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IMMUNOBIOLOGY
From the Blood Research Institute, The Blood Center of
Southeastern Wisconsin, Milwaukee, WI; and the Departments of
Microbiology, Pharmacology, and Cellular Biology, Medical College of
Wisconsin, Milwaukee, WI.
Platelet Endothelial Cell Adhesion Molecule-1
(PECAM-1, CD31) is a 130-kd member of the immunoglobulin gene
superfamily that is expressed on the surface of platelets, endothelial
cells, myeloid cells, and certain lymphocyte subsets. PECAM-1
has recently been shown to contain functional immunoreceptor
tyrosine-based inhibitory motifs (ITIMs) within its cytoplasmic
domain, and co-ligation of PECAM-1 with the T-cell antigen
receptor (TCR) results in tyrosine phosphorylation of
PECAM-1, recruitment of Src homology 2 domain-containing protein tyrosine phosphatase-2 (SHP-2), and
attenuation of TCR-mediated cellular signaling. To determine the
molecular basis of PECAM-1 inhibitory signaling in
lymphocytes, the study sought to (1) establish the importance of the
PECAM-1 ITIMs for its inhibitory activity, (2) determine
the relative importance of SHP-2 versus SHP-1 in mediating the
inhibitory effect of PECAM-1, and (3) identify the protein
tyrosine kinases required for PECAM-1 tyrosine
phosphorylation in T cells. Co-ligation of wild-type PECAM-1
with the B-cell antigen receptor expressed on chicken DT40 B cells
resulted in a marked reduction of calcium mobilization The strength of the signal transduced by the T-cell
antigen receptor (TCR) is influenced by the cellular context in which antigen is recognized (reviewed in1). One of the variables that provides context for antigen recognition is the expression of
inhibitory receptors on T-cell targets. Inhibitory receptors possess,
within their cytoplasmic domains, an immunoreceptor tyrosine-based inhibitory motif (ITIM), which is characterized by the presence of a
tyrosine residue surrounded by a hydrophobic residue at position It has been proposed that Platelet Endothelial Cell Adhesion Molecule-1
(PECAM-1, CD31) may regulate signals transduced by antigen
receptors (reviewed in3). PECAM-1 possesses a
dual ITIM that, on tyrosine phosphorylation, supports the binding of SHP-2.4,5 In T cells, PECAM-1 becomes tyrosine
phosphorylated and binds SHP-2 in response to engagement of the
(TCR),6 and heightened levels of PECAM-1
tyrosine phosphorylation and SHP-2 binding are observed in response to
cross-linking of PECAM-1 alone or together with the
TCR.7 In previous studies, we have used PECAM-1-expressing T-cell lines to show that
PECAM-1 functions as an inhibitory receptor, in that
co-ligation of PECAM-1 with the TCR results in attenuation of
TCR-induced release of calcium from intracellular stores.7
These studies did not, however, address whether either tyrosine
phosphorylation of the PECAM-1 ITIMs or subsequent
binding of SHP-2 were required for the inhibitory function of
PECAM-1. The recent finding that mutant forms of cytotoxic T-lymphocyte antigen 4 (CTLA-4), which fail to become tyrosine phosphorylated and bind SHP-2, continue to inhibit T-cell
activation8,9 make this possibility an important
consideration. In addition, several studies6,10-12 have
reported that, in addition to SHP-2, the related PTP, SHP-1, can bind
to the tyrosine-phosphorylated PECAM-1 ITIMs, albeit with
lower affinity. Pumphrey et al12 have reported that SHP-2,
SHP-1, and SHIP bind to the tyrosine-phosphorylated PECAM-1
ITIMs. Although each of these phosphatases may be capable of
binding to the PECAM-1 ITIMs, their relative ability to
support the inhibitory function of PECAM-1 has not yet been addressed.
