(Received for publication, October 2, 1996, and in revised form, January 7, 1997)
From the Blood Research Institute, Blood Center of
Southeastern Wisconsin, Milwaukee, Wisconsin 53233-2121 and the
§ Departments of Cellular Biology and Pharmacology, Medical
College of Wisconsin, Milwaukee, Wisconsin 53226
Platelet/endothelial cell adhesion molecule-1 (PECAM-1) is a homophilic adhesion receptor that mediates leukocyte/endothelial cell interactions that take place during transendothelial migration. Recent reports have shown that the binding of certain anti-PECAM-1 antibodies results in up-regulation of integrin function on the surface of leukocytes and platelets, suggesting that PECAM-1 may be capable of transmitting information into the cell following its engagement. PECAM-1 isolated from resting or activated but nonaggregated platelets was phosphorylated predominantly on serine residues; however, PECAM-1 derived from activated, aggregated platelets was strongly phosphorylated on tyrosine. Synthetic tyrosine-phosphorylated peptides derived from five different regions within the cytoplasmic domain of PECAM-1 were screened for their ability to associate with cytoplasmic signaling molecules. The protein-tyrosine phosphatase SHP-2 was found to interact specifically with two different PECAM-1 phosphopeptides containing highly conserved phosphatase-binding motifs on PECAM-1 with the sequences VQpY663TEV and TVpY686SEV. More important, SHP-2 bound not only PECAM-1 phosphopeptides, but also became associated with full-length cellular PECAM-1 during the platelet aggregation process, and this interaction was mediated by the amino-terminal Src homology 2 domains of the phosphatase. Since SHP-2 normally serves as a positive regulator of signal transduction, its association with activated PECAM-1 suggests a number of potential mechanisms by which PECAM-1 engagement might be coupled to integrin activation in vascular cells.
PECAM-11 is a 130-kDa member of the immunoglobulin gene superfamily that is expressed on platelets and leukocytes and is also present at high concentration at the intercellular junctions of endothelial cells (for a review, see Refs. 1 and 2). Because of its presence on the surface of these vascular cells, PECAM-1 has been implicated in mediating a number of cell/cell interactions, including those that take place during cell migration (3), transendothelial migration of monocytes and neutrophils (4), and following antigenic stimulation of transmigrating lymphocytes (5). Although multiple mechanisms have been proposed for PECAM-1-mediated adhesion events, recent studies suggest that PECAM-1 molecules on adjacent cells are able to interact homophilically with each other, utilizing amino-terminal Ig homology domains 1 and 2 to effect these cellular associations (6, 7).
In addition to serving as a cell adhesion receptor, several lines of evidence suggest that PECAM-1 may be capable of transmitting signals into the cell following its engagement. Tanaka et al. (8) were the first to show that antibody-mediated engagement of PECAM-1 on the surface of lymphocytes could lead to up-regulation of integrin function, a finding that has been reproduced in lymphokine-activated killer cells (9), CD34+ hematopoietic progenitor cells (10), monocytes and neutrophils (11), and natural killer cells (12). Although stimulation of Fc receptors by bound antibody may be contributing to cellular activation in some of these studies, it is likely that PECAM-1 dimerization itself may be capable of transducing as yet undefined signals into the cell, a process that could be mimicked by homophilic PECAM-1/PECAM-1 interactions that are thought to occur between leukocytes and endothelial cells during the process of transendothelial migration.
The mechanism by which PECAM-1 engagement might lead to downstream signaling events is unknown. Previous studies have shown that, following cellular activation, PECAM-1 becomes phosphorylated (13, 14), but the target for this phosphorylation event appeared to be serine residues within the cytoplasmic domain (13). A more recent report by Modderman et al. (15) showed that PECAM-1 derived from resting platelets that had been preincubated with the membrane-permeable tyrosine phosphatase inhibitor pervanadate became tyrosine-phosphorylated. Precisely when this might occur, if at all, during normal platelet physiology remained to be determined, but it was clear from their studies that the balance of intracellular kinases and phosphatases was likely to play a role in regulating both the stoichiometry of PECAM-1 phosphorylation and the residues that might become phosphorylated during different stages of cellular activation.
