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J. Biol. Chem., Vol. 276, Issue 51, 48549-48553, December 21, 2001

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Molecular Events in Transmembrane Signaling via E-selectin

SHP2 ASSOCIATION, ADAPTOR PROTEIN COMPLEX FORMATION AND ERK1/2 ACTIVATION^*

Yenya Hu, Brian Szente^§, Jeanne-Marie Kiely^¶, and Michael A. Gimbrone Jr.

From the ^¶ Vascular Research Division, Departments of Pathology, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02132

Received for publication, June 14, 2001, and in revised form, September 6, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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-1),¹ tumor necrosis factor-, or bacterial endotoxin. It is detectable in vivo at the sites of inflammation (1-3). 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 (4-6). 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 (7). 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 (5, 7); however, the molecular mechanisms involved have not been well defined. The cytoplasmic domain of E-selectin consists of 32 amino acids (1), 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 (7).

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 (8). SHP2 associates with tyrosine-phosphorylated epidermal growth factor receptor, the platelet-derived growth factor receptor (9), insulin receptor (10), and with the T and B cell receptors (11). It becomes tyrosine phosphorylated upon cell stimulation and can become associated with other adapter proteins (12-14). Moreover, SHP2 has been show to have a positive effect on ERK1/2 signaling pathway (15), (16).

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.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Reagents-- Medium 199, RPMI 1640, and Dulbecco's phosphate-buffered saline were obtained from M. A. Bioproducts (Walkersville, MD). Fetal bovine serum (FBS) was purchased from Life Technologies, Inc. (Grand Island, NY). Endothelial cell growth factor was obtained from Biomedical Technologies (Stoughton, MA). Paraformaldehyde (laboratory grade) was purchased from Fisher Scientific (Springfield, NJ). Recombinant human IL-1 was a gift from Biogen (Cambridge, MA). Antibodies (anti-SHP2, PY99, Shc, Grb2, and Sos) and Protein A/G-PLUS-agarose was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). RC20 was from Transduction Lab (San Diego, CA). Anti-phospho-ERK1/2 was from New England Biolab (Beverly, MA). Anti-E-selectin antibodies, H18/7 and H4/18, were made in our laboratory (17) and anti-HLA complex I molecule, W6/32, was from the American Type Culture Collection (ATCC, Rockville, MD). WT and mutant SHP2 cDNA were prepared as previously described.

Cultured Cells-- Human umbilical cord vein endothelial cells (HUVEC) were isolated and established in culture as previously described (18). Primary cultures were serially passaged (1:3 split ratio) and maintained in Medium 199 buffered with 25 mmol/liter HEPES buffer and supplemented with 20% fetal bovine serum (FBS), endothelial cell growth factor (25 µg/ml), and porcine intestinal heparin (50 µg/ml). For experimental use, subcultured (passage 2 or 3) endothelial cells were plated on gelatin-coated 35- or 100-mm tissue culture dishes (Difco Laboratories, Detroit, MI). HL-60, a human promyelocytic leukocyte cell line, was obtained from the American Type Culture Collection (ATCC) and grown in RPMI 1640 medium supplemented with 10% FBS, 100 units/ml penicillin, 100 µg/ml streptomycin, and 20 mM L-glutamine. JY human lymphocytic cells, kindly provided by Dr. T. A. Springer (Center for Blood Research, Boston, MA), were maintained in RPMI 1640 medium supplemented with 10% FBS, 100 units/ml penicillin, 100 µg/ml streptomycin, and 20 mM L-glutamine. COS-7 cells, a monkey kidney fibroblast cell line, was obtained from the American Type Culture Collection (ATCC) and grown in Dulbecco's modified Eagle's medium supplemented with 10% FBS, 100 units/ml penicillin, 100 µg/ml streptomycin, and 20 mM L-glutamine.