PECAM-1 does not possess intrinsic kinase activity, and the
range of the PTKs responsible for tyrosine phosphorylating the ITIMs within the cytoplasmic domain of PECAM-1 is not
known. TCR-mediated signal transduction involves the activity of
members of the Src, Syk, and Tec families of PTKs (reviewed
in1,13). Several in vitro studies have shown that
exogenous c-Src can phosphorylate glutathione-S-transferase
(GST)-fusion proteins containing the PECAM-1 cytoplasmic
domain5,14,15 or native PECAM-1 isolated from
bovine aortic endothelial cells.15 In addition,
PECAM-1 has been shown to become tyrosine phosphorylated in
COS-1 cells that have been transiently co-transfected with
pp60c-Src, p56lck,
p59fyn, and p53lyn.10 The
conclusion that Src family PTKs are involved in PECAM-1 tyrosine phosphorylation is supported by the recent observation that
PECAM-1 tyrosine phosphorylation is reduced or abolished on
exposure of stimulated platelets to the Src family PTK inhibitor, PP2.16 Among the Src family PTKs present in T cells,
p56lck plays the best-documented role in TCR-mediated
signal transduction in mature T lymphocytes.1 Whether
p56lck functions in PECAM-1 signaling, however,
is not yet known.
In the present investigation, we sought to determine whether an
ITIM-less form of PECAM-1 expressed in the DT40 chicken
B-cell line might be able to attenuate calcium mobilization
and whether protein phosphatases other than SHP-2 might be
able to functionally complement the inhibitory activity of a
PECAM-1/SHP-2 signaling complex. We also investigated
the importance of p56lck in PECAM-1 tyrosine
phosphorylation in T lymphocytes. Our results suggest that the
molecular requirements for the inhibitory function of PECAM-1
include intact ITIMs, SHP-2, and, in T cells, the Src family PTK,
p56lck.
Cell lines and cell culture
Generation and characterization of PECAM-1
transfectants
Antibodies To stimulate wild-type and mutant Jurkat T cells, the human CD3 -specific antibody (Ab), UCHT1 (Pharmingen, San Diego, CA), containing no azide and low endotoxin, was used. Wild-type and mutant
DT40 B cells were stimulated with mouse anti-chicken B-cell antigen
receptor (BCR), clone M-1 (Southern Biotechnology, Birmingham, AL). An
F(ab')2 fragment of the murine anti-human PECAM mAb,
PECAM-1.3, used for cross-linking experiments, was prepared
by using an Immunopure IgG1 F(ab')2 Preparation Kit
purchased from Pierce Biotechnology (Rockford, IL). Azide-free
F(ab')2 goat antimouse (GAM) immunoglobulin G (IgG) was
purchased from Accurate Chemical and Scientific (Westbury, NY) or ICN
(Aurora, OH). Fluorescein isothiocyanate (FITC)-conjugated GAM used in
the flow cytometric analyses was purchased from Pharmingen.
PTK expression in T cells was evaluated by Western analysis, using mouse monoclonal antibodies (mAbs) specific for c-Src and p56lck from Santa Cruz Biotechnology (Santa Cruz, CA) and p59fyn from Transduction Laboratories (San Diego, CA). SHP-2 was detected by using a rabbit polyclonal SHP-2-specific Ab purchased from Santa Cruz Biotechnology. Horseradish peroxidase (HRP)-conjugated GAM and goat antirabbit (GAR) IgG (Jackson Immunoresearch, West Grove, PA) were used as secondary reagents for detection of bound mouse mAb and rabbit polyclonal Ab, respectively. Methods for induction and assessment of PECAM-1 tyrosine phosphorylation have been previously described.7 Calcium mobilization assays All procedures for measurement of intracellular calcium concentrations by fluorescence spectrofluorometry were performed in the dark. DT40 cells (5 × 106/mL) were incubated for 45 minutes at 37°C with 3 µM Fura-2AM (Molecular Probes, Eugene, OR) in calcium mobilization