The purpose of this investigation was to determine whether PECAM-1 could become tyrosine-phosphorylated in response to agonist-induced cellular activation and subsequent cell/cell interactions. In addition, we sought to identify cytoplasmic signaling molecules that might associate with the cytoplasmic domain of PECAM-1 and to provide a mechanistic link between PECAM-1 engagement and the subsequent activation of cell-surface integrins that had been observed in previous investigations. Using human platelets as a model, we show that PECAM-1 becomes tyrosine-phosphorylated during the platelet aggregation process and that this creates docking sites for the protein-tyrosine phosphatase SHP-2. The interaction between SHP-2 and PECAM-1 is dependent upon integrin-mediated platelet/platelet interactions and occurs via the Src homology 2 (SH2) domains of the phosphatase and highly conserved phosphatase-binding motifs encompassing phosphotyrosines 663 and 686 within the cytoplasmic domain of PECAM-1.
Phenylmethylsulfonyl fluoride, prostaglandin E1, Triton X-100, bovine serum albumin, leupeptin, streptavidin-agarose, cyanogen bromide-activated Sepharose, dimethyl sulfoxide, pyridine, aprotinin, and reduced glutathione were purchased from Sigma. Protein G-Sepharose and glutathione-Sepharose 4B were from Pharmacia Biotech (Uppsala). Recombinant N- and C-terminal SH2 domains of the protein-tyrosine phosphatase SHP-2, expressed as a GST fusion protein (GST-N-SH2-C-SH2), were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Polyvinylidene difluoride membranes were obtained from Millipore Corp. (Bedford, MA). Sodium dodecyl sulfate, glycine, prestained broad range standards, Affi-Gel 10, Tween 20, and TEMED were from Bio-Rad. The enhanced chemiluminescence Western blotting detection kit was obtained from Amersham Life Science, Inc. [32P]Orthophosphate was obtained from DuPont NEN. TLC plates were purchased from Merck. Fresh platelet concentrates (<2 days old) were provided by volunteer donors to The Blood Center of Southeastern Wisconsin (Milwaukee, WI).
AntibodiesThe properties of the anti-PECAM-1 murine monoclonal antibodies PECAM-1.2 (specific for extracellular Ig homology domain 6) and PECAM-1.3 (specific for Ig homology domain 1) have been previously described (6). Horseradish peroxidase-conjugated PY-20 (an anti-phosphotyrosine murine monoclonal antibody) was obtained from Zymed Laboratories, Inc. (South San Francisco). Polyclonal antibodies directed to the N- and C-terminal SH2 domains of the protein-tyrosine phosphatase SHP-2 were obtained from Santa Cruz Biotechnology. This antibody specifically recognizes a single SHP-2 band in cell lysates and does not cross-react with SHP-1. Horseradish peroxidase-conjugated anti-rabbit IgG was obtained from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA). Normal rabbit IgG and normal mouse IgG1 were obtained from Sigma.
Preparation of Washed PlateletsPlatelets were obtained from normal healthy volunteers who had not taken any antiplatelet medication in the preceding 10 days. Whole blood was anticoagulated with acid citrate/dextrose, pH 4.6, in the presence of 50 ng/ml prostaglandin E1 according to previously described methods (13). Platelet-rich plasma was obtained by centrifugation at 250 × g for 15 min at room temperature, and platelets were obtained by sedimentation at 1000 × g for 15 min. The platelet pellet was washed twice in Ringer citrate/dextrose buffer (108 mM NaCl, 38 mM KCl, 1.7 mM NaHCO3, 21.2 mM sodium citrate, 27.8 mM glucose, and 1.1 mM MgCl2, pH 6.5) in the presence of 50 ng/ml prostaglandin E1. Finally, the platelet pellet was resuspended in Ringer citrate/dextrose buffer, pH 7.4, at a concentration of 1 × 109 platelets/ml.
Platelet Activation/Aggregation StudiesWashed platelets
were activated by the addition of 7 µM TRAP in the
absence of stirring. In some instances, F(ab)2 fragments of PECAM-1.2 were used to activate platelets resuspended in 2 mM CaCl2, 1 mM MgCl2,
and 100 µg/ml fibrinogen. Platelet aggregation was induced
by adding 7 µM TRAP to stirred platelets (1000 rpm) in
the presence of 2 mM CaCl2, 1 mM
MgCl2, and 100 µg/ml fibrinogen for 3-10 min at 37 °C
in a four-channel automated platelet analyzer (BioData Corp., Horsham,
PA). In some experiments, platelets were preincubated for 5 min at
37 °C with 0.5 mM RGDW peptide prior to activation by
TRAP.
