Association of p59fyn with the T Lymphocyte
Costimulatory Receptor CD2
BINDING OF THE Fyn Src HOMOLOGY (SH) 3 DOMAIN IS REGULATED BY
THE Fyn SH2 DOMAIN*
Huamao
Lin
§,
Jill E.
Hutchcroft
,
Christopher E.
Andoniou¶,
Malek
Kamoun
,
Hamid
Band¶, and
Barbara E.
Bierer
**
From the
Department of Pediatric Oncology,
Dana-Farber Cancer Institute, § Committee on Immunology,
Division of Medical Sciences, and ¶ Lymphocyte Biology Section,
Department of Rheumatology and Immunology, Brigham and Woman's
Hospital, Boston, Massachusetts 02115, the
Department of
Pathology and Laboratory Medicine, University of Pennsylvania School of
Medicine, Philadelphia, Pennsylvania 19104, and the ** Department of
Medicine, Harvard Medical School, Boston, Massachusetts 02115
 |
ABSTRACT |
Human CD2 is a 50-55-kDa cell surface receptor
specifically expressed on the surface of T lymphocytes and NK cells.
Stimulation of human peripheral blood T cells with mitogenic pairs of
anti-CD2 monoclonal antibodies (mAbs) is sufficient to induce
interleukin-2 production and T cell proliferation in the absence of an
antigen-specific signal through the T cell receptor. CD2 has been shown
previously to associate physically with the Src family protein-tyrosine
kinases p56lck and p59fyn. We now report that
stimulation of T cells with mitogenic pairs of anti-CD2 mAbs enhanced
the association of the Fyn polypeptide with the CD2 complex, whereas
stimulation with single anti-CD2 mAb had minimal effect. Using
glutathione S-transferase (GST) fusion proteins, we found
that CD2 bound to the Src homology (SH) 3 domain of Fyn. Interestingly,
the CD2-Fyn association was negatively regulated by the Fyn SH2 domain;
CD2 bound poorly to GST fusion proteins expressing both the SH2 and SH3
domains of Fyn. However, the inhibitory effect of the Fyn SH2 domain on
binding of the Fyn SH3 domain to CD2 was relieved by peptides
containing a phosphorylated YEEI sequence that bound directly to the
Fyn SH2 domain. In addition, we found that the ability of the Fyn SH2
domain to precipitate tyrosine-phosphorylated proteins, including the
CD3
chain, was enhanced after T cell stimulation with mitogenic
pairs of CD2 mAbs. Finally, overexpression of a mutated Fyn
molecule, in which the ability of the Fyn SH2 domain to bind
phosphotyrosine-containing proteins was abrogated, inhibited
CD2-induced transcriptional activation of the nuclear factor of
activated T cells (NFAT), suggesting a functional involvement of the
Fyn SH2 domain in CD2-induced T cell signaling. We thus propose that
stimulation through the CD2 receptor leads to the tyrosine
phosphorylation of intracellular proteins, including CD3
itself,
which in turn bind to the Fyn-SH2 domain, allowing the direct
association of the Fyn SH3 domain with CD2 and the initiation of
downstream signaling events.
 |
INTRODUCTION |
The activation of T lymphocytes is initiated by engagement of the
T cell receptor (TCR)1 by
peptide embedded in MHC molecules expressed on the surface of
antigen-presenting cells (APCs) (1, 2). In addition to the TCR-CD3
complex, T cells also express cell surface coreceptors such as CD4,
CD8, CD28, LFA-1, and CD2 that bind to their cognate ligands on APCs.
The binding of the co-receptors with their ligands appears to modulate
the avidity of T cell/APC interactions. Moreover, coreceptor ligation
may also initiate intracellular signaling pathways that regulate T cell
activation (3-6).
CD2 is a 50-55-kDa glycoprotein expressed on a majority of thymocytes,
T cells, and NK cells. A number of ligands of human CD2 have now been
identified including CD58, CD48, CD59, and a novel carbohydrate
structure associated with CD15 (7-12). CD2 engagement has been shown
either to synergize with or to inhibit antigen-induced T cell responses
depending, in part, upon the specific experimental system (13-16).
Unlike many other coreceptors, stimulation of T cells with certain
pairs of anti-CD2 mAbs can lead to IL-2 production and T cell
proliferation in the absence of direct ligation of the TCR-CD3 complex
(17). However, it appears that optimal CD2-induced T cell activation
requires the expression of components of the CD3 complex such as
CD3
, even though direct engagement of the TCR-CD3 complex is not
necessary (18). Recent reports have shown that CD2 ligation is capable of reversing T cell anergy (19) and regulating T cell responsiveness to
IL-12 (20), further implicating CD2 as a critical regulator of the
immune response.
The molecular determinants of CD2-mediated T cell activation are
currently under investigation. A number of proteins, including CD3
and CD3
chains, phosphoprotein p29/30, Lck, Fyn, CD4, CD5, CD8, p85
subunit of phosphatidylinositol 3-kinase, CD45, and
and
tubulins have been shown to co-immunoprecipitate with CD2 (21-28). The
functional outcome and biological significance of these protein
associations remain to be determined. The 116-amino acid cytoplasmic
tail of CD2 contains no tyrosine that upon phosphorylation could
mediate interaction with SH2 domain-containing signaling elements.
Mutational and deletional analyses of human CD2 have shown that
repeated proline-rich domains, potentially able to mediate binding to
SH3-containing proteins, are important for CD2 signaling (29-31). In
this report, we have examined the association of CD2 with Fyn, a Src
family protein-tyrosine kinase (PTK). We find that stimulation with
mitogenic anti-CD2 mAbs increased the association of Fyn with CD2 and
that it was the SH3 domain of Fyn that bound to the CD2 cytoplasmic
domain. The Fyn SH2 domain was unable to bind to CD2 directly;
nevertheless, it inhibited the binding of the Fyn SH3 domain to CD2.
The inhibitory effect of the Fyn SH2 domain on Fyn SH3 binding to CD2
was able to be reversed by specific phosphotyrosine-containing
peptides. In addition, the binding of the Fyn SH2 domain to
tyrosine-phosphorylated proteins, including CD3
chains, was enhanced
after stimulation with mitogenic combinations of anti-CD2 mAbs.
Finally, we demonstrated that overexpression of a Fyn loss-of-function
SH2 domain mutant, but not wild-type Fyn or a Fyn loss-of-function SH3
domain mutant, inhibited CD2-induced NFAT transcriptional activity.
Taken together, our data suggest that the SH2 domain of Fyn regulates
the CD2-Fyn association in an activation-dependent manner;
multimolecular complexes of CD2-CD3
-Fyn may thus quantitatively
regulate the amount of Fyn polypeptide in the CD2 complex.
 |
EXPERIMENTAL PROCEDURES |
Cell Culture--
The T cell leukemia cell line Jurkat clone J77
was a gift of K. Smith (Cornell University, New York, NY). Jurkat cells
transformed with SV40 large tumor (T) antigen were also used in
transient transfection experiments. Lymphocytes were grown in RPMI 1640 (Mediatech, Herndon, VA) supplemented with 10% heat-inactivated fetal
bovine serum (Sigma), 100 units/ml penicillin (Life Technologies, Inc.), 100 µg/ml streptomycin (Life Technologies, Inc.), 2 mM glutamine (Life Technologies, Inc.), 10 mM
HEPES, pH 7.3, and 50 µM 2-mercaptoethanol (Sigma),
termed 10% fetal calf serum medium. Resting human peripheral blood T
cells were isolated from normal volunteers by centrifugation through
Ficoll (Organon Teknika, Durham, NC), plastic adherence, and nylon wool
filtration. Contaminating red cells were lysed with Tris-buffered
ammonium chloride. The purified human T cells were cultured in 10%
fetal calf serum medium and used within 24 h of isolation.