Generation of E-selectin Tyrosine Mutants-- A PCR-directed mutagenesis approach was used to generate specific mutants of E-selectin. Two tyrosine mutants (Tyr⁶⁰³  Phe, Tyr⁶⁰⁸  Phe) were generated as follows. A forward primer F2 (5'-GGTTTGGTGAGGTGCTCCATTC-3') and two different reverse primers, R8 (5'-GCGTTAACTTAAAGGATGAAAGAAGGCTTTTGG-3') and R19 (5'-GCGTTAACTTAAAGGATGTAAGAAGGCTTTTGGAAGCTTCC-3') were used to amplify two fragments using wild-type E-selectin cDNA as the template. Each reverse primer (R8 and R19) contained both a stop codon and a HpaI restriction site at its 3' end in order to make mutant E-selectin cytoplasmic domain fragments. In both cases, the amplified fragments were ligated into the Topo-TA cloning vector (Invitrogen Corp., San Diego, CA), and the mutated sequences were confirmed by sequence analysis. The fragments were gel purified, and prepared by sequential digestion with EcoRI and HpaI. They were then used to replace the corresponding EcoRI/HpaI fragments of the wild-type E-selectin cDNA in the expression vector, pCDM8 (Invitrogen).

Transfection of COS-7 Cells with WT-E-selectin, WT-SHP2, Catalytic Inert SHP2 (muSHP2), and the Point Mutants, Tyr⁶⁰³  Phe and Tyr⁶⁰⁸  Phe-- WT-E-selectin was co-transfected with the empty vector, PJ3, or WT-SHP2 (wSHP2) or mutant SHP2 (muSHP2), or point mutants Tyr⁶⁰³  Phe or Tyr⁶⁰⁸  Phe (1 µg/condition) was transfected into subconfluent (70% confluency) COS-7 cells, using LipofectAMINE reagent (42 µg) in OPTI-MEM I reduced serum medium (Life Technologies, Inc., Gaithersburg, MD). Forty-eight to 72 h post-transfection, the COS-7 cells were subjected to either fixed HL-60 cell adhesion or cell surface cross-linking for the time indicated in the figures and assayed for ERK1/2 activity using a mAb (phospho-ERK1/2) from New England Biolabs.

Immunoprecipitation-- After treatment, cells were rinsed with ice-cold Dulbecco's phosphate-buffered saline and lysed in either Triton lysis buffer (for E-selectin and SHP2 association) (1% Triton X-100, 10 mM Tris, pH 7.5, 100 mM NaCl, 50 mM NaF, 1 mM Na₃VO₄, 2 mM phenylmethlysulfonyl fluoride, 21 µg/ml aprotinin) (19) or Nonidet lysis buffer (for adapter proteins) (25 mM HEPES, 0.1% SDS, 0.5% sodium deoxycholate, 3% Nonidet P-40, 125 mM NaCl, 10 mM NaF, 10 mM Na₃VO₄, 10 mM sodium pyrophosphate, 20 µg/ml aprotinin, 20 µg/ml leupeptin) (20) on ice for 5 min. Cells were then scraped off the plates and collected in microcentrifuge tubes. The total cell lysates were centrifuged at 14,000 rpm for 15 min at 4 °C and the supernatant was pre-cleared with protein A/G-agarose for 1 h at 4 °C. Aliquots of the supernatant were subjected to immunoprecipitation with various antibodies overnight at 4 °C. Twenty-five µl of protein A/G-agarose beads were added to the incubation for an additional 1 h at 4 °C. The immunocomplex was then washed three times with the Triton lysis buffer or Nonidet lysis buffer and resuspended in 50 µl of the same buffer. Twenty-five microliters of 3 × sample buffer was added and samples were boiled at 100 °C for 5 min. Each tube was then vortexed and centrifuged at 14,000 rpm for 2 min.

In Vitro Immunocomplex Phosphatase Assay-- After treatment, cells were lysed on ice with Triton buffer for 5 min and centrifuged at 14,000 rpm for 15 min at 4 °C. The supernatant was then pre-cleared with protein A/G-agarose for 1 h at 4 °C and the resultant supernatant was subjected to immunoprecipitation with H18/7 overnight at 4 °C. Twenty-five microliters of protein A/G-agarose beads were added to the incubation for an additional 1 h at 4 °C. The immunocomplex was then washed three times with the Triton lysis buffer and re-suspended in 50 µl of the phosphatase assay buffer (80 mM MOPS, 10 mM EDTA, 10 mM dithiothreitol, 1 mM Na₃VO₄, pH 7.0) (19) and incubated at 37 °C for 30 min. After the reaction, 25 µl of 3 × SDS buffer were added to the beads and the proteins were eluted from the beads by boiling for 5 min.