Platelets subjected to either activation or aggregation were lysed by the addition of 0.5 ml of Triton lysis buffer (2% Triton X-100, 10 mM EGTA, 15 mM HEPES, 145 mM NaCl, 0.1 mM MgCl2, 1 mM phenylmethylsulfonyl fluoride, 20 µg/ml leupeptin, and 2 mM sodium orthovanadate, pH 7.4), and the lysate was slowly rocked for 1 h at 4 °C. Triton-soluble and -insoluble (cytoskeletal) fractions were separated by centrifugation at 15,000 × g for 5 min at 4 °C as described previously (13). In some experiments, samples were lysed by the addition of an equal volume of ice-cold 2 × radioimmune precipitation assay buffer (final concentration = 20 mM Tris, pH 7.4, 1 mM EGTA, 158 mM NaCl, 0.1% (w/v) SDS, 1% (w/v) sodium deoxycholate, 1 mM Na3VO4, 2 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 1% (v/v) Triton X-100).
Immunoprecipitation AnalysisFollowing fractionation of the 15,000 × g Triton-soluble platelet supernatant, lysates were precleared with 50 µl of a 50% slurry of CNBr-activated Sepharose for 30 min at 4 °C and then centrifuged at 4000 rpm for 5 min. Precleared lysates were incubated overnight with either PECAM-1.3 (10 µg/ml) or normal mouse IgG1 (10 µg/ml). Immune complexes were recovered using 50 µl of a 50% slurry of protein G-Sepharose and washed four times by brief centrifugation in immunoprecipitation buffer (50 mM Tris, pH 7.4, containing 150 mM NaCl and 2% Triton X-100). Bound proteins were eluted from the beads in SDS reducing sample buffer, boiled for 10 min, resolved by 12.5% SDS-PAGE, transferred to a polyvinylidene difluoride membrane, and analyzed by immunoblotting with a 1:5000 dilution of either horseradish peroxidase-conjugated PY-20 or polyclonal antibodies directed against either the C- or N-terminal SH2 domain of SHP-2. In the latter two cases, the membrane was additionally incubated for 1 h with horseradish peroxidase-conjugated goat anti-rabbit IgG (1:25,000). Immunoreactivity was then determined by chemiluminescence detection according to the manufacturer's instructions (Amersham Life Science, Inc.).
Peptide SynthesisPECAM-1 peptides were synthesized using a Model 9050 Pepsynthesizer (Millipore Corp.) with Fmoc chemistry as described previously (16). Minor modifications to the procedure included the incorporation of phosphotyrosine residues during peptide synthesis. Fmoc-Tyr(PO3tBu2)-OH (Quality Controlled Biochemicals, Hopkinton, MA) was side chain-protected with a t-butyl group and was introduced as the free acid. It was activated with 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate and 0.3 M 1-hydroxylbenzotriazole hydrate in dimethylacetamide. Prior to cleavage from the resin, peptides were biotinylated at the amino terminus by incubation with a 1.5-fold molar excess of NHS-LC-biotin (Pierce) dissolved in dimethylacetamide containing 20% 4-dimethylaminopyridine to facilitate coupling. All peptides were >85% purity and were analyzed by electrospray mass spectroscopy (Protein Structure and Carbohydrate Facility, University of Michigan, Ann Arbor, MI) to confirm the expected molecular mass for both the nonphosphorylated and phosphorylated PECAM cytoplasmic tail peptides. Lyophilized biotinylated peptides were finally dissolved at a final concentration of 1 mg/ml in 10 mM sodium phosphate and 150 mM NaCl, pH 7.4, containing 0.3% dimethyl sulfoxide and were stored at 4 °C until use.
Peptide Binding StudiesPeptides (10 µg/ml) were incubated with 1.5 mg of 15,000 × g Triton-soluble platelet proteins overnight at 4 °C with constant mixing. Fifty µl of streptavidin-agarose beads were then added to the peptide mixture and incubated for an additional hour at 4 °C. The beads were washed five times in immunoprecipitation buffer by centrifugation at 4000 rpm for 5 min. Bound proteins were eluted in SDS sample buffer, boiled 10 min, and resolved by 12.5% SDS-PAGE.