Antibodies and Peptides--
The anti-CD2 mAbs T112
and T113 (the kind gift of E. Reinherz, Dana Farber Cancer
Institute, Boston, MA), anti-CD2 mAb 9.1 (the kind gift of B. Dubont,
Memorial Sloan-Kettering Cancer Institute, New York, NY), the
anti-CD3
mAb 6B10.2 (Santa Cruz Biotechnology, Santa Cruz, CA), the
anti-phosphotyrosine mAb 4G10 (the kind gift of T. Roberts, Dana Farber
Cancer Institute), the anti-CD3
mAb OKT3 (ATTC, Rockville, MD), the
anti-CD43 antibody L10 (32), the anti-MHC class I mAb W6/32 (ATTC), the
rabbit anti-mouse IgG antisera (Southern Biotechnology Associates,
Birmingham, AL), the polyclonal anti-GST antibody (Santa Cruz
Biotechnology), the polyclonal anti-Lck antisera (Upstate
Biotechnology, Lake Placid, NY), and the polyclonal anti-Fyn antibody
(Santa Cruz Biotechnology) were used as indicated. The mAb 3G12, raised
against the -ATSQHPPPPPGHRSQ- sequence derived from the human CD2
cytoplasmic tail, was generated in the laboratory of Dr. M Kamoun
(University of Pennsylvania School of Medicine, Philadelphia, PA). The
phosphotyrosyl peptide EPQpYEEIPIYL (here termed -pYEEI-) and its
unphosphorylated analog EPQYEEIPIYL (here termed -YEEI-) were
synthesized and purified as described (33).
cDNA Constructs and Fusion Proteins--
The wild-type Fyn
construct, a loss-of-function mutated Fyn construct in which the Arg
was replaced with Lys at amino acid 176 in the Fyn SH2 domain (denoted
Fyn SH2*), and a loss-of-function mutated Fyn construct in which the
Pro was replaced with Val in at amino acid 134 in the Fyn SH3 domain
(denoted Fyn SH3*) were the kind gift of R. Perlmutter (Merck-Research
Institute, Rahway, NJ). Wild-type or mutated Fyn constructs were
subcloned into pALTER vector (Promega, WI) and used for transfection
into Jurkat cells. The GST fusion constructs containing different
domains of Fyn have been described previously (33). Escherichia
coli DH5
cells were transformed with PGEX2T.K containing the
Fyn SH2 domain (termed GST-FynSH2), the Fyn SH3 domain (termed
GST-FynSH3), the Fyn SH3 domain mutant (termed GST-FynSH3W119K) in
which a tryptophan (W) at amino acid 119 was replaced by a lysine (K),
and the Fyn SH3-SH2 domains (termed GST-FynSH3SH2).
Transformed E. coli were induced with 0.4 mM of
isopropyl-1-thio-
-D-galactopyranoside (Sigma). The cells
were resuspended in Tris-buffered saline (150 mM NaCl, 10 mM Tris, pH 8.0) containing 1% Triton X-100 and lysed by
sonication. The GST fusion proteins were affinity-purified using
glutathione-Sepharose beads (Amersham Pharmacia Biotech). Immobilized
proteins were aliquoted and stored as a 50% slurry (v/v) in
Tris-buffered saline with 0.1% Triton X-100 at
80 °C. In some
experiments, fusion proteins were also eluted from the beads according
to the manufacturer's instruction (Amersham Pharmacia Biotech).
Cell Stimulation, Immunoprecipitations, and in Vitro Kinase
Assays--
Jurkat cells or human purified T cells (1-2 × 107 as indicated) were resuspended in 0.5 ml of buffer A
(RPMI 1640 supplemented with 100 units/ml penicillin, 100 µg/ml
streptomycin, 2 mM glutamine, 10 mM HEPES, pH
7.3), incubated with 0.5 µl of T112 and 0.5 µl of
T113 ascites on ice for 15 min, and then transferred to
37 °C for 5 min, unless otherwise indicated. The cells were lysed by addition of 0.5 ml of cold lysis buffer B (2% Brij 97, 150 mM NaCl, 25 mM Tris, pH 7.5, 2 mM
EDTA, 2 mM Na3VO4, 20 µg/ml
leupeptin, 20 µg/ml aprotinin, and 2 mM
phenylmethylsulfonyl fluoride) on ice for 20 min. Lysates were
clarified by centrifugation at 15,000 × g for 10 min
at 4 °C. Equivalent amounts of mAbs were added to unstimulated cells
after lysis to normalize the final antibody concentrations.
In CD2 immunoprecipitations, 20 µl of 1:1 ratio of protein A:protein
G slurry and 0.5 µl of T112 and 0.5 µl of
T113 ascites were added together to the clarified cell
lysates and incubated at 4 °C for 2 h. The beads were then
washed three times with washing buffer C (0.1% Brij 97, 150 mM NaCl, 25 mM Tris, pH 7.5, 1 mM Na3VO4). In the in vitro kinase
reaction, the washed beads were incubated in 50 µl of kinase buffer D
(10 mM MnCl2, 5 mM
p-nitrophenyl phosphate, 25 mM Hepes, pH 7.5, and 10 µCi of [
-32P]ATP) for 15 min at room
temperature. Kinase reactions were stopped by addition of 2× SDS
sample buffer. Phosphoproteins were separated by SDS-polyacrylamide gel
electrophoresis (SDS-PAGE), transferred to polyvinylidene difluoride
(PVDF), and visualized by autoradiography. In reimmunoprecipitation
experiments, co-immunoprecipitated proteins were dissociated by boiling
in 50 µl of 2% SDS for 10 min, after which the beads were separated
and removed by filtration through microspin tubes (Costar, MA).
Dissociated proteins were renatured by the addition of 900 µl of
Tris-buffered saline with 1% Nonidet P-40, reimmunoprecipitated with
the indicated antibodies, separated by SDS-PAGE, transferred to PVDF
membranes, and then visualized by autoradiography or immunoblotted as
indicated.
In the experiments using immobilized GST fusion proteins, fusion
proteins previously bound to glutathione-Sepharose beads were added to
the clarified cell lysates and incubated at 4 °C for 2 h. The
beads were then washed three times with washing buffer C and subjected
to deglycosylation and Western blot analysis, as detailed below. In the
peptide binding experiments, both GST fusion proteins and cell lysates
were incubated separately with 50 µM -YEEI- or -pYEEI-
peptides for 30 min at 4 °C, prior to GST precipitation.
Protein Deglycosylation and Western Blotting--
CD2
immunoprecipitates or GST fusion protein precipitates were boiled in 30 µl of denaturing buffer E (100 mM Tris, pH 8.0, 10 mM EDTA, 0.5% SDS, 1%
-mercaptoethanol), renatured in
1% Nonidet P-40 and 50 µM Na3PO4
and then incubated with 500 units of PNGase F (New England Biolabs) at
37 °C for 2 h. Deglycosylated proteins were boiled in SDS
sample buffer, separated by SDS-PAGE, transferred to PVDF, and probed
with primary antibodies followed by horseradish peroxidase-conjugated
secondary antibodies. Polypeptides recognized in the Western blot were
detected using the ECL method according to manufacturer's instructions
(Amersham Pharmacia Biotech).
Transient Transfection and Luciferase Assays--
SV40 large
tumor (T) antigen-transformed Jurkat cells (1 × 107)
were incubated with 20 µg of vector, wild-type or mutated Fyn constructs as indicated, with 5 µg of a reporter plasmid p3xNFAT-luc (34), carrying the luciferase gene driven by three tandem repeats of
the distal NFAT sequences derived from the IL-2 promoter, for 15 min at
room temperature. Cells were then electroporated at 250 V, 800 microfarads (Life Technologies, Inc.). After electroporation, the cells
were transferred to 10% fetal calf serum medium and incubated at
37 °C for 12 h. Transfected cells were stimulated for 6 h
with 1:1,000 dilution of T112 and T113, a
mitogenic pair of anti-CD2 mAbs, or with PMA (10 ng/ml) plus ionomycin
(2 µM). Cells were washed with PBS, and samples were
prepared using the Enhanced Luciferase Kit (Analytic Luminescent
Laboratory, San Diego, CA), according to the manufacturer's
instructions. The relative luciferase units are presented as the
percentage of the maximal stimulation for each transfection condition
induced by PMA plus ionomycin.
 |
RESULTS |
Stimulation with Mitogenic Pairs of Anti-CD2 mAbs Increased the
Association of CD2 with Phosphorylated Lck and Fyn--
The Src family
tyrosine kinases Lck and Fyn have been shown to associate with CD2
(21), yet how these key proteins are regulated in
CD2-dependent signaling events remains to be established.