Immunoblotting-- Aliquots (25 µl) of immunoprecipitates were separated on a 7.5 or 12% SDS-polyacrylamide electrophoresis gel and then transferred to a nylon membrane (Millipore, Bedford, MA). Membranes were blocked with either 5% bovine serum albumin (when anti-phosphorylated tyrosine antibody was used) or 5% nonfat milk in TTBS (20 mM Tris, 138 mM NaCl, 0.5% Tween 20, pH 7.6) for 1 h at room temperature and then incubated with various primary antibodies (1:1000 diluted in blocking buffer), including RC20, SHP2, PY99, Shc, Grb2, and Sos, for an additional 1 h at room temperature. After three washes with TTBS, membranes were incubated with a horseradish peroxidase-conjugated polyclonal goat anti-mouse or goat-anti-rabbit antibody (1:1000) (Santa Cruz Biotechnology, Inc.) in TTBS for an additional hour at room temperature, and again washed three times in TTBS. The labeled proteins were visualized using an enhanced chemiluminscence kit (Amersham Bioscience, Inc.).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cross-linking Cell Surface E-selectin Molecules Induce Tyrosine Phosphorylation on E-selectin and Its Association with SHP2-- There are two tyrosine residues on the cytoplasmic domain of E-selectin that could potentially play a role in mediating cytosolic events associated with transmembrane signals. To examine whether these tyrosine residues actually become phosphorylated as a consequence of perturbation of the extracellular domain of E-selectin, we compared the state of tyrosine phosphorylation of E-selectin following antibody-mediated cross-linking of cell surface E-selectin (Fig. 1A, upper panel). Tyrosine phosphorylation of E-selectin was detectable 2-30 min after cross-linking of cell surface E-selectin (2 min, 1-fold; 15 min, 4-fold; 30 min, 3-fold increase over IL-1 alone, densitometry results), but not as much after cross-linking of HLA class I molecules (2 min, no change; 15 min, 2-fold; 30 min, 1-fold increase over IL-1 alone). Comparable amounts of E-selectin were present in these immunocomplexes (Fig. 1A, lower panel).

View larger version (47K): [in this window] [in a new window]   Fig. 1.   E-selectin becomes tyrosine phosphorylated and associated with SHP2 upon antibody-mediated cross-linking. A, HUVEC monolayers were activated with IL-1 (10 units/ml, 4 h, 37 °C) and treated with H4/18 (a mouse mAb against E-selectin, 10 µg/ml, 30 min, 4 °C) or W6/32 (a mouse mAb against HLA class I molecules, 10 µg/ml, 30 min, 4 °C), followed by a goat anti-mouse IgG (GAM-IgG) (1:200, 37 °C) for the times indicated (0, 2, 15, and 30 min) to induce selective cell surface molecular cross-linking. Tyrosine phosphorylation of E-selectin was detected by sequential immunoprecipitation with a second E-selectin-specific mouse mAb, H18/7, and immunoblotting with a phosphotyrosine monoclonal antibody (RC20) (upper panel). The same blot was stripped and blotted with a goat anti-mouse E-selectin antibody (lower panel). B, HUVEC surface E-selectin molecules were cross-linked with mAb H4/18, followed by a goat anti-mouse IgG for the times (0, 2, 15, and 30 min) indicated, and SHP2 association with E-selectin detected by sequential immunoprecipitation and immunoblotting. C, COS-7 cells were transfected with WT-E-selectin, Tyr603  Phe mutant, or Tyr608  Phe mutant. Following selective cross-linking (H4/18+IgG or H+G) of cell surface E-selectin for 2 min, SHP2 association with E-selectin was detected by sequential immunoprecipitation and immunoblotting. C, COS-7 cells; Mock, mock transfected COS-7 cells.

It has been shown that the protein-tyrosine phosphatase SHP2 becomes associated with tyrosine-phosphorylated receptors and regulates signaling from cell surface receptors (21, 22). Therefore we examined the association between E-selectin and SHP2 following antibody-mediated cross-linking of cell surface E-selectin. Co-immunoprecipitation experiments revealed that SHP2 became associated with E-selectin, as early as 2 min after cross-linking and was still detectable at 30 min (Fig. 1B). The time difference between the E-selectin tyrosine phosphorylation (15 min) and SHP2 association with E-selectin (2 min) could have been attributed to that the anti-SHP2 antibody was more sensitive to detect SHP2 than the anti-phosphotyrosine antibody to detect E-selectin phosphorylation. To determine which tyrosine residue(s) on the cytoplasmic domain of E-selectin were responsible for the observed association between SHP2 and E-selectin, we generated two single tyrosine point mutants, Tyr⁶⁰³  Phe and Tyr⁶⁰⁸  Phe, and transfected these mutants into COS-7 cells. Forty-eight to 72 h post-transfection, cell surface E-selectin molecules were cross-linked using a monoclonal antibody specific for E-selectin (H4/18), and the association between SHP2 and E-selectin was again examined using co-immunoprecipitation. As seen in Fig. 1C, mutation of Tyr⁶⁰³ essentially ablated the association of SHP2 with E-selectin induced by the cell surface E-selectin cross-linking. The Tyr⁶⁰⁸ mutant exhibited no difference in SHP2 association under either basal or cross-linking conditions. A fluorescence immunoassay documented that equivalent amounts of E-selectin were expressed at the cell surface in both the Tyr⁶⁰³- and Tyr⁶⁰⁸-transfected cells (data not shown).