Phosphoamino Acid AnalysisWashed platelets were resuspended at 1 × 109 platelets/ml in 138 mM NaCl, 2.7 mM KCl, 5 mM glucose, and 50 mM HEPES, pH 7.4, and incubated at 37 °C unstirred with 0.5 mCi/ml 32Pi (carrier-free sodium orthophosphate) for 3 h. 32P-Labeled platelets were then sedimented by centrifugation and resuspended in gel filtration buffer (137 mM NaCl, 2.7 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 3.3 mM NaH2PO4, and 20 mM HEPES, pH 7,.4 with 0.1% (w/v) glucose and 0.1% (w/v) bovine serum albumin). Following the addition of 500 µl of 2 × radioimmune precipitation assay buffer containing 1 mM sodium orthovanadate, 32P-labeled platelet proteins were immunoprecipitated using PECAM-1.3-Affi-Gel 10 beads, separated by SDS-PAGE under reducing conditions, and transferred to a polyvinylidene difluoride membrane. Labeled bands were identified by autoradiography, excised, submerged in 6 N HCl, and boiled for 60 min at 110 °C. The supernatant containing the phosphoamino acids was dried, and the residue was dissolved in 10 µl of water. Two-µl aliquots of the 32P-labeled samples and unlabeled phosphoamino acid standards (1 mg/ml) were dotted sequentially onto cellulose TLC plates and air-dried. Hydrolysates were resolved by electrophoresis in two dimensions: first at pH 1.9 in 0.58 M formic acid and 1.36 M acetic acid (1000 V for 20 min) and then at pH 3.5 in 0.87 M acetic acid, 0.5% (v/v) pyridine, and 0.5 mM EDTA (500 V for 25 min). TLC plates were dried at 80 °C, stained with ninhydrin to reveal the migration of the unlabeled standards, and then subjected to autoradiography to detect comigrating 32P-labeled amino acids (17).
Previous studies have shown that the cytoplasmic
domain of PECAM-1 becomes phosphorylated on serine residues following
activation of platelets with either thrombin or phorbol ester (13). To examine whether one or more of the five tyrosine residues within the
PECAM-1 cytoplasmic domain might also become phosphorylated under
certain physiological conditions, we compared the phosphorylation state
of PECAM-1 in human platelets that were 1) resting; 2) activated with
TRAP without stirring (i.e. nonaggregated); or 3)
TRAP-activated, stirred, and fully aggregated. Following detergent
lysis in the presence of the tyrosine phosphatase inhibitor vanadate
(see "Experimental Procedures"), the phosphorylation state of
PECAM-1 was evaluated both immunochemically and biochemically. As shown
in Fig. 1A, PECAM-1 was
32P-labeled in both resting and activated platelets
(lanes 1 and 2), but incorporation of
[32P]orthophosphate into PECAM-1 was greatest under
conditions where aggregation had been allowed to occur (lane
3). To determine which residues within the cytoplasmic domain of
PECAM-1 had become phosphorylated under each of these three conditions,
the three bands shown in the top row of Fig. 1A were excised
from the gel and acid-hydrolyzed, and phosphoamino acids were analyzed
by two-dimensional electrophoresis. As shown in Fig. 1B,
PECAM-1 derived from resting platelets was slightly phosphorylated on
serine (panel 1), and the level of serine phosphorylation
increased 2-3-fold following platelet activation (panel 2),
as reported previously (13). No labeling of tyrosine residues was
observed in either resting or activated cells (panels 1 and
2), even when the film was overexposed (data not shown). Interestingly, whereas 32P had been incorporated solely on
serine residues in PECAM-1 derived from resting or activated,
nonaggregated platelets, phosphotyrosine was readily detectable in
PECAM-1 derived from activated, aggregated platelets. These
findings were confirmed immunologically in immunoblot analysis
employing the anti-phosphotyrosine-specific monoclonal antibody PY-20
(Fig. 1C, lanes 1-3). Interestingly, the
addition of RGD peptide before TRAP stimulation blocked tyrosine
phosphorylation of PECAM-1 (lane 4), suggesting that this
event required prior integrin-mediated platelet aggregation. Together,
these data indicate that PECAM-1 initially becomes phosphorylated on
one or more serine residues upon platelet activation, followed by
tyrosine phosphorylation sometime during the platelet aggregation
process.