We examined whether extracellular ligation of CD2 altered the in vitro kinase activity of CD2-associated intracellular signaling proteins including Lck and Fyn. Different cell surface receptor complexes were immunoprecipitated from Jurkat T cells. The immune complexes were then incubated in kinase buffer containing
[
-32P]ATP, and the resulting in vitro
phosphorylated proteins were separated by SDS-PAGE, transferred to PVDF
membrane, and visualized by autoradiography (Fig.
1). Several phosphorylated proteins were present in CD2 immunoprecipitates prepared from resting Jurkat cells
including prominent phosphoproteins in the 50-60-kDa size range (Fig.
1, lane 4). A number of phosphoproteins were also found in
CD3 immune complexes; no apparent kinase activity was detected in
association with MHC class I molecules or CD43 (Fig. 1, lanes
1 and 3). Stimulation with a mitogenic combination of CD2 mAbs (T112 + T113) increased the total
amount of CD2-associated kinase activity (Fig. 1, lane 5).
There was enhanced phosphorylation of a number of proteins, including
proteins migrating at 28, 40, 50-60, 120, and 150 kDa.

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Fig. 1.
T cell stimulation using mitogenic
combinations of anti-CD2 mAbs increased CD2-associated protein kinase
activities. Jurkat cells were either unstimulated ( ) or
incubated with T112 and T113, a mitogenic pair
of anti-CD2 mAbs, at 37 °C for 5 min (+) prior to lysis. The cells
were then lysed in Brij 97 lysis buffer, and the post-nuclear lysates
were precipitated with anti-MHC-I (lane 1), anti-CD3
(lane 2), anti-CD43 (lane 3), anti-CD2
(lanes 4 and 5) as indicated. The immune
complexes were incubated with kinase buffer and
[ -32P]ATP to phosphorylate associated proteins. The
in vitro phosphorylated proteins were separated by SDS-PAGE
and detected by autoradiography.
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When comparable studies were performed using purified human T cells,
the phosphoprotein profiles from resting and anti-CD2 stimulated cells
were somewhat different than those of Jurkat cells. Nevertheless, CD2
stimulation increased the phosphorylation of CD2-associated proteins in
the range of 50-60 kDa (Fig. 2, lanes 1 and 2). We performed
reimmunoprecipitation experiments to determine if these phosphoproteins
included Lck and Fyn, which migrate at a similar size range. Equivalent
amounts of CD2 protein were immunoprecipitated from human purified T
cells without or with pre-stimulation at 37 °C with the mitogenic
combination of anti-CD2 mAbs T112 + T113. After
in vitro phosphorylation (lanes 1 and
2), the CD2 complexes were dissociated by boiling and then subjected to reimmunoprecipitation using antisera directed against either Fyn or Lck. Ligation of CD2 led to a modest increase in the
amount of phosphorylated Lck and to a pronounced increase in the amount
of phosphorylated Fyn associated with CD2 (Fig. 2, lanes
5-8). We further characterized the latter interaction.

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Fig. 2.
Stimulation with mitogenic pairs of anti-CD2
mAb increased the association of CD2 with phosphorylated Lck and
Fyn. Purified human T cells either unstimulated (lanes
1, 3, 5, and 7) or stimulated
with the anti-CD2 mAbs T112 and T113 for 5 min
(lanes 2, 4, 6, and 8) were
lysed; CD2 was then precipitated from the cell lysates. The immune
complexes were phosphorylated in the presence of
[ -32P]ATP. Proteins associated with CD2 were either
separated by SDS-PAGE (lanes 1 and 2) or
dissociated from the immune complexes and reimmunoprecipitated with
either protein A alone (lane 3 and 4), or protein
A plus anti-Fyn (lanes 5 and 6) or anti-Lck
(lanes 7 and 8) antibodies. The in
vitro phosphorylated proteins were separated by SDS-PAGE and
detected by autoradiography.
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CD2-dependent Stimulation Quantitatively Increased the
Amount of Fyn Associated with CD2--
We determined whether the
increase in phosphorylated Fyn associated with CD2 was secondary to
quantitative changes in the amount of Fyn polypeptide recruited to the
CD2 complex. Human T cells were stimulated with mitogenic combination
of anti-CD2 mAbs for varying times at 37 °C, after which CD2 was
immunoprecipitated from cell lysates. The Fyn associated with CD2 was
detected by Western blot (Fig.
3A, upper panel).
Although minimal Fyn was associated with CD2 in the resting cells (Fig.
3A, lane 1), the amount of Fyn associated with
CD2 increased after stimulation of the cells with mitogenic pairs of
anti-CD2 mAbs (T112 + T113) in a
time-dependent fashion (Fig. 3A, lanes
2-5). The amount of Fyn associated with CD2 was maximal at about
5 min after stimulation (Fig. 3A, lane 4) and
decreased by about 15 min (Fig. 3A, lane 5).
Similar results were observed in experiments performed in Jurkat cells
(data not shown). In contrast to mitogenic pairs of anti-CD2 mAbs
stimulation, using either of the single anti-CD2 mAb T112
or T113 alone only marginally altered the amount of CD2-Fyn association (Fig. 3B).

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Fig. 3.
Mitogenic pairs of anti-CD2 mAbs stimulation
increased the amount of Fyn associated with CD2. A,
purified human T cells were stimulated with the anti-CD2 mAbs
T112 and T113 at 37 °C for the indicated
periods of time before lysis in Brij 97 lysis buffer. CD2 was then
precipitated from the cell lysates. The immune complexes were treated
with PNGase F before separation by SDS-PAGE. CD2 (lower
panel) and Fyn (upper panel) were visualized by Western
blot using the anti-CD2 mAb 3G12 and anti-Fyn antibodies respectively,
as described under "Experimental Procedures." B,
unstimulated T cells (lane 1) or cells stimulated with
anti-CD2 mAb T112 (lane 2), T113
(lane 3), or T112 and T113
(lane 4) were lysed and CD2 was precipitated from cell
lysates. The immune complexes were treated with PNGase F before
separation by SDS-PAGE. CD2 (lower panel) or Fyn
(upper panel) were visualized by Western blot as in
B. C, CD2 precipitated from T cell lysates were
either treated with PNGase F (lane 2) or untreated
(lane 1) before detection by Western blot using the anti-CD2
mAb 3G12. The proteins running at approximately 50 and 25 kDa are heavy and light chains of the precipitating
antibody, respectively. PNGase F-treated deglycosylated CD2 migrates at
approximately 40 kDa and is indicated by the
arrow.
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A trivial explanation for the above result could be that different
amounts of CD2 were precipitated from resting versus
stimulated cells, or at different time points, but that the
stoichiometry of the Fyn association to CD2 was unchanged. We therefore
developed a procedure to establish the amount of precipitated CD2. CD2
is a highly glycosylated protein that migrates as a diffuse band of
approximate 50-60 kDa in SDS-PAGE and is indistinguishable from Ig heavy chains following Western blot (Fig. 3C,
lane 1). However, after treatment of the precipitated
material with PNGase F to remove N-linked carbohydrates
residues, CD2 migrated as a distinct band of 40 kDa in
SDS-PAGE. The deglycosylated CD2 was then detected by the anti-CD2 mAb
3G12, which recognizes the cytoplasmic tail of CD2 (Fig. 3C,
lane 2). Quantitatively similar amounts of CD2 were
precipitated at different time points following stimulation (Fig. 3,
A and B, lower panel). Taken together,
these results suggest that stimulation with mitogenic pairs of anti-CD2
mAbs led to a time-dependent increase in the amount of Fyn
polypeptide associated with CD2.