Tyrosine Residue 603 Is Essential for E-selectin-dependent ERK1/2 Activation-- We had previously shown that cross-linking cell surface E-selectin resulted in the activation of ERK1/2 (7). To determine whether the two tyrosine residues in the cytoplasmic domain of E-selectin play any role in the subsequent ERK1/2 activation, we transfected these single tyrosine point mutants into COS-7 cells, cross-linked cell surface E-selectin and measured the level of phosphorylated ERK1/2. As seen in Fig. 2, the mutation, Tyr⁶⁰³  Phe resulted in a complete loss of ERK1/2 activity, while the Tyr⁶⁰⁸  Phe mutant had no impact on the activation of ERK1/2 (upper panel). Equal amounts of ERK1/2 protein were present in each sample as determined by stripping and re-probing using an antibody that recognizes total ERK1/2 (lower panel). A fluorescence immunoassay documented that comparable levels of cell surface E-selectin were expressed on both types of transfected COS-7 cells (data not shown). Therefore, tyrosine residue 603 in the cytoplasmic domain of E-selectin appears to play an essential role in the E-selectin-dependent activation of ERK1/2.

View larger version (14K): [in this window] [in a new window]   Fig. 2.   Tyrosine 603 is essential to the E-selectin-dependent ERK1/2 activation. COS-7 cells were transfected with WT-E-selectin, Tyr603  Phe mutant or Tyr608 Phe mutant. Cell surface E-selectin molecules were cross-linked with H4/18 (H), followed by GAM-IgG (G) for 30 min at 37 °C. Phospho-ERK1/2 was measured as an index of ERK1/2 activation. C, COS-7 cells; Mock, mock transfected COS-7 cells.

The Catalytic Domain of SHP2 Plays an Important Role in the E-selectin-dependent ERK1/2 Activation-- To determine whether SHP2 functions in E-selectin-dependent ERK1/2 activation, we co-transfected WT-E-selectin together with either wild type-SHP2 (wSHP2) (Fig. 3, lanes 5 and 6) or a mutant SHP2 (muSHP2) (lanes 7 and 8), which lacks catalytic activity, into COS-7 cells. As control, we co-transfected the empty vector, PJ3, with WT-E-selectin (lanes 3 and 4). Cell surface E-selectin molecules then were selectively cross-linked using an E-selectin mAb (H4/18) and phosphorylated ERK1/2 was measured as an index of the ERK1/2 activity. Fig. 3 shows that co-transfection of muSHP2 with WT-E-selectin resulted in an inhibition of cross-link-mediated ERK1/2 activation. Comparable amounts of both wSHP2 and muSHP2 were detected by Western blot (data not shown). Thus, this dominant negative experiment indicated that the catalytic property of SHP2 plays an important role in downstream E-selectin-dependent ERK1/2 activation.

View larger version (14K): [in this window] [in a new window]   Fig. 3.   E-selectin-dependent ERK1/2 activation is dependent upon the catalytic domain of SHP2. Lane 1, COS-7 cells. Lane 2, mock transfected COS-7 cells. COS-7 cells were transfected with WT-E-selectin and the empty vector, PJ3, in lanes 3 and 4. COS-7 cells were transfected with WT-E-selectin and wild type-SHP2 (WT-SHP2) (lanes 5 and 6) or with WT-E-selectin and mutant SHP2 (catalytically inert) (mSHP2) (lanes 7 and 8). Lanes 3, 5, and 7, no cell surface E-selectin cross-linking. Lanes 2, 5, and 8, cell surface E-selectin molecules were cross-linked with H4/18, followed by GAM-IgG, as described in the legend to Fig. 1. Phospho-ERK1/2 was measured as an index of activated ERK1/2.

