From the Departments of Immunology and
¶ Medicine, University of Toronto, Toronto, Ontario M5S
1A2, Canada and § Ontario Cancer Institute/Princess Margaret Hospital,
Toronto, Ontario M5G 2M9, Canada
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ABSTRACT |
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CD28 provides a costimulatory signal that results in optimal activation of T cells. The signal transduction pathways necessary for CD28-mediated costimulation are presently unknown. Engagement of CD28 leads to its tyrosine phosphorylation and subsequent binding to Src homology 2 (SH2)-containing proteins including the p85 subunit of phosphatidylinositol 3'-kinase (PI3K); however, the contribution of PI3K to CD28-dependent costimulation remains controversial. Here we show that CD28 is capable of binding the Src homology 3 (SH3) domains of several proteins, including Grb2. The interaction between Grb2 and CD28 is mediated by the binding of Grb2-SH3 domains to the C-terminal diproline motif present in the cytoplasmic domain of CD28. While the affinity of the C-terminal SH3 domain of Grb2 for CD28 is greater than that of the N-terminal SH3 domain, optimal binding requires both SH3 domains. Ligation of CD28, but not tyrosine-phosphorylation, is required for the SH3-mediated binding of Grb2 to CD28. We propose a model whereby the association of Grb2 with CD28 occurs via an inducible SH3-mediated interaction and leads to the recruitment of tyrosine-phosphorylated proteins such as p52shc bound to the SH2 domain of Grb2. The inducible interaction of Grb2 to the C-terminal region of CD28 may form the basis for PI3K-independent signaling through CD28.
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INTRODUCTION |
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Engagement of the T cell receptor
(TCR)1 by the major
histocompatibility complex-peptide complex in the absence of
costimulatory molecules is insufficient to induce production of
cytokines and can render the T cells unresponsive to further antigenic
challenge (1). CD28 is preeminent among a group of receptors, including 4-1BB and CD43, that can provide costimulatory signals to T cells (2-4). CD28 is a type 1 transmembrane protein of the Ig superfamily, which is expressed on the cell surface as a glycosylated homodimer (5).
CD28 costimulation of TCR-dependent responses increases IL-2 production (6, 7), prevents the induction of anergy (8), and
renders T cells resistant to apoptotic cell death (9-11). These
effects are mediated by increased transcription of cytokine genes
through the activation of a composite NF-B and AP-1 transcriptional
element (12, 13), the stabilization of cytokine mRNAs (14), and the
expression of the survival protein, BclxL (9). TCR-mediated
T cell activation is sensitive to the immunosuppressive drug
cyclosporin, while those pathways activated by CD28 are not, suggesting
that costimulatory pathways are distinct from those activated by the
TCR (7). Early biochemical events induced through CD28 include tyrosine
phosphorylation (15, 16) and activation of PI3K (17-19) and acidic
sphingomyelinase (20). The identity of the signal transduction pathways
that are required for CD28-mediated costimulation are presently
unknown.
The cytoplasmic domain of CD28 contains no recognized intrinsic enzymatic activity; however, CD28 has been reported to associate with signaling proteins following ligation. Phosphorylation of CD28 on tyrosine 173 within the motif YMNM present in the cytoplasmic domain provides a binding site for the SH2 domain of the p85 subunit of PI3K (17-19); however, conflicting results regarding the requirement for PI3K in CD28-dependent costimulation have been published. Mutant forms of CD28 that are unable to bind to PI3K demonstrate an absolute requirement for PI3K in mediating CD28 signals in mouse T cell hybridoma cell lines (17). Further, wortmannin, a potent inhibitor of PI3K, inhibits costimulation through CD28 in human peripheral T cells (21, 22). In contrast to these studies, CD28 can provide costimulatory signals in the absence of PI3K activation in Jurkat cells and purified mouse T cells (23-26). Moreover, activation of PI3K by ectopically expressed CD19, a potent activator of PI3K in B cells, in conjunction with TCR ligation is insufficient to induce IL-2 transcription in Jurkat cells (23). These studies demonstrate that PI3K activation is neither necessary nor sufficient for CD28-mediated costimulation in certain cellular systems and suggest that alternative signaling pathways are involved in costimulation. In support of this view, deletion of 10 amino acids in the C-terminal portion of CD28, remote from the PI3K binding site, attenuates costimulation (26, 27). Until now, no signaling proteins that bind to this site have been identified.