The SH3 Domain, but Not the SH2 or the Amino-terminal Unique
Domain, of Fyn Was Able to Bind to CD2--
The Fyn molecule contains
an amino-terminal unique domain (termed NT), an SH2 domain, an SH3
domain, and a catalytic kinase domain. The NT, SH2. and SH3 domains of
Fyn mediate protein-protein interactions and it is believed that these
interactions regulate the subcellular localization and
substrate-binding characteristics of Fyn (35). GST fusion proteins
containing the NT (termed GST-FynNT), SH2 (termed GST-FynSH2) or SH3
(termed GST-FynSH3) domains of Fyn were expressed in bacteria and
purified on glutathione-Sepharose (33); their ability to bind to CD2
was tested (Fig. 4A). Neither GST-FynNT nor GST-FynSH2 was able to precipitate CD2 from either resting or CD2 stimulated cells (Fig. 4A, lanes
2-5). In contrast, GST-FynSH3 was able to precipitate CD2 from T
cell lysates in an activation-independent manner (Fig. 4A,
lanes 6 and 7). The binding of GST-FynSH3 to CD2
was demonstrated in unstimulated cells; binding was not increased after
stimulating with mitogenic pairs of anti-CD2 mAbs. Furthermore, the
specific binding of GST-FynSH3 and CD2 was abrogated by a point
mutation in the ligand-binding pocket of the SH3 domain in which a
tryptophan is replaced by a lysine at position 119 (GST-FynSH3W119K)
(Fig. 4B, lanes 4 and 5). These
results suggest that the binding of CD2 to Fyn was mediated through the
Fyn SH3 domain, and that binding required Fyn SH3 sequences essential
for binding to proline-rich sequence motifs (36, 37).

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Fig. 4.
The SH3 domain, but not the SH2, or the
amino-terminal domain, of Fyn was able to bind to CD2.
A, unstimulated Jurkat cells (lanes 2,
4, 6, and 8) or cells stimulated with
the anti-CD2 mAbs T112 and T113 (lanes
1, 3, 5, and 7) for 5 min were
lysed and then precipitated with glutathione bead-bound GST (lane
1), GST-FynNT (lanes 2 and 3), GST-FynSH2
(lanes 4 and 5) or GST-FynSH3 fusion proteins
(lanes 6 and 7). CD2 was precipitated directly
using mAb 9.1 and served as a positive control (lane 8). The
precipitates were treated with PNGase F and analyzed by Western blot
using the anti-CD2 mAb 3G12. B, unstimulated Jurkat T cells
(lanes 2 and 4) or cells stimulated
with anti-CD2 mAbs T112 and T113
(lanes 1, 3, and 5) were
lysed and precipitated with glutathione beads bound GST, GST-FynSH3, or
the Fyn SH3 domain mutant GST-FynSH3W119K, as described under
"Experimental Procedures." The precipitates were treated with
PNGase F, separated by SDS-PAGE, and analyzed by Western blot using the
anti-CD2 mAb 3G12.
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The SH2 Domain of Fyn Negatively Regulated the Binding of the SH3
Domain to CD2--
The association of CD2 with Fyn was increased in
cells stimulated in vivo with mitogenic combinations of
anti-CD2 mAbs (Fig. 3B). However, the association of the SH3
domain of Fyn with CD2 appeared unchanged after stimulation (Fig.
4A, lanes 6 and 7). In
vitro binding of Fyn SH2 domain to the Fyn SH3 domain has been demonstrated (33); it has been suggested that interaction of the SH2
and SH3 domains might interfere with binding to other ligands (33). We
therefore tested whether the binding of the Fyn SH3 domain to CD2 was
regulated by other domains of Fyn, and, specifically, whether the Fyn
SH2 domain inhibited the binding of the Fyn SH3 domain to CD2. GST
fusion proteins containing both the SH2 and SH3 domains of Fyn (termed
GST-FynSH3SH2) were used (Fig.
5A). The Fyn SH3SH2 domain
bound poorly to CD2 compared with the Fyn SH3 domain alone (Fig.
5A, lower panel, lane 2 and 3). In contrast, GST-FynSH3SH2 and GST-FynSH3 bound to the
phosphoprotein Cbl (Fig. 5A, upper panel,
lanes 2 and 3), demonstrating the ability of the
GST-FynSH3SH2 protein to interact with target proteins. These data
suggest that the presence of the Fyn SH2 domain was able to inhibit the
binding of the Fyn SH3 domain to CD2. In addition, the inhibitory
effect of the Fyn SH2 domain on Fyn SH3 was not universal because the
Fyn SH3SH2 domain was able to precipitate other polypeptides such as
Cbl.

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Fig. 5.
The SH2 domain of Fyn negatively regulated
the binding of the SH3 domain to CD2. A, Jurkat T cell
lysates were precipitated with glutathione bead-bound GST (lane
1), GST-FynSH3 (lane 2), or GST-FynSH3SH2 fusion
proteins (lane 3). The precipitates were treated with PNGase
F, separated by SDS-PAGE, and analyzed by Western blot using the
anti-CD2 mAb 3G12 (lower panel) or anti-Cbl antisera
(upper panel). B, Jurkat T cell lysates were
incubated with glutathione bead-bound GST (lane 1),
GST-FynSH3 (lane 2), or GST-FynSH3SH2 fusion proteins in the
absence (lane 3) or presence of -pYEEI- (lane 4)
or -YEEI- peptide (lane 5), as described under
"Experimental Procedures." Precipitated proteins were then treated
with PNGase F, separated by SDS-PAGE, and analyzed by Western blot
using anti-CD2 mAb 3G12. B, Jurkat cells were either
unstimulated ( ) or stimulated with a mitogenic pair of anti-CD2 mAbs
T112 and T113 (+) prior to lysis. Cell lysates
were incubated with eluted GST-FynSH3SH2 proteins before anti-CD2
immunoprecipitation was carried out. The precipitates were treated with
PNGase F, separated by SDS-PAGE, and analyzed by Western blot using the
anti-CD2 mAb 3G12 (lower panel) or anti-GST antibody
(upper panel).
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It has been suggested that the interaction of the Fyn SH2 and SH3
domains functionally interferes with binding to other ligands; an
11-mer phosphorylated peptide EPQpYEEIPIYL (termed -pYEEI-), a high
affinity ligand of Fyn SH2 domain, has been shown to be able to abolish
Fyn SH2-SH3 interactions (33). We therefore tested whether the same
peptide was able to reverse the inhibitory effect of the Fyn SH2 domain
on the binding of the Fyn SH3 domain to CD2 (Fig. 5B). As
expected, the GST-FynSH3SH2 bound to CD2 poorly (Fig. 5B,
lane 2); however, preincubation with the -pYEEI- peptide
restored the ability of the GST-FynSH3SH2 domain to bind to CD2 (Fig.
5B, lane 4). Phosphorylation of the tyrosine in
the peptide was required to reverse the inhibition, because the
unphosphorylated -YEEI- peptide had no effect (Fig. 5B,
lane 3). Coomassie Blue staining demonstrated that similar
amounts of the GST-FynSH3SH2 fusion proteins were used in all lanes
(data not shown).
Stimulation via CD2 Enhanced the Association of the FynSH3SH2
Domain to Tyrosyl-phosphorylated Proteins, One of Which was the CD3
chain--
We sought to demonstrate one practical example of a
phosphorylated polypeptide able to bind to the Fyn SH2 domain and
thereby potentially able to modify the ability of the Fyn SH3SH2 domain to bind to CD2. In unstimulated Jurkat cells, the GST-FynSH3SH2 domain
was able to bind to a number of tyrosyl-phosphorylated proteins as
detected by blotting with the anti-phosphotyrosine mAb 4G10 (Fig.