SHP2 Associated with E-selectin Is Enzymatically Active and Becomes Tyrosine Phosphorylated-- SHP2 is a protein-tyrosine phosphatase. To exam whether the SHP2 associated with the cytoplasmic domain of E-selectin is enzymatically active, E-selectin was immunoprecipitated from IL-1-activated, mAb H18/7 cross-linked HUVEC and subjected to immunocomplex phosphatase assays (Fig. 4). The longer cell surface E-selectin was cross-linked, the lower the resultant level of tyrosine phosphorylation of E-selectin in the immunocomplex. Comparable amounts of E-selectin and SHP2 were detectable in the immunocomplex (lower panels). These data suggest that protein-tyrosine phosphatase SHP2 in the E-selectin-containing immunoprecipitates is catalytically active, and that E-selectin can be one of the substrates for SHP2 in vitro.

View larger version (40K): [in this window] [in a new window]   Fig. 4.   SHP2 associated with E-selectin is catalytically active. Cell surface E-selectin molecules were selectively cross-linked with a mouse mAb H4/18 and by GAM-IgG (see Fig. 1, legend), for the times (1, 2, and 5 min) indicated. E-selectin phosphorylation on tyrosine residues was detected by immunoprecipitation/immunoblotting. Comparable amounts of E-selectin (middle panel) and SHP2 (lower panel) were detectable in these immunocomplexes.

There are two tyrosine residues on the COOH terminus of SHP2 (8). We therefore examined if SHP2 became tyrosine phosphorylated in an E-selectin-dependent manner. After the adhesion of fixed HL-60 cells, or mAb cross-linking of cell surface E-selectin, SHP2 was immunoprecipitated from the total cell lysates and immunoblotted with the phosphotyrosine antibody, PY99. As seen in Fig. 5, SHP2 showed significantly increased tyrosine phosphorylation after only 2 min of fixed HL-60 cell adhesion or cell surface E-selectin cross-linking. This suggested that SHP2 not only could act as a tyrosine phosphatase, but also could provide a possible docking site via its phosphorylated tyrosine residues for the binding of other adapter proteins.

View larger version (20K): [in this window] [in a new window]   Fig. 5.   SHP2 is tyrosine phosphorylated following HL-60 adhesion or cell surface E-selectin cross-linking. All samples were treated with IL-1 (10 units/ml, 4 h, 37 °C). Paraformaldehyde-fixed HL-60 cells (2 × 106/ml) were allowed to adhere to activated HUVEC monolayers at 4 °C for 30 min, and then the monolayers were warmed to 37 °C for the times (2, 15, and 30 min) indicated on the figure. Alternatively, cross-linking of cell surface E-selectin with H4/18, followed by goat-anti-mouse IgG, was performed as described in the legend to Fig. 1, for the times indicated.

Shc, Grb2/Sos, and Ras Form an E-selectin-dependent Signaling Complex-- It has been shown that SHP2 itself can act as an adapter protein, via its phosphorylated tyrosine residues as docking sites for downstream adapter proteins, such as Shc and Grb2 (22). Therefore we utilized co-immunoprecipitation/immunoblotting to examine the association between SHP2, Shc, and other adapter proteins. As seen in Fig. 6A, SHP2 became associated with Shc after 2 min of cell surface E-selectin cross-linking, but was no longer associated after 15 or 30 min. Increased Shc/Grb2 association was evident after 2 min of the adhesion of fixed HL-60, but not of JY cells (a control for non-E-selectin-dependent adhesion), and mAb-induced cross-linking of cell surface E-selectin, but not HLA class I molecules (Fig. 6B). As reported for the other cell types, Grb2 and Sos formed a constitutive complex in IL-1-activated HUVEC (23-25) (Fig. 6C).

