R. Dixon J.E. Larner A.C. J. Biol. Chem. 1996; 271: 15862-15865Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). In our system, we demonstrate by co-immunoprecipitation that SHP2 itself becomes tyrosine phosphorylated and associates with Shc upon cross-linking of cell surface E-selectin (Figs. 5 and 6 A). This further results in the formation of an E-selectin-dependent signaling complex, which includes Shc, Grb2, and Sos (Fig. 6, B and C). These adapter proteins form a macromolecular complex to bridge SHP2 to downstream signals. Previously we had described, that upon cross-linking cell surface E-selectin, Ras was activated and became associated with Raf-1 and phospho-MEK (7Hu Y. Kiely J.M. Szente B.E. Rosenzweig A. Gimbrone Jr., M.A. J. Immunol. 2000; 165: 2142-2148Crossref PubMed Scopus (73) Google Scholar). Here, we show that Sos becomes associated with Ras in an E-selectin-dependent manner (Fig. 6 D). This suggests that the E-selectin-dependent signaling complex, which includes SHP2, Shc, Grb2, and Sos, is physically related to the Ras/Raf-1/MEK/ERK1/2 pathway. In summary, we have shown that E-selectin can transduce signals across the endothelial cell cytoplasmic membrane, via the phosphorylation of a specific tyrosine residue in its cytoplasmic domain. This results in the association of the cytoplasmic domain of E-selectin with SHP2, a protein-tyrosine phosphatase. SHP2, in turn, acts as both a positive effector to the downstream E-selectin-dependent ERK1/2 activation and an adapter protein to bridge between E-selectin and a downstream adapter complex, comprised of Shc, Grb2, and Sos. These events thus outline a molecular mechanism by which cross-linking of E-selectin during leukocyte adhesive interactions on the surface of an activated endothelial cell, can initiate a transmembrane signaling cascade within that endothelial cell. The downstream consequences of these signaling events, including the modulation of endothelial gene expression, may have multifunctional implications for the pathology of inflammation. We thank Dr. Benjamin Neel for providing valuable reagents and offering critical advice (Beth Israel Deaconess Medical Center, Boston, MA) and Kay Case for expert assistance in cell culture.
We have demonstrated previously that murine interferon-γ (MuIFN-γ) binds to the extracellular domain of the receptor a chain through its N-terminus and subsequently to the cytoplasmic domain of the receptor via its C-terminus. Binding of the C-terminus to the cytoplasmic domain of the receptor is thought to occur following endocytosis of the IFN-γ-receptor complex. In fact, the MuIFN-γ C-terminus peptide, MuIFN-γ(95-133), has full agonist activity on macrophages where it is internalized through pinocytosis. Here we examine the structural elements required for the agonist activity of MuIFN-γ(95-133). Disruption of the α helical structure of the peptide by proline substitutions or truncation of the helix resulted in significant loss of binding or loss of antiviral activity or both and induction of MHC class II molecules. Further, removal of the polycationic sequence RKRKR in the tail beyond the helical structure also resulted in loss of agonist activity. Thus, we have isolated the functional site on MuIFN-γ to the C-terminus and have shown that its helical structure and polycationic tail are required for binding to the cytoplasmic domain of the receptor and induction of biologic activity.
The tyrosine kinase JAK2 is an integral part of the signal transduction pathways of a number of cytokines and growth factors, including IFN-gamma. Previously, we identified a species-nonspecific binding site for the C terminus of IFN-gamma, encompassed by IFN-gamma peptide IFN-gamma(95-133), on the membrane proximal region of the cytoplasmic domain of the IFN-gamma R alpha-chain. Using both a radioligand binding assay and coimmunoprecipitation with antireceptor antiserum, we were able to demonstrate specific interaction of JAK2 with the murine IFN-gamma R(MIR) alpha-chain. Furthermore, this interaction is increased by the addition of murine IFN-gamma or its C-terminal peptide, muIFN-gamma(95-133). We also identified two regions of the cytoplasmic domain of the receptor that interact with JAK2 using synthetic peptides of the MIR alpha-chain in receptor competition studies. These regions are encompassed by receptor peptide MIR(283-309), which is adjacent to the membrane proximal region at which the C terminus of IFN-gamma binds, and receptor peptide MIR(404-432), which lies near the C terminus of the receptor, encompassing a potentially important phosphorylation site. These data show site-specific interaction between JAK2 and IFN-gamma with the IFN-gamma R and have broader implications for the role of the IFN-gamma ligand in the IFN-gamma signal transduction pathway. Furthermore, the data support previous studies that demonstrated that intracellular IFN-gamma plays a role in cell activation.
Abstract E-selectin, a cytokine-inducible adhesion molecule, supports rolling and stable arrest of leukocytes on activated vascular endothelium. Previous studies have suggested that this transmembrane protein can also transduce signals into the endothelial cell. We now demonstrate activation of the mitogen-activated protein kinase (MAPK) signaling cascade in cultured HUVEC in response to E-selectin-dependent leukocyte adhesion and Ab-mediated cross-linking of cell surface E-selectin. Adhesion of increasing numbers of HL60 cells to IL-1β-activated HUVEC stimulated robust increases in MAPK activity that were abrogated by an E-selectin blocking Ab. Cross-linking of cell surface E-selectin with Abs, as a mimic of multivalent ligand engagement, strongly stimulated MAPK/extracellular signal-related kinase (ERK) kinase (MEK)-dependent MAPK activation and concomitant up-regulation of mRNA for c-fos, an immediate early response gene, whereas Ab cross-linking of HLA class I molecules (present at comparable density) failed to do so. Coimmunoprecipitation documented Ras, Raf-1 and, phospho-MEK complex formation. Unactivated HUVEC transduced with a full-length adenoviral E-selectin construct also exhibited cross-link-induced MAPK activation, macromolecular complex formation, and c-fos up-regulation, whereas HUVEC transduced with a cytoplasmic domain deletion mutant failed to respond. These observations indicate that E-selectin can transduce an activating stimulus via the MAPK cascade into the endothelial cell during leukocyte adhesion.
The c-ski locus extends a minimum of 65 kb in the chicken genome and is expressed as multiple mRNAs resulting from alternative exon usage. Four exons comprising approximately 1.5 kb of cDNA sequence have been mapped within the chicken c-ski locus. However, c-ski cDNAs include almost 3 kb of sequence for which the exon structure was not defined. From our studies using the polymerase chain reaction and templates of RNA and genomic DNA, it is clear that c-ski cDNAs are encoded by a minimum of eight exons. A long 3' untranslated region is contiguous in the genome with the distal portion of the ski open reading frame such that exon 8 is composed of both coding and noncoding sequences. Exons 2 and 3 are separated by more than 25 kb of genomic sequence. In contrast, exons 3 through 8, representing more than half the length of c-ski cDNA sequences, are closely linked within 10 kb in the chicken genome.