6A, lane 3);
stimulation of the cells with anti-CD2 mAbs quantitatively increased
the tyrosyl-phosphorylated proteins detected. GST alone was unable to
bind to any detectable phosphorylated proteins (Fig. 6A,
lanes 1 and 2). Again, Coomassie Blue staining of
the membrane demonstrated that comparable amounts of GST fusion proteins were used in the different lanes (data not shown). A tyrosyl-phosphorylated protein of approximately 20 kDa was able to bind
to the SH2 domain but not to the SH3 domain of Fyn (Fig. 6B,
lanes 3-6); the molecular weight approximated that of
CD3
. Because CD3
chains have been shown to associate with CD2
(24), we tested whether the 20-kDa protein that bound to Fyn SH3SH2 was
CD3
. Proteins bound to the FynSH3SH2 domain were dissociated from
the complex by boiling in SDS buffer and subjected to
re-immunoprecipitation using anti-CD3
antibodies (Fig.
6C, lanes 1 and 2) or control (Fig.
6C, lanes 3 and 4). CD3
was present
in the Fyn SH3SH2 complex in both unstimulated and stimulated cells.
However, the amount of CD3
that bound to FynSH3SH2 was significantly
increased upon anti-CD2 stimulation (Fig. 6C, lanes
1 and 2).

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Fig. 6.
FynSH3SH2 bound to increased amount of
tyrosyl-phosphorylated proteins after stimulation, one of which was
CD3 chain. A, unstimulated Jurkat cells
(lanes 1 and 3) or cells stimulated
with anti-CD2 mAbs T112 and T113 (lanes
2 and 4) for 5 min were lysed and then precipitated
with glutathione bead-bound GST (lanes 1 and 2),
or GST-FynSH3SH2 fusion proteins (lane 3 and 4).
The associated proteins were separated by SDS-PAGE and analyzed by
Western blot using the anti-phosphotyrosine mAb 4G10. B,
unstimulated Jurkat cells (lanes 1, 3,
5, 7, and 9) or cells stimulated with
anti-CD2 mAbs T112 and T113 for 5 min
(lanes 2, 4, 6, 8, and
10) were lysed and then precipitated with glutathione
bead-bound GST (lanes 1 and 2), GST-FynSH2
(lanes 3 and 4), GST-FynSH3 (lanes 5 and 6), or
GST-FynSH3SH2 fusion proteins (lanes 7 and 8).
CD3 were immunoprecipitated using anti-CD3 mAb and served as a
positive control (lanes 9 and 10). The
precipitates were separated by SDS-PAGE and analyzed with Western blot
using anti-phosphotyrosine mAb 4G10. C, unstimulated Jurkat
cells (lanes 1 and 3) or cells stimulated with
anti-CD2 mAb T112 and T113 (lanes 2 and 4) were lysed and then precipitated with glutathione
bead-bound GST-FynSH3SH2 fusion proteins as described in A.
After precipitation, the beads were washed and boiled in 2% SDS
buffer. Dissociated proteins were re-immunoprecipitated with protein A
plus anti-CD3 antibody (lanes 1 and 2) or
protein A alone (lanes 3 and 4).
Re-immunoprecipitated proteins were separated by SDS-PAGE and analyzed
by Western blot using mAb 4G10 to detect tyrosyl-phosphorylated
proteins.
|
|
Overexpression of Fyn Mutant with Loss-of-function Mutation in the
SH2 Domain Inhibited CD2-induced NFAT Transcriptional Activity--
We
have shown that CD2 is able to associate with the Fyn SH3 domain and
that Fyn SH2 domain negatively regulates CD2-Fyn SH3 association. We
reasoned that a loss-of-function Fyn SH2 mutant containing an intact
SH3 domain would compete with endogenous Fyn for binding to CD2 (and
other ligands), and may therefore interfere with CD2-induced signal
transduction. To test the functional involvement of Fyn in CD2-mediated
signaling, we transfected Jurkat cells transiently with wild-type Fyn
or a mutated Fyn construct containing point mutations in either the SH2
(Fyn SH2*) or SH3 domain (Fyn SH3*) that abolished the ability to bind
to phosphotyrosine-containing or proline-rich proteins, respectively.
Jurkat cells were cotransfected with the reporter plasmid p3xNFATluc,
carrying the luciferase gene driven by three tandem repeats of the NFAT
sequences derived from the distal IL-2 promoter, to monitor NFAT
transcriptional activation (Fig. 7). The
expression of wild-type Fyn and mutant Fyn molecules were comparable as
detected by Western blot (data not shown). In the absence of
stimulation, the basal level of NFAT activity was low in transfectants
expressing vector alone. Overexpression of wild-type Fyn, Fyn SH3*
(and, to a lesser extent, Fyn SH2*) enhanced the basal level NFAT
activity, albeit minimally. Mitogenic pairs of anti-CD2 mAbs greatly
enhanced NFAT-driven transcription. At optimal concentrations of
anti-CD2 stimulation, overexpression of wild-type Fyn or Fyn SH3*
induced comparable NFAT transcription as vector-alone transfection.
Overexpression of Fyn SH2*, however, inhibited CD2-induced NFAT
transcriptional activity, suggesting that the Fyn SH2 domain was
functionally important in CD2-induced signal transduction pathways.
Because the kinase domains were intact in wild-type and mutated Fyn
molecules, the inhibition by Fyn SH2* of CD2-mediated signaling was
mediated, at least in part, through the non-catalytic domain of
Fyn.

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|
Fig. 7.
Overexpression of Fyn mutant with
loss-of-function mutation in the SH2 domain inhibited CD2-induced NFAT
transcriptional activity. SV40 large T antigen-transformed Jurkat
cells were transiently transfected with vector, wild-type Fyn, a
loss-of-function mutated Fyn construct in which the Arg was replaced
with Lys at amino acid 176 in the Fyn SH2 domain (denoted Fyn SH2*), or
a loss-of-function mutated Fyn construct in which the Pro was replaced
with Val at amino acid 134 in the Fyn SH3 domain (denoted Fyn SH3*). A
reporter plasmid p3xNFATluc, carrying the luciferase gene driven by
three tandem repeats of the distal NFAT sequences derived from the IL-2
promoter, was co-transfected into Jurkat cells to monitor the
transcriptional activity of NFAT, as described under "Experimental
Procedures." Twelve hours after transfection, 106 cells
were left unstimulated, stimulated with mitogenic pairs of anti-CD2
mAbs, or stimulated with PMA (10 ng/ml) plus ionomycin (2 µM) for 6 h. Cells were then washed and then lysed,
and the soluble extract was assayed for luciferase activity. The
relative luciferase units were presented for each transfection
condition as the percentage of maximal stimulation induced by PMA plus
ionomycin.
|
|
 |
DISCUSSION |
In this report, we have investigated the association of CD2 with
Src family PTK, and, specifically, the molecular regulation of Fyn
binding to CD2. All Src family members share a similar NH2-terminal myristoylated domain that permits membrane
localization, an SH3 domain that binds to proline rich sequences, an
SH2 domain that interacts with tyrosyl-phosphorylated proteins, and a
COOH-terminal kinase domain (35). Although the exact substrates of Src
PTK are unknown, a number of intracellular proteins such as rasGAP, the
p85 subunit of phosphatidylinositol 3-kinase, Syk, ZAP-70, and Cbl have
been shown to associate with Src PTK in T cells (35, 38, 39). T cells
lacking the expression of Lck (40) or expressing a dominant negative
form of Fyn (41) are defective in response to TCR stimulation.
Furthermore, the involvement of Src family PTK in CD2-mediated
signaling has been suggested in many studies. Lck and Fyn have both
been reported to be associated with CD2 in immunoprecipitation studies
(21, 25). CD2 signaling is defective in the Lck-negative Jurkat variant
cell line JCam.1 (42); CD2-dependent signaling is also
defective in cell lines deficient in expression of CD45 (43), a
transmembrane protein-tyrosine phosphatase believed to regulate the
kinase activities of Src family members (44). Finally, Fyn has been
shown to colocalize with CD2 in immunofluorescence studies (45).