Interaction of the Protein-tyrosine Phosphatase SHP-2 with Tyrosine-phosphorylated Peptides Derived from the Cytoplasmic Domain of PECAM-1
The cytoplasmic domain of PECAM-1 is structurally and
functionally complex, being encoded by eight distinct exons (exons
9-16) that can be alternatively spliced to yield different PECAM-1
isoforms (18-20). Regions of the cytoplasmic domain encoded by exons
9, 11, and 13-15 each contain a tyrosine residue that could have become phosphorylated during platelet aggregation. To determine whether
any of these might be involved in targeting one or more cytoskeletal or
cytosolic signaling molecules to the cytoplasmic domain of PECAM-1 in
activated, aggregated platelets, we synthesized a matched series of
biotinylated 11-amino acid peptides corresponding to a specific region
of the PECAM-1 cytoplasmic domain, each, with the exception of
PECAM-(594-604) (residue 594 is the first amino acid of the
cytoplasmic domain), containing the potential tyrosine phosphorylation
site in the center, with and without phosphate (Fig.
2A). These biotinylated peptides were mixed
with detergent lysates of resting human platelets, and bound proteins were recovered using streptavidin-agarose beads. Immunoblot analysis using a series of antibodies specific for signaling molecules known to
be present in human platelets identified SHP-2, a 70-kDa protein-tyrosine phosphatase, bound to PECAM-1-(658-668) and
PECAM-1-(681-691) (regions encoded by exons 13 and 14 of the
PECAM-1 gene, respectively), but only when the peptides
themselves were tyrosine-phosphorylated at Tyr663 and
Tyr686 (Fig. 2B). Interaction of SHP-2 with each
of the two biotinylated peptides was specific, as formation of the
complex could be competitively inhibited by a 20-50-fold molar excess
of nonbiotinylated PECAM-1 phosphopeptide (Fig. 2, C and
D).
SH2 Domains of SHP-2 Mediate Binding to PECAM-1-(658-668) and PECAM-1-(681-691)
SHP-2 is one of several intracellular
cytosolic protein-tyrosine phosphatases that are able to localize to
specific sites within the cell and has been shown to interact with a
number of activated growth factor receptors via its SH2 domains located at the amino terminus of the molecule (depicted schematically in Fig.
3A). To examine whether the SH2 domains of
SHP-2 were responsible for associating with the two biotinylated
PECAM-1 phosphopeptides identified above, the tyrosine-phosphorylated or nonphosphorylated forms were incubated with a GST fusion protein containing both the amino- and carboxyl-terminal SH2 domains of SHP-2,
and complexes were captured with streptavidin-agarose beads. As shown
in Fig. 3 (B and C, left panels),
nonphosphorylated peptides failed to associate with either GST alone
(lanes 1-3) or the GST-N-SH2-C-SH2 fusion protein
(lanes 4-6). In contrast, phosphopeptides encompassing Tyr663 and Tyr686 bound avidly to the GST
fusion protein containing the two SH2 domains of SHP-2 (Fig. 3,
B and C, right panels, lanes
4-6). This interaction was specific, as the phosphopeptides did
not interact with the GST protein alone (right panels,
lanes 1-3). These data demonstrate that the interaction of
SHP-2 with these two PECAM-1 cytoplasmic domain phosphopeptides is
directly mediated by one or both of the amino-terminal SH2 domains of
the phosphatase.
SHP-2 Binds PECAM-1 in Activated, Aggregated Human Platelets
Although PECAM-1 becomes phosphorylated on tyrosine
residues during platelet aggregation (Fig. 1), and SHP-2 binds to
synthetic phosphopeptides corresponding to two small discrete regions
within the cytoplasmic domain of PECAM-1 (Fig. 2), the ability of SHP-2 to associate with cellular PECAM-1 in an activated human platelet remained to be demonstrated. To address this issue, PECAM-1 was immunoprecipitated from detergent lysates of platelets that had been
(a) treated with buffer (resting), (b)
TRAP-activated but not aggregated, or (c) TRAP-activated and
fully aggregated, and the resulting immunoprecipitate was probed for
the presence of SHP-2 by immunoblot analysis. As shown in Fig.