assay (CMA) buffer (PBS containing 20 mM HEPES [pH 7.2], 5 mM glucose, 0.025% bovine serum albumin, and 1 mM CaCl2). Cells were then adjusted to a final concentration of 1 × 106 cells/mL with the addition of CMA buffer and were incubated at room temperature for 10 minutes in the presence of Abs at the indicated final concentrations. Preliminary dose response studies were performed to identify a dose of mAb directed against the chicken BCR (100 ng/mL) that induced a measurable but suboptimal increase in the intracellular calcium concentration only when cross-linked with F(ab')2 fragments of GAM as measured by a change in the fluorescent properties of Fura-2AM (data not shown). To remove unbound Abs, cells were washed 3 times at room temperature with 4 mL of CMA buffer. Cell suspensions (2 mL) were transferred to a cuvette equipped with a magnetic stir bar and placed in a sample chamber, maintained at 37°C, of an SLM 8100 spectrofluorometer (SLM-Aminco, Urbana, IL). To cross-link surface-bound Abs, GAM F(ab')2 was added at a final concentration of 50 µg/mL, which was determined in preliminary experiments to be super-optimal. Intracellular calcium concentration was assessed by measuring the intensity of light emitted at 510 nm on excitation at 340 and 380 nm every 3 seconds for 1 to 2 minutes. Results are presented as the ratio of the intensity of 510 nm light on excitation at 340, relative to 380, nm as a function of time.
Previous studies have shown that PECAM-1 becomes
tyrosine phosphorylated, binds SHP-2, and attenuates release of calcium
from intracellular stores on its co-ligation with the TCR. The goal of
the present investigation was to characterize the molecular requirements for the observed inhibitory activity of PECAM-1. We first sought to determine whether the inhibitory activity of PECAM-1 depends on tyrosine residues found within its
cytoplasmic ITIMs. Studies in other cell systems have shown that
PECAM-1 becomes tyrosine phosphorylated on residues 663 and
686,21 and sequences around these tyrosine residues
conform to an ITIM (reviewed in3). To test the importance
of these 2 tyrosines for the inhibitory function of PECAM-1,
we made use of an ITIM-less mutant construct in which the tyrosine
residues at positions 663 and 686 of the PECAM-1 cytoplasmic
domain were substituted with phenylalanine residues.21
Because the Jurkat T-cell line that we have used in previous studies
constitutively expresses PECAM-1, we expressed PECAM-1 constructs in chicken DT40 B cells,22
which are PECAM-1 negative (data not shown). As shown in
Figure 1, wild-type and ITIM-less forms
of PECAM-1 were expressed at similar levels on the surface of
transfected DT40 cells. To determine whether the tyrosine residues
within the PECAM-1 ITIMs were required to modulate antigen receptor-initiated signal transduction, we co-ligated wild-type
or ITIM-less PECAM-1 with the BCR and measured the degree of
calcium mobilization in Fura-2AM-loaded DT40 transfectants. As shown
in Figure 2, cross-linking the BCR alone
induced a rapid rise in intracellular calcium in B cells expressing
either wild-type (Figure 2A) or ITIM-less (Figure 2B) forms of
PECAM-1. Similar to what has been observed for the T-cell
antigen receptor,7 co-ligation of wild-type
PECAM-1 with the BCR resulted in dose-dependent attenuation
of calcium mobilization (Figure 2A), whereas ITIM-less PECAM-1 was unable to attenuate calcium mobilization at any
concentration of anti-PECAM-1 Ab tested (Figure 2B). From
these results, we conclude that the ability of PECAM-1 to
interfere with calcium mobilization induced by antigen receptor
cross-linking is dependent on the presence of intact
ITIMs.