View larger version (39K): [in this window] [in a new window]   Fig. 6.   The formation of an E-selectin-dependent adapter protein complex. HUVEC were treated with IL-1 (10 units/ml, 4 h, 37 °C) as control (C, control). A, cell surface E-selectin molecules were cross-linked by the mouse mAb, H18/7 (10 µg/ml, 30 min, 4 °C), followed by a goat anti-mouse IgG (1:200, 37 °C) for the times (0, 2, 15, and 30 min) indicated. The amounts of Shc associated with SHP2 were detected by co-immunoprecipitation. B, paraformaldehyde-fixed HL-60 (2 × 106/ml) or JY cells (non-E-selectin-dependent adhesion) were allowed to adhere to activated HUVEC monolayers for 30 min at 4 °C and then transferred to 37 °C for 2 min. Alternatively, cell surface E-selectin or HLA class I molecules were selectively cross-linked with mAb H18/7 (H+G) or W6/32 (W+G), respectively, as described in the legend to Fig. 1, legend. C, cell surface E-selectin molecules were cross-linked with H18/7 and a goat anti-mouse IgG for the times (0, 2, 15, and 30 min) indicated and the association of Grb2 and Sos was detected by co-immunoprecipitation. D, paraformaldehyde-fixed HL-60 and JY cells (2 × 106/ml) were allowed to adhere to activated HUVEC for 30 min at 4 °C and warmed to 37 °C for 2 min. Cell surface E-selectin and HLA class I molecules were cross-linked using a mAb H18/7 (H+G) or W6/32 (W+G), respectively, for 2 min. The association of Ras and Sos was detected by co-immunoprecipitation.

We previously had reported that Ras·Raf-1·Phospho-MEK form an E-selectin-dependent macrocomplex (7). It has been described that Sos, a guanine nucleotide-releasing factor, can recruit Ras to the cytoplasmic membrane to carry out Ras/Raf-1/MEK signaling (20, 25). To determine whether the events we describe above were related to Ras, we immunoprecipitated Sos from total HUVEC cell lysates and immunoblotted for Ras (Fig. 6D). Increased Ras association with Sos was apparent following either adhesion of HL-60 or antibody-induced cross-linking of cell surface E-selectin. This suggests that Ras is associated with upstream adapter proteins in an E-selectin-dependent manner.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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 (20, 26-31). 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⁺ (4, 32). 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 (5), dephosphorylation of serine residues in the cytoplasmic domain of E-selectin (6), and activation of ERK1/2, as well as the formation of a Ras·Raf·MEK macrocomplex (7).

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 -receptor and platelet endothelial cell adhesion molecule)-1 (33, 34). 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. 1B) 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. 1C), 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-1-activated HUVEC, did not generate any changes in tyrosine phosphorylation on E-selectin (Fig. 1A). 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 (5, 7). 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 (6). There have been reports that serine/threonine dephosphorylation may be involved in the subsequent tyrosine phosphorylation (35). 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 (36, 37), epidermal growth factor (38), and fibroblast growth factor (13), 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 (12, 16, 22, 39). 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 6A). 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 (7). Here, we show that Sos becomes associated with Ras in an E-selectin-dependent manner (Fig. 6D). 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.

	ACKNOWLEDGEMENTS

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.

	FOOTNOTES

^* This work was supported by National Institutes of Health Grant P01-HL36028 (to M. A. G., Jr.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Current address: Cellular and Molecular Laboratory, Division of Cardiac & Thoracic Surgery, Dept. of Surgery, Vanderbilt University Medical Center, Nashville, TN 37232. E-mail: yenya.hu@surgery.mc.vanderbilt.edu.

^§ Current address: SmithKline Beecham Pharmaceuticals, Oncology Research Department, 709 Swedeland Rd., UW 2532, King of Prussia, PA 19406.

To whom correspondence should be addressed: Vascular Research Division, Dept. of Pathology, Brigham and Women's Hospital, 221 Longwood Ave., LMRC-401 Boston, MA 02115. Tel.: 617-732-5901; Fax: 617-732-5933; E-mail: mgimbrone@rics.bwh.harvard.edu.

Published, JBC Papers in Press, October 15, 2001, DOI 10.1074/jbc.M105513200

	ABBREVIATIONS

The abbreviations used are: IL-1, interleukin 1b; ERK1/2, extracellular signal-regulated kinase; Ras, rat sarcoma virus; Raf, proto-oncogene; MEK, mitogen-activated protein kinase/ERK1/2; SH2, Src homology domain 2; SHP2, SH2-containing protein-tyrosine phosphatase 2; Shc, SH2-domain-containing 2-collagen-related; Grb2, growth factor receptor-bound protein 2; Sos, son of sevenless; HUVEC, human umbilical vein endothelial cells; MOPS, 4-morpholinepropanesulfonic acid; FBS, fetal bovine serum; mAb, monoclonal antibody.

	REFERENCES

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