4, a small amount of SHP-2 became associated with
PECAM-1 following platelet activation, but this increased greatly when
platelet aggregation was allowed to occur. Similar to the PECAM-1
phosphopeptide studies described for Fig. 3, the binding of SHP-2 to
full-length PECAM-1 was mediated by the SH2 domains of the phosphatase
(Fig. 5). In parallel with the requirements for tyrosine
phosphorylation of PECAM-1 (Fig. 1C), the association of
SHP-2 with PECAM-1 required integrin-mediated platelet/platelet
interactions, as the addition of 0.5 mM RGDW peptide to the
cuvette before TRAP-induced platelet activation completely blocked
formation of the PECAM-1·SHP-2 complex. Cross-linking of PECAM-1 on
the platelet surface using an F(ab)2 fragment of the
anti-PECAM-1 monoclonal antibody PECAM-1.2 also resulted in a small
degree of SHP-2 binding (Fig. 4, last lane), consistent with
the known ability of this antibody to expose P-selectin on the platelet
surface and to elicit conformational changes in the integrin
IIb
3 (21). Together, these data provide
strong evidence that the association of SHP-2 with PECAM-1 is not
merely an in vitro phenomenon, but occurs in vivo
during the platelet aggregation process.
There is increasing evidence to suggest that, following
interaction with the extracellular matrix or with other cells,
cell-surface adhesion receptors may be capable of transmitting signals
across the plasma membrane. For example, cross-linking of certain
integrins with antibodies or adhesion of cells to specific integrin
ligands can stimulate tyrosine phosphorylation of multiple cytosolic
components, increase cytoplasmic pH and ionized calcium, initiate
phosphoinositide synthesis, and modify patterns of gene expression
(22-24). Integrins do not contain intrinsic kinase or phosphatase
activity within their cytoplasmic domains, nor do they harbor SH2 or
SH3 domains that could serve to recruit signaling molecules following
ligand binding. Studies suggest, rather, that outside-in signaling
events may be brokered by the accumulation and assembly of specific
cytoskeletal (25) and signaling (26-28) molecules at sites of integrin
clustering. The molecular basis underlying at least some of these
interactions has recently been suggested by the work of Law et
al. (29), who showed that the 3-subunit of the
integrin
IIb
3 becomes tyrosine-phosphorylated in response to thrombin-induced platelet aggregation, creating potential docking sites for the SH2-containing adapter proteins Grb2 and Shc.
This mode of signal transduction may represent a general paradigm that
is used by PECAM-1 as well. Although the cytoplasmic domain of PECAM-1
lacks demonstrated catalytic activity, it contains numerous potential
sites for phosphorylation of serine, threonine, and tyrosine residues
(30). We found that PECAM-1, like the integrin
3-subunit, becomes tyrosine-phosphorylated during the platelet aggregation process (Fig. 1). Once "activated" by tyrosine phosphorylation, PECAM-1 associated with the 70-kDa protein-tyrosine phosphatase SHP-2 (Figs. 2 and 4). SHP-2 (also known as SHPTP-2, Syp,
PTP1D, PTP2C, and SH-PTP3 (31)) is a ubiquitously expressed protein-tyrosine phosphatase that is composed of two SH2 domains in the
amino-terminal third of the protein, followed by a catalytic phosphatase domain and a 76-amino acid carboxyl terminus that can
become phosphorylated by receptor tyrosine kinases (32-35). Previous
studies have shown that the SH2 domains of SHP-2 direct its interaction
with the cytoplasmic domains of a number of tyrosine-phosphorylated (i.e. activated) growth factor receptors, including the
platelet-derived growth factor receptor (34-36), the epidermal growth
factor receptor (34, 35, 37), p145c-Kit (35), and HER2-neu
(35). SHP-2 has also been linked to the insulin signal transduction
pathway through its association with insulin receptor substrate-1 (36).
SHP-2 signaling has not, however, previously been linked to cell
adhesion. Thus, our finding that tyrosine-phosphorylated PECAM-1 serves
as a docking site for the SH2 domains of SHP-2 in activated human
platelets (Figs. 3, 4, 5) provides the first evidence that SHP-2 may also
propagate transmembrane signals that emanate from cell/cell
interactions.