We and others have shown that Fyn is able to associate physically with
CD2 (Fig. 2, and Ref. 24). We have demonstrated here that the binding
of CD2 to Fyn was mediated by the SH3 domain of Fyn and required the
specific amino acids within the Fyn SH3 domain that have been shown to
mediate binding to proline-rich ligands (Fig. 4, A and
B). The human CD2 cytoplasmic tail has five different
proline-rich sequences; these regions are largely conserved among
human, rat, and mouse species (46). Rat CD2 has been reported to bind
to Lck; binding of Lck to CD2 was shown to be mediated by the SH3
domain of Lck (31), but the regulation of Lck SH3 binding was not
further explored. It will be important to determine the specific
sequences of CD2 involved in SH3 binding of Fyn in comparison to Lck
and perhaps to other signaling proteins that cooperatively participate
in CD2 signaling and to correlate these structural studies with
function. These studies are currently under way.
The CD2-Fyn association was dramatically increased in vivo
in cells stimulated with mitogenic combinations of anti-CD2 mAbs compared with unstimulated cells or cells stimulated with a single anti-CD2 antibody alone (Fig. 3B). However, by in
vitro analysis using GST fusion proteins, the association of the
SH3 domain of Fyn with CD2 appeared robust but unchanged after
stimulation. We demonstrated further that the Fyn SH2 domain negatively
regulated the Fyn SH3 domain binding to CD2 (Fig. 5A).
Regulation of SH3 domain interaction to ligand has been suggested
previously (33, 47, 48). The initial crystallographic structure of the
minimal Lck SH3-SH2 domain revealed that the SH2 and SH3 domain of Lck could form intermolecular dimers (49). However, structural analysis of
the entire Src molecule revealed that dimerization occurs not by
intermolecular associations, but by intramolecular associations between
the SH3 domain of Src and the linker region located between the SH2 and
kinase domains (50). Intramolecular folding of Src renders the kinase
inactive; this analysis left open the possibility that intermolecular
SH2-SH3 interactions were possible following stimulation of kinase
activity. The structural analysis of Hck, another member of the Src
kinase family, also supports an intramolecular association (47). In
addition, the multidimensional NMR study of Itk, a T cell-specific
tyrosine kinase belonging to the Tec tyrosine kinase family, has
demonstrated an intramolecular interaction between the Itk SH3 domain
and the proline-rich sequence of Itk (51). Finally, direct interactions
between isolated SH2 and SH3 domains of Fyn in vitro have
also been reported (33), suggesting that the SH2-SH3 interaction may
decrease the binding of the Fyn SH3 domain to a subset of unidentified
ligands (33). Our observations support a role for CD2 as one of the
"regulated" ligands of the Fyn SH3 domain. Occupancy of the SH2
domain with phosphorylated ligands following T cell activation may
compete for Fyn SH2 binding and allow the binding of Fyn SH3 domain to
CD2 and other proline-rich ligands (33). Our observation that the
binding of Fyn SH3-SH2 domain to CD2 was restored upon treatment with
tyrosyl-phosphorylated peptide (Fig. 5B) is consistent with
the model. It is also worth noting that the inhibitory effect of the
Fyn SH2 domain on the Fyn SH3 domain to bind to ligands is not
universal; the binding of Fyn SH3 domain to Cbl was largely unchanged
even in the presence of the Fyn SH2 domain (Fig. 5A). The
affinity of Cbl toward the Fyn SH3 domain may be higher than that of
CD2, allowing it to overcome the inhibitory effect of Fyn SH2 domain.
Alternatively, the Fyn SH2 domain binding to tyrosyl-phosphorylated
sequences on Cbl may help to relieve the Fyn SH2-dependent
inhibition of Fyn SH3 domain binding (39).
It has been shown that CD2-mediated activation of T cells requires the
CD3
chains or other CD3 complex components and that CD3
physically associates with CD2 (18, 24). We have demonstrated increased
binding of the Fyn SH2 domain to CD3
after anti-CD2 stimulation
(Fig. 6, B and C), suggesting that the
trimolecular complex may serve to amplify the early CD2-mediated
activation signals. CD3
contains multiple
EX2YX2L/IX7YX2L/I
motifs, termed immune receptor tyrosine-based activation motifs
(ITAMs). ITAMs are phosphorylated upon activation and may serve as
potential ligands of the Fyn SH2 domain (18, 52, 53). Lck has been shown to be able to phosphorylate CD3
(54); whether Lck catalyzed CD3
phosphorylation in vivo is not clear. In unstimulated
cells, minimal amount of Fyn were associated with CD2 (Fig. 3A).
Anti-CD2 mAbs stimulation resulted in the tyrosyl phosphorylation of a number of intracellular proteins, including CD3
, mediated by a
tyrosine kinase, potentially Lck. Phosphorylation of CD3
rendered the CD3
able to bind to the Fyn SH2 domain (Fig. 6B);
occupancy of the Fyn SH2 domain by ligand appeared to be able to
reverse the inhibitory effect of the Fyn SH2 domain on the binding of the Fyn SH3 domain to CD2. We propose that as a result of occupancy of
the Fyn SH2 domain, possibly by CD3
itself, quantitatively more Fyn
is able to be recruited to the CD2 complex, and, cooperatively, recruitment of Fyn (and other PTKs) may potentially catalyze further tyrosyl phosphorylation of CD3
and other downstream effectors. Thus,
formation of the multimolecular CD2 complex, including CD3
and Fyn,
appears to result in amplification of intracellular signaling. Although
our data suggest the functional involvement of CD3
in CD2-mediated
signaling, it does not exclude the involvement of other CD3 components.
Other CD3 components containing the ITAM motif(s), including CD3
,
have been shown to play a role in CD2-mediated signaling (55).
The association of CD2 with Src family PTK, including Lck and Fyn, has
been demonstrated previously (21, 23, 25). CD2-induced signal was
abolished in Jurkat cells lacking Lck expression (42). However, the
functional significance of Fyn in CD2-induced signal transduction has
not been addressed. We show here that a loss-of-function Fyn SH2*
mutant inhibited CD2-mediated NFAT-transcriptional activity, suggesting
a critical role of Fyn in the CD2-mediated signal pathway. Furthermore,
the function of Fyn in CD2-induced pathway was, at least in part,
mediated by the non-catalytic domain, as the kinase domain was intact
in Fyn SH2* mutant. This result is consistent with our model in that
the Fyn SH2* molecule (with an intact SH3 domain) will compete with
endogenous Fyn for binding to CD2 but, unable to bind to or recruit
phosphotyrosine-containing signaling molecules, will therefore inhibit
CD2-induced signaling.
CD2 is able to associate with both Fyn and Lck. Our data suggest that
each kinase plays independent roles in CD2-induced transduction pathway. Lck and Fyn are not functionally interchangeable; although thymocyte development was arrested at an early stage in mice with a
homozygous deletion of Lck expression (56), thymocyte development was
grossly normal in mice lacking Fyn expression (57, 58) or bearing a
dominant negative form of Fyn (59). Lck but not Fyn has been reported
to associate with CD4 and CD8 (60, 61), whereas Fyn but not Lck has
been shown to bind the phosphoprotein p120/p130 FYB (62). The SH3
domain of Fyn has been shown to precipitate quantitatively higher
amounts of Cbl and phosphatidylinositol 3-kinase than the SH3 domain of
Lck from cell lysates (39, 63); the functional significance of these
differences are not known. The characterization of Pyk2, a downstream
molecule of Fyn but not Lck (64), further suggested that Fyn and Lck
may lead to divergent signal transduction pathways. In certain
immunofluorescence studies, CD2 co-localized with Fyn but not with Lck
after anti-CD2 mAbs treatment (45). Finally, in some T cell lines
rendered anergic, the kinase activity of Fyn was elevated, whereas that of Lck was unchanged or diminished (65, 66). It is of note that CD2
stimulation has been shown to be able to reverse anergy and T cell
unresponsiveness (19); the recruitment of Fyn by CD2 may play a role in
reversing the anergic state.
 |
ACKNOWLEDGEMENT |
We thank Jacqueline Slavik for purification of
human peripheral T lymphocytes.
 |
FOOTNOTES |
*
This work was supported by the National Institutes of
Health.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.