Next, we sought to determine whether SHP-2 is required for the
inhibitory activity of PECAM-1. Although SHP-2 is the
major phosphatase associated with tyrosine-phosphorylated
PECAM-1,4-7 Hua et al11 have reported
that phosphopeptides containing the PECAM-1 ITIM tyrosine
residues or intact tyrosine-phosphorylated PECAM-1 can
precipitate both SHP-1 and SHP-2. In addition, Pumphrey et
al12 have reported that GST-fusion proteins containing the SH2 domains of SHP-1, SHP-2, or SHIP can interact with
tyrosine-phosphorylated PECAM-1. To determine whether
phosphatases other than SHP-2 can form a functional complex with
PECAM-1 capable of mediating its inhibitory activity, we made
use of an SHP-2-deficient variant of DT40 cells.23 As
shown in Figure 3A, wild-type DT40 cells express both SHP-2 and SHP-1, whereas SHP-2-negative DT40 cells contain normal levels of SHP-1 but no SHP-2. Transfection of wild-type PECAM-1 resulted in similar cell surface expression in both
wild-type and SHP-2-negative DT40 cells (Figure 3B), although the
range of PECAM-1 expression in wild-type DT40 cells (left
panel) was broader than that observed in SHP-2-negative cells (right
panel). As shown in Figure 4A,
co-ligation of PECAM-1 with the BCR in SHP-2-positive,
wild-type DT40 transfectants resulted in a dose-dependent attenuation
of calcium mobilization. In contrast, PECAM-1 was unable to
attenuate calcium mobilization at any concentration of
anti-PECAM-1 Ab tested in SHP-2-deficient DT40 cells (Figure 4B), despite abundant levels of the related PTP, SHP-1 (Figure 3A).
Failure of PECAM-1 to inhibit BCR-induced calcium
mobilization in SHP-2-deficient DT40 cells is unlikely to be
attributable to minor differences in PECAM-1 expression
between these 2 cell lines, as wild-type DT40 cells expressing much
lower levels of PECAM-1 (Figure 1A) inhibit calcium
mobilization to a similar extent as do wild-type DT40 cells expressing
higher levels of PECAM-1 (compare Figure 2A with Figure 4A).
From these results, we conclude that the ability of PECAM-1
to modulate antigen receptor signaling is dependent on the presence of
SHP-2.
Last, we sought to determine the requirement for individual members of
the Src family of PTKs for PECAM-1 tyrosine phosphorylation in T cells. The major Src family PTK activated on ligation of the TCR
in mature T cells is p56lck.1 Thus, we asked
whether p56lck is required for PECAM-1 tyrosine
phosphorylation in T cells. To address this question, we made use of a
p56lck-deficient variant Jurkat T-cell line, JCaM1.6.
JCaM1.6 cells, as expected, expressed no p56lck, but they
did express normal levels of pp60Src and p59fyn
(Figure 5A) and had normal levels of
PECAM-1 at the cell surface (Figure 5B). As previously
observed,7 PECAM-1 became tyrosine phosphorylated in wild-type Jurkat cells stimulated by cross-linking of
either the TCR alone, PECAM-1 alone, or by co-cross-linking of PECAM-1 and the TCR (Figure
6, top). In contrast, PECAM-1
failed to become tyrosine phosphorylated under any of these conditions in p56lck-deficient JCaM1.6 cells (Figure 6, middle).
Finally, PECAM-1 tyrosine phosphorylation was restored in
JCaM1.6 cells that were stably transfected with a
p56lck-encoding plasmid and in which p56lck
expression was reconstituted (Figure 6, bottom). From these results, we
conclude that p56lck is required for PECAM-1
tyrosine phosphorylation in response to cross-linking of either the TCR
or PECAM-1, and that, under these conditions, abundant levels
of c-Src and p59fyn are unable to functionally compensate
for the lack of p56lck.