SHP-2 has been shown to associate through its SH2 domains with the
activated platelet-derived growth factor receptor at
Tyr(P)1009 (38, 39). The sequence specificity for this
interaction has been examined in great detail (40-42), and studies to
date indicate that the sequence recognition elements surrounding the
Tyr(P) residue that are required for high affinity binding of the
N-terminal SH2 domain of SHP-2 include Val at position 2, a
-branched residue (Thr/Val/Ile) at position +1, and a hydrophobic
residue (Val/Leu/Ile) at position +3. Of the five tyrosine residues
found within the cytoplasmic domain of PECAM-1, only the two encoded by
exons 13 and 14, with the sequences VQpY663TEV and
TVpY686SEV, fulfill these requirements. Sequence alignment
of each of these sequences among various species (Fig.
6) reveals absolute conservation of the four key
residues surrounding PECAM-1 Tyr663 that constitute the
SHP-2-binding motif. The residues surrounding Tyr686,
however, only loosely conform to the consensus sequence, suggesting that the C-terminal site on the PECAM-1 cytoplasmic domain may constitute only a low affinity binding site for SHP-2. It should be
pointed out that while we have shown that tyrosine-phosphorylated peptides encompassing residues 658-668 and 681-691 of the PECAM-1 cytoplasmic domain bind SHP-2 (Figs. 2 and 3) and that SHP-2 binds tyrosine-phosphorylated PECAM-1 (Figs. 4 and 5), further studies employing mutant forms of PECAM-1 will be required to determine the
binding site on PECAM-1 for SHP-2 in vivo. In this regard, it is notable that deletion of exon 14 results in the loss of the
ability of PECAM-1-transfected fibroblasts to bind PECAM-1-negative cells (7, 20, 43), a function that we attribute to loss of
PECAM-1-mediated signal transduction (see below). Whether
Tyr686 becomes phosphorylated and acts synergistically with
Tyr663 to localize SHP-2 to activated PECAM-1 or whether it
serves as an independent docking site for the recruitment of additional signaling molecules is the subject of current investigation in our
laboratory.
The catalytic activity of SHP-2 can be regulated both by its physical interaction with activated (i.e. tyrosine-phosphorylated) receptor tyrosine kinases (39) as well as by receptor tyrosine kinase-mediated phosphorylation of SHP-2 itself (34, 35, 37). Unlike the platelet-derived growth factor receptor, which can both bind to and phosphorylate SHP-2, PECAM-1 is incapable of phosphorylating SHP-2 at its carboxyl terminus following their interaction. Preliminary studies suggest, however, that PECAM-1 might be able to directly activate SHP-2 by virtue of its binding, as tyrosine-phosphorylated peptides comprising PECAM-1-(658-668) and PECAM-1-(681-691) stimulate the phosphatase activity of SHP-2.2 One scenario whereby PECAM-1 might initiate cellular signaling, therefore, might take advantage of its ability to localize SHP-2 to the inner face of the plasma membrane and activate it, thereby bringing the phosphatase into proximity with nearby phosphorylated signaling molecules. One particularly attractive set of substrates for SHP-2 may be protein-tyrosine kinases, such as pp60src, which are constitutively bound to the membrane via post-translationally added fatty acid side chains and which normally exist in an inactive state due to intramolecular interactions with a negative regulatory phosphotyrosine residue (44). Whether PECAM-1/SHP-2 interactions promote cellular signaling by dephosphorylating Src family kinases is not yet known.
SHP-2 has the capacity to be a multifunctional signaling molecule.
C-terminal to its phosphatase domain, human SHP-2 contains three
tyrosine residues, and the sequence around two of these (Y542TNI and Y580ENV), when
tyrosine-phosphorylated, conforms to the consensus binding site for
the SH2 domain of the adapter protein Grb2 (40). In fact, tyrosine
phosphorylation of SHP-2 induced by platelet-derived growth factor (45,
46), interleukin-3 and granulocyte-macrophage colony-stimulating factor
(47), and steel factor (48) has been shown to result in the direct
binding of Grb2 in vivo. Since Grb2 normally exists bound,
via its SH3 domains, to the guanine nucleotide-releasing factor Sos
(49-56), it has been proposed that SHP-2 functions as an adapter
protein, linking the activation of these transmembrane growth factor
and cytokine receptors to the Ras signaling pathway in hematopoietic
cells. In addition, Welham et al. (47) have found that SHP-2
coprecipitates with the p85 subunit of phosphatidylinositol 3-kinase,
although the molecular nature of this interaction is not well
understood. Whether SHP-2 integrates signals that derive from PECAM-1
homophilic interactions with either the Ras or phosphatidylinositol
3
-kinase pathway in platelets and other vascular cells remains to be
determined.