To whom correspondence should be addressed. Present address:
NHLBI, National Institutes of Health, Bldg. 10, Rm. 5D49, 10 Center
Dr., Bethesda, MD 20892. Tel.: 301-402-6786; Fax: 301-480-1792; E-mail:
biererb{at}nih.gov.
1
The abbreviations used are: TCR, T cell
receptor; GST, glutathione S-transferase; mAb, monoclonal
antibody; APC, antigen-presenting cell; MHC, major histocompatibility
complex; IL, interleukin; PTK, protein-tyrosine kinase; PAGE,
polyacrylamide gel electrophoresis; PVDF, polyvinylidene difluoride;
PNGase F, peptide:N-glycosidase F; NFAT, nuclear factor of
activated T cells; PMA, phorbol 12-myristate 13-acetate; ITAM, immune
receptor tyrosine-based activation motif; NT domain, amino-terminal
unique domain.
 |
REFERENCES |
-
Marrack, P.,
and Kappler, J.
(1987)
Science
238,
1073-1079[Medline]
[Order article via Infotrieve]
-
Davis, M. M.,
and Bjorkman, P. J.
(1988)
Nature
334,
395-402[CrossRef][Medline]
[Order article via Infotrieve]
-
Bierer, B. E.,
Sleckman, B. P.,
Ratnofsky, S. E.,
and Burakoff, S. J.
(1989)
Annu. Rev. Immunol.
7,
579-599[CrossRef][Medline]
[Order article via Infotrieve]
-
June, C. H.,
Ledbetter, J. A.,
Linsley, P. S.,
and Thompson, C. B.
(1990)
Immunol. Today
11,
211-216[CrossRef][Medline]
[Order article via Infotrieve]
-
Allison, J. P.
(1994)
Curr. Opin. Immunol.
6,
414-419[CrossRef][Medline]
[Order article via Infotrieve]
-
Davis, S. J.,
and van der Merwe, P. A.
(1996)
Immunol. Today
17,
177-187[CrossRef][Medline]
[Order article via Infotrieve]
-
Selvaraj, P.,
Plunkett, M. L.,
Dustin, M.,
Sanders, M. E.,
Shaw, S.,
and Springer, T. A.
(1987)
Nature
326,
400-403[CrossRef][Medline]
[Order article via Infotrieve]
-
Hünig, T.,
Tiefenthaler, G.,
Meyer zum Büschenfelde, K.-H.,
and Meuer, S. C.
(1987)
Nature
326,
298-301[CrossRef][Medline]
[Order article via Infotrieve]
-
Hahn, W. C.,
Menu, E.,
Bothwell, A. L. M.,
Sims, P. J.,
and Bierer, B. E.
(1992)
Science
256,
1805-1807[Medline]
[Order article via Infotrieve]
-
Deckert, M.,
Kubar, J.,
Zoccola, D.,
Bernard-Pomier, G.,
Angelisova, P.,
Horejsi, V.,
and Bernard, A.
(1992)
Eur. J. Immunol.
22,
2943-2948[Medline]
[Order article via Infotrieve]
-
Sandrin, M. S.,
Mouhtouris, E.,
Vaughan, H. A.,
Warren, H. S.,
and Parish, C. R.
(1993)
J. Immunol.
151,
4606-4613[Abstract/Free Full Text]
-
Warren, H. S.,
Altin, J. G.,
Waldron, J. C.,
Kinnear, B. F.,
and Parish, C. R.
(1996)
J. Immunol.
157,
2866-2873
-
Bierer, B. E.,
Peterson, A.,
Gorga, J. C.,
Herrmann, S. H.,
and Burakoff, S. J.
(1988)
J. Exp. Med.
168,
1145-1156[Abstract]
-
Moingeon, P.,
Chang, H.-C.,
Wallner, B. P.,
Stebbins, C.,
Frey, A. Z.,
and Reinherz, E. L.
(1989)
Nature
339,
312-314[CrossRef][Medline]
[Order article via Infotrieve]
-
Miller, G. T.,
Hochman, P. S.,
Meier, W.,
Tizard, R.,
Bixler, S. A.,
Rosa, M. D.,
and Wallner, B. P.
(1993)
J. Exp. Med.
178,
211-222[Abstract]
-
Gückel, B.,
Berek, C.,
Lutz, M.,
Altevogt, P.,
Schirrmacher, V.,
and Kyewski, B. A.
(1991)
J. Exp. Med.
174,
957-967[Abstract]
-
Meuer, S. C.,
Hussey, R. E.,
Fabbi, M.,
Fox, D.,
Acuto, O.,
Fitzgerald, K. A.,
Hodgdon, J. C.,
Protentis, J. P.,
Schlossman, S. F.,
and Reinherz, E. L.
(1984)
Cell
36,
897-906[Medline]
[Order article via Infotrieve]
-
Howard, F. D.,
Moingeon, P.,
Moebius, U.,
McConkey, D. J.,
Yandava, B.,
Gennert, T. E.,
and Reinherz, E. L.
(1992)
J. Exp. Med.
176,
139-145[Abstract]
-
Boussiotis, V. A.,
Freeman, G. J.,
Griffin, J. D.,
Gray, G. S.,
Gribben, J. G.,
and Nadler, L. M.
(1994)
J. Exp. Med.
180,
1665-1673[Abstract]
-
Gollob, J. A.,
Li, J.,
Reinherz, E. L.,
and Ritz, J.
(1995)
J. Exp. Med.
182,
721-731[Abstract]
-
Bell, G. M.,
Bolen, J. B.,
and Imboden, J. B.
(1992)
Mol. Cell. Biol.
12,
5548-5554[Abstract]
-
Schraven, B.,
Ratnofsky, S.,
Gaumont, Y.,
Lindegger, H.,
Kirchgessner, H.,
Bruyns, E.,
Moebius, U.,
and Meuer, S. C.
(1994)
J. Exp. Med.
180,
897-906[Abstract]
-
Schraven, B.,
Wild, M.,
Kirchgessner, H.,
Siebert, B.,
Wallich, R.,
Henning, S.,
Samstag, Y.,
and Meuer, S. C.
(1993)
Eur. J. Immunol.
23,
119-123[Medline]
[Order article via Infotrieve]
-
Beyers, A. D.,
Spruyt, L. L.,
and Williams, A. F.
(1992)
Proc. Natl. Acad. Sci. U. S. A.
89,
2945-2949[Abstract]
-
Carmo, A. M.,
Mason, D. W.,
and Beyers, A. D.
(1993)
Eur. J. Immunol.
23,
2196-2201[Medline]
[Order article via Infotrieve]
-
Shimizu, Y.,
Mobley, J. L.,
Finkeistein, L. D.,
and Chan, A. S. H.
(1995)
J. Cell Biol.
131,
1867-1880[Abstract]
-
Schraven, B.,
Samstag, Y.,
Altevogt, P.,
and Meuer, S. C.
(1990)
Nature
345,
71-74[CrossRef][Medline]
[Order article via Infotrieve]
-
Offringa, R.,
and Bierer, B. E.
(1993)
J. Biol. Chem.
268,
4979-4988[Abstract/Free Full Text]
-
Hahn, W. C.,
Rosenstein, Y.,
Calvo, V.,
Burakoff, S. J.,
and Bierer, B. E.
(1992)
Proc. Natl. Acad. Sci. U. S. A.
89,
7179-7183[Abstract]
-
Chang, H.-C.,
Moingeon, P.,
Lopez, P.,
Krasnow, H.,
Stebbins, C.,
and Reinherz, E.