PECAM-1 was originally assigned to the family of immunoglobulin-like cellular adhesion molecules (CAMs) based on sequence similarity between its 6 extracellular immunoglobulin domains and those of other immunoglobulin domain-containing CAMs.24 However, more recent studies have revealed features of PECAM-1 that implicate it as a member of the immunoglobulin-like, ITIM-containing inhibitory receptor family (reviewed in3). Thus, PECAM-1 possesses ITIMs in its cytoplasmic domain,24 the PECAM-1 ITIMs, on tyrosine phosphorylation support binding of the cytoplasmic protein tyrosine phosphatase, SHP-2,4 and perhaps SHP-111 and SHIP,12 and tyrosine-phosphorylated PECAM-1 to which SHP-2 is bound attenuates calcium release from intracellular stores on co-ligation with the TCR.7 These previous observations have provided circumstantial evidence in favor of inclusion of PECAM-1 within the family of immunoglobulin-like, ITIM-containing inhibitory receptors. The present studies provide firmer evidence for this relationship by demonstrating that the PECAM-1 ITIMs and SHP-2 are required for its inhibitory function. In addition, we clarify the role of the Src family PTKs by showing that, in T lymphocytes, PECAM-1 tyrosine phosphorylation depends on the presence of p56lck. In the present study, we expand on previous observations that the
PECAM-1 ITIMs become phosphorylated on tyrosine residues and bind SHP-2 by verifying that the presence of the PECAM-1
ITIMs is required for its inhibitory activity. Studies of another
important regulator of T-cell activity, CTLA-4, have taught us that
this is an important molecular connection to establish. The
observations that (1) treatment of T cells with soluble anti-CTLA-4 Abs
results in sequestration of CTLA-4 away from the TCR and augmentation of T-cell responses,25,26 (2) T-cell responses are
inhibited on co-ligation of CTLA-4 with the TCR,25 and (3)
CTLA-4 deficiency results in fatal lymphoproliferative
disease27 support the role of CTLA-4 as an inhibitor of
T-cell activation. The findings that CTLA-4 possesses cytoplasmic
ITIMs that bind SHP-2 in a tyrosine phosphorylation-dependent
manner suggested that the CTLA-4 ITIMs might be required for its
inhibitory activity.28 However, studies have revealed that
mutant forms of CTLA-4, in which the ITIM tyrosine residues are
substituted with phenylalanine, retain full ability to bind
SHP-229 and interfere with T-cell
responses,8,9,30 suggesting that the inhibitory activity
of CTLA-4 has more to do with its ability to compete with CD28 for
binding to B/7.1 and B/7.2 than with its ability to become tyrosine
phosphorylated and recruit a cytoplasmic PTP. Whereas PECAM-1
is unlike CTLA-4 in its dependence on ITIM phosphorylation and PTP
binding for inhibitory activity, it is similar to other members of the
immunoglobulin-like, ITIM-containing inhibitory receptor family, such
as Fc Our finding that PECAM-1 does not possess inhibitory activity in the absence of SHP-2 suggests that the reported interactions between PECAM-1 and either SHP-16,10,11 or SHIP12 are insufficient to support inhibitory function. The inability of PECAM-1/SHP-1 complexes to inhibit cellular responses may be attributable to the relatively low affinity of the PECAM-1/SHP-1 interaction. In support of this possibility, Sagawa et al6 found that SHP-1 precipitated less tyrosine-phosphorylated PECAM-1 than did SHP-2. In addition, Hua et al11 found that SHP-1 binds tyrosine-phosphorylated PECAM-1 with about 5-fold lower affinity than does SHP-2. Alternatively, a possible explanation for the inability of PECAM-1 to inhibit cellular responses in the presence of ample amounts of SHP-1 but no SHP-2 may be that targets of the antigen receptor signal transduction pathway that are dephosphorylated by PECAM-1/SHP-2 may not be dephosphorylated by PECAM-1/SHP-1 complexes. In support of this possibility, catalytic domain swapping studies have shown that the catalytic domains of SHP-1 and SHP-2 have different substrate specificities.34,35 Experiments are currently under way to identify the components of the TCR signal transduction pathway whose level of tyrosine phosphorylation is reduced on co-ligation of PECAM-1/SHP-2 complexes with the TCR relative to that observed on cross-linking of the TCR alone. Once the targets for dephosphorylation by PECAM-1/SHP-2 are identified, it should be possible to determine whether they are also substrates for dephosphorylation by SHP-1 bound to PECAM-1. We found that, in the absence of p56lck, PECAM-1