In resting platelets, the majority of PECAM-1 molecules are either free
within the plane of the membrane or associated with the underlying
membrane skeleton (13). During platelet aggregation, however, >50% of
PECAM-1 receptors become associated with the actin cytoskeleton. In
contrast, SHP-2 exists either free in the cytosol or associated with
the membrane skeleton, with virtually none becoming attached
to the cytoskeleton in activated human platelets (57). In our studies
(Fig. 4), we found that SHP-2 became associated with PECAM-1 present in
the 15,000 × g supernatant of activated,
detergent-solubilized platelets, i.e. those PECAM-1 receptors that were either linked to the membrane skeleton or free
within the plane of the membrane. While we have not yet determined the
stoichiometry of SHP-2:PECAM-1 in any of these subcellular fractions,
it is tempting to speculate that a subpopulation of PECAM-1, perhaps
attached to the membrane skeleton of human platelets, serves a unique
functional role in the assembly of signaling complexes during the
IIb
3-mediated platelet aggregation
process. Two other nontransmembrane protein-tyrosine phosphatases
present in platelets, PTP1B and SHP-1, have been found to be associated
with the 15,000 × g detergent-insoluble cytoskeleton
during platelet aggregation, where they are thought to participate in
the dephosphorylation of tyrosine-phosphorylated proteins that have
become associated within the cytoskeleton (57). However, we have found
no evidence for their association with the cytoplasmic domain of
PECAM-1 to date.
Finally, we predict that bidirectional signaling through PECAM-1 may be
of functional importance in other vascular cells. We have shown that
PECAM-1 can function downstream from integrin engagement, as
tyrosine phosphorylation of PECAM-1 and its subsequent association with
SHP-2 are each dependent upon prior integrin-mediated platelet/platelet
contact (Figs. 1 and 4). The relationship between PECAM-1- and
integrin-mediated signaling, however, appears to be able to work in
both directions. The ability of PECAM-1, upon its engagement, to serve
as an amplifier of integrin-mediated cell adhesion (8-12) provides one
cogent series of examples in which PECAM-1 functions
upstream of integrins, serving primarily as an agonist
receptor rather than as a cell adhesion molecule per se.
"PECAM-1-mediated" binding to the integrin
v
3 of lymphokine-activated killer cells
(58) and monocyte-like U937 cells (59) and the interaction of
PECAM-1-transfected L-cells with PECAM-1-negative murine L-cell
fibroblasts (60, 61) may represent additional examples of adhesive
interactions that are enabled by outside-in signal transduction through
PECAM-1. This process may be relevant to both inflammation and
thrombosis during the process of selectin-mediated leukocyte (62) and
platelet (63) rolling on activated endothelium. Homophilic interactions
between PECAM-1 located at endothelial cell intercellular junctions and
either platelet or leukocyte PECAM-1 might act in concert with
selectin-mediated signaling to promote integrin activation and
subsequent tight adhesion necessary for transendothelial migration.
Whether the anti-inflammatory effects of anti-PECAM-1 antibodies
observed in a number of in vitro (4) and in vivo
(64, 65) models are due, in part, to inhibition of PECAM-1-mediated
outside-in signaling remains to be determined. Further studies aimed at
identifying the mechanisms by which PECAM-1/SHP-2 interactions broker
these events and the signaling molecules that participate in these
complex cellular processes should shed additional light on the role of
the cell adhesion and cell signaling molecule PECAM-1 in vascular cell biology.
We are grateful to Drs. James Augustine and Cheryl Hillery for helpful discussions and to Norman Mermelstein for technical advice. We also thank Trudy M. Holyst for expert assistance in the design and production of PECAM phosphopeptides. Dr. Mark Zukowski and Barbara Karin-Tamir (Amgen Corp.) generously provided the amino acid sequence of rat PECAM-1 prior to its publication.