(1989)
J. Exp. Med.
169,
2073-2083[Abstract]
-
Bell, G. M.,
Fargnoli, J.,
Bolen, J. B.,
Kish, L.,
and Imboden, J. B.
(1996)
J. Exp. Med.
183,
169-178[Abstract]
-
Remold-O'Donnell, E.,
Kenney, D. M.,
Parkman, R.,
Cairns, L.,
Savage, B.,
and Rosen, F. S.
(1984)
J. Exp. Med.
159,
1705-1723[Abstract]
-
Panchamoorthy, G.,
Fukazawa, T.,
Stolz, L.,
Payne, G.,
Reedquist, K.,
Shoelson, S.,
Songyang, Z.,
Cantley, L.,
Walsh, C.,
and Band, H.
(1994)
Mol. Cell. Biol.
14,
6372-6385[Abstract]
-
Rooney, J. W., Y., L. S.,
Glimcher, L. H.,
and Hoey, T.
(1995)
Mol. Cell. Biol.
15,
6299-6243[Abstract]
-
Chan, A. C.,
Desai, D. M.,
and Weiss, A.
(1994)
Annu. Rev. Immunol.
12,
555-592[CrossRef][Medline]
[Order article via Infotrieve]
-
Feng, S.,
Chen, J. K.,
Yu, H.,
Simon, J. A.,
and Schreiber, S. L.
(1994)
Science
266,
1241-1247[Medline]
[Order article via Infotrieve]
-
Weng, Z.,
Thomas, S. M.,
Rickles, R. J.,
Taylor, J. A.,
Brauer, A. W.,
Seidel-Dugan, C.,
Michael, W. M.,
Dreyfuss, G.,
and Brugge, J. S.
(1994)
Mol. Cell. Biol.
14,
4509-4521[Abstract]
-
Prasad, K. V. S.,
Janssen, O.,
Kapeller, R.,
Raab, M.,
Cantley, L. C.,
and Rudd, C. E.
(1993)
Proc. Natl. Acad. Sci. U. S. A.
90,
7366-7370[Abstract]
-
Reedquist, K. A.,
Fukazawa, T.,
Druker, B.,
Panchamoorthy, G.,
Shoelson, S. E.,
and Band, H.
(1994)
Proc. Natl. Acad. Sci. U. S. A.
91,
4135-4139[Abstract]
-
Straus, D. B.,
and Weiss, A.
(1992)
Cell
70,
585-593[Medline]
[Order article via Infotrieve]
-
Fusaki, N.,
Semba, K.,
Katagiri, T.,
Suzuki, G.,
Matsuda, S.,
and Yamamoto, T.
(1994)
Int. Immunol.
6,
1245-1255[Abstract]
-
Hubert, P.,
Lang, V.,
Debré, P.,
and Bismuth, G.
(1996)
J. Immunol.
156,
4322-4332
-
Koretzky, G. A.,
Picus, J.,
Schultz, T.,
and Weiss, A.
(1991)
Proc. Natl. Acad. Sci. U. S. A.
88,
2037-2041[Abstract]
-
McFarland, E. D.,
Hurley, T. R.,
Pingel, J. T.,
Sefton, B. M.,
Shaw, A.,
and Thomas, M. L.
(1993)
Proc. Natl. Acad. Sci. U. S. A.
90,
1402-1406[Abstract]
-
Gassmann, M.,
Amrein, K. E.,
Flint, N. A.,
Schraven, B.,
and Burn, P.
(1994)
Eur. J. Immunol.
24,
139-144[Medline]
[Order article via Infotrieve]
-
Hahn, W. C.,
and Bierer, B. E.
(1993)
J. Exp. Med.
178,
1831-1836[Abstract]
-
Sicheri, F.,
Moarefi, I.,
and Kuriyan, J.
(1997)
Nature
385,
602-609[CrossRef][Medline]
[Order article via Infotrieve]
-
Ravichandran, K. S.,
Lorenz, U.,
Shoelson, S. E.,
and Burakoff, S. J.
(1995)
Mol. Cell. Biol.
15,
593-600[Abstract]
-
Eck, M.,
Atwell, S.,
Shoelson, S. E.,
and Harrison, S. C.
(1994)
Nature
368,
764-769[CrossRef][Medline]
[Order article via Infotrieve]
-
Xu, W.,
Harrison, S. C.,
and Eck, M.
(1997)
Nature
385,
595-601[CrossRef][Medline]
[Order article via Infotrieve]
-
Andreotti, A. H.,
Bunnell, S. C.,
Feng, G.,
Berg, L. J.,
and Schreiber, S. L.
(1997)
Nature
385,
93-97[CrossRef][Medline]
[Order article via Infotrieve]
-
Reth, M.
(1989)
Nature
338,
383-384[Medline]
[Order article via Infotrieve]
-
Irving, B. A.,
Chan, A. C.,
and Weiss, A.
(1993)
J. Exp. Med.
177,
1093-1103[Abstract]
-
Iwashima, M.,
Irving, B. A.,
van Oers, N. S. C.,
Chan, A. C.,
and Weiss, A.
(1994)
Science
263,
1136-1139[Medline]
[Order article via Infotrieve]
-
Steeg, C.,
von Bonin, A.,
Mittrucker, H.,
Malissen, B.,
and Fleischer, B.
(1997)
Eur. J. Immunol.
27,
2233-2238[Medline]
[Order article via Infotrieve]
-
Molina, T. J.,
Kishihara, K.,
Siderovski, D. P.,
van Ewijk, W.,
Narenfran, A.,
Timms, E.,
Wakeham, A.,
Paige, C. J.,
Hartmann, K. U.,
Veillette, A.,
Davidson, D.,
and Mak, T. W.
(1992)
Nature
357,
161-164[CrossRef][Medline]
[Order article via Infotrieve]
-
Appleby, M. W.,
Gross, J. A.,
Cooke, M. P.,
Levin, S. D.,
Qian, X.,
and Perlmutter, R. M.
(1992)
Cell
70,
751-763[Medline]
[Order article via Infotrieve]
-
Stein, P. L.,
Lee, H.,
Rich, S.,
and Soriano, P.
(1992)
Cell
70,
741-750[Medline]
[Order article via Infotrieve]
-
Cooke, M. P.,
Abraham, K. M.,
Forbush, K. A.,
and Perlmutter, R. M.
(1991)
Cell
65,
281-291[Medline]
[Order article via Infotrieve]
-
Rudd, C. E.,
Trevillyan, J. M.,
Wong, L. L.,
Dasgupta, J. D.,
and Schlossman, S. F.
(1988)
Proc. Natl. Acad. Sci. U. S. A.
85,
5190-5194[Abstract]
-
Veillette, A.,
Bookman, M. A.,
Horak, E. M.,
and Bolen, J. B.
(1988)
Cell
55,
301-308[Medline]
[Order article via Infotrieve]
-
da Silva, A. J.,
Li, Z.,
de Vera, C.,
Canto, E.,
Findell, P.,
and Rudd, C. E.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
7493-7498[Abstract/Free Full Text]
-
Kapeller, R.,
Prasad, K. V. S.,
Jassen, O.,
Hou, W.,
Schaffhausen, B. S.,
Rudd, C. E.,
and Cantley, L. C.
(1994)
J. Biol. Chem.
269,
1927-1933[Abstract/Free Full Text]
-
Qian, D.,
Lev, S.,
van Oers, N. S. C.,
Dikic, I.,
Schlessinger, J.,
and Weiss, A.
(1997)
J. Exp. Med.
185,
1253-1259[Abstract/Free Full Text]
-
Gajewski, T. F.,
Qian, D.,
Fields, P.,
and Fitch, F. W.
(1994)
Proc. Natl. Acad. Sci. U. S. A.
91,
38-42[Abstract]
-
Quill, H.,
Riley, M. P.,
Cho, E. A.,
Casnelie, J. E.,
Reed, J. C.,
and Torigoe, T.
(1992)
J. Immunol.
149,
2887-2893[Abstract/Free Full Text]
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