fails to become tyrosine phosphorylated on cross-linking of the TCR
and/or PECAM-1 in T cells and that, on reconstitution of
p56lck expression, PECAM-1 tyrosine
phosphorylation in response to these stimuli is restored. This result
is consistent with previous studies that demonstrate the ability of Src
family PTKs to phosphorylate PECAM-1 in in vitro kinase
assays,5,15 on overexpression in bovine aortic endothelial
cells,14 or on transient transfection in COS-1
cells10 and also with a study showing that
PECAM-1 tyrosine phosphorylation was reduced or abolished by
the Src family PTK inhibitor, PP2.16 One possible
explanation for this finding is that PECAM-1 is a direct
substrate of a Src family PTK but that, of the many Src family PTKs
shown to be capable of mediating PECAM-1 tyrosine
phosphorylation, only p56lck has the appropriate target
specificity under these conditions in T cells. Alternatively, it may be
that other members of the Src family of PTKs could phosphorylate
PECAM-1 in T cells but fail to do so because they do not
become activated on TCR or PECAM-1 cross-linking. This
conclusion is consistent with the finding that p56lck is
the major Src family PTK activated in mature T cells on ligation of the
TCR (reviewed in1). However, the extent to which
PECAM-1 cross-linking induces activation of each of the Src
family kinases is currently unknown. Our results do not, however,
exclude the possibility that PECAM-1 is phosphorylated by a
non-Src family kinase whose activation is dependent on the activity of
p56lck. In T cells, the major Syk family PTK downstream of
p56lck in TCR signaling is the In summary, we have found that the ability of PECAM-1 to function as an inhibitory receptor in T lymphocytes is dependent on the presence of intact ITIMs within its cytoplasmic domain, requires the recruitment and activation of SHP-2, and, in T cells, involves the activity of the Src family PTK, p56lck. Further definition of the signaling components affected by the PECAM-1/SHP-2 signaling complex is expected to provide important insights into the way this member of the immunoglobulin-ITIM family functions in blood and vascular cells.
We are grateful to Dr David Straus (University of Chicago) for generously providing p56lck-reconstituted JCaM1.6 cells, to Sara Hoffman for generating the data derived from this cell line and shown in Figure 6, and to Matthew Armstrong for technical assistance provided in the early phases of these studies.
Submitted June 28, 2000; accepted December 4, 2000.
Supported by grant P01 HL44612 from the National Institutes of Health and by the Blood Center Research Foundation.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Debra K. Newman, Blood Research Institute, The Blood Center of Southeastern Wisconsin, 638 N 18th St, Milwaukee, WI 53233; e-mail: dknewman{at}bcsew.edu.
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S. Yamasaki, K. Nishida, M. Hibi, M. Sakuma, R. Shiina, A. Takeuchi, H. Ohnishi, T. Hirano, and T. Saito Docking Protein Gab2 Is Phosphorylated by ZAP-70 and Negatively Regulates T Cell Receptor Signaling by Recruitment of Inhibitory Molecules J. Biol. Chem., November 21, 2001; 276(48): 45175 - 45183. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
similar to
previous observations in T cells. In contrast, co-ligation of an
ITIM-less form of PECAM-1 had no inhibitory effect.
Furthermore, wild-type PECAM-1 was unable to attenuate
calcium mobilization in SHP-2-deficient DT40 variants despite abundant
levels of SHP-1 in these cells. Finally, PECAM-1 failed to
become tyrosine phosphorylated in p56lck-deficient Jurkat T
cells. Together, these data provide important insights into the
molecular requirements for PECAM-1 regulation of antigen
receptor signaling.
(Blood. 2001;97:2351-2357)
2
and a leucine or valine residue at the +3 position (reviewed in
PECAM-1). Antibody-pretreated cells
were then stained with FITC-conjugated GAM IgG and analyzed by flow
cytometry. Transfectants chosen for analysis expressed equivalent
levels of cell surface PECAM-1.
RIIB,
-associated protein of 70 kd (ZAP-70).