More recently, Grb2 has been implicated in CD28 signaling (28, 29). Grb2 is a linker protein that utilizes both SH2- and SH3- dependent interactions to bind to a diverse repertoire of signaling proteins. The canonical function of Grb2 is to stabilize an intermolecular complex between receptor tyrosine kinases, such as epidermal growth factor receptor, Met, and Flt3, and the positive regulator of the Ras pathway, Sos. Grb2 binds constitutively to Sos through its SH3 domains and inducibly binds to tyrosine-phosphorylated receptors via its SH2 domain (30-35). Activation of the Ras signaling pathway is a critical step during T cell activation (36, 37). In TCR-stimulated cells, there is a rapid formation of a complex between Sos/Grb2 and a 36-kDa membrane protein, LAT, that is a substrate for the TCR-induced tyrosine kinases (38-40). Antibody-mediated aggregation of CD28 can also activate Ras (41). Grb2 has been shown to bind to CD28 following ligation of CD28 (28, 29). Under these circumstances, the Grb2-CD28 association was in part mediated by the Grb2-SH2 domain binding to the CD28 PI3K binding site, Tyr173 (28, 29). This interaction may be responsible for CD28-dependent Ras activation (29). The SH3 domains of Grb2 also bind the product of the protooncogene c-cbl in T cells; however, the role of this interaction during T cell activation is not known (42-45).
CD28 contains two potential SH3-binding diproline motifs, one of which is contained in part by the C-terminal region required for costimulation. SH3 domains bind to short peptide sequences rich in proline residues, which adopt a left-handed type II polyproline helix conformation. Two proline residues are presented as a bidentate hydrophobic contact surface that binds to a shallow hydrophobic groove common to SH3 domains (46). In this report, we demonstrate that in addition to binding SH2-containing signaling molecules, CD28 is an SH3-binding protein. Following ligation, CD28 binds to the SH3 domains of Grb2 and Itk as well as to the WW domain of YAP.
The association between Grb2 and CD28 occurs via an SH3-proline interaction involving the diproline motif embedded in the C-terminal portion of the cytoplasmic domain of CD28. The interaction between CD28 and Grb2-SH3 domains is phosphotyrosine-independent and does not require a functional SH2 domain. The SH3-mediated interaction between CD28 and Grb2 allows the SH2 domain of Grb2 to bind to phosphotyrosine-containing proteins such as p52shc. We propose a model in which Grb2 functions in a heretofore uncharacterized manner to couple CD28 to tyrosine-phosphorylated proteins involved in CD28-mediated costimulation.
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EXPERIMENTAL PROCEDURES |
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Cell Lines--
The murine thymoma cell lines VCD28 and
VCD2810 expressing human CD28 were a kind gift from D. Couez (INSERM
U298, Angers, France). COS-7 and 293T cells were purchased from ATCC.
All cells were grown in Dulbecco's modified Eagle's medium
supplemented with 10% fetal bovine serum (Life Technologies, Inc.) at
37 °C in a humidified atmosphere containing 5% CO2.
G418 (Life Technologies) was added at 2 mg/ml to cells transfected with
CD28. Rat2 cells expressing the Fms-Flt3 chimeric receptor (FF3) have
been described elsewhere (47).
Antibodies-- Anti-human CD28 mAb 9.3 ascites was a kind gift from P. Linsley (Bristol-Meyers Squibb Pharmaceutical Co., Seattle, WA). Anti-CD28 serum was produced in rabbits by immunizing with a synthetic peptide corresponding to the C-terminal 18 amino acids of CD28 coupled to keyhole limpet hemocyanin. Anti-Grb2-SH2 serum was produced in rabbits immunized with GST-Grb2-SH2. The antibodies were purified on protein A-Sepharose (Amersham Pharmacia Biotech), and coupled to CNBr-activated beads (Bio-Rad) according to the manufacturer's directions. Uncoupled CNBr sites were quenched by alternate washes in high and low pH followed by incubation in 100 mM Tris-HCl, pH 8.0. Purified rabbit Ig purchased from Jackson Laboratories was also coupled to CNBr beads and was used as a nonspecific Ig control in Fig. 4B. Anti-Flt3 serum was produced in rabbits against a TrpE-Flt3 fusion protein and has been described previously (47). Anti-Grb2, anti-Cbl, and anti-Myc were purchased from Santa Cruz Biotechnology (Santa Cruz, CA); anti-Sos was purchased from Transduction Laboratory; and the anti-phosphotyrosine mAb 4G10 was purchased from Upstate Biotechnology (Lake Placid, NY).
GST Fusion Proteins--
Full-length Grb2; Grb2 mutants P49L,
R86K, G203R, and P49L/G203R; or individual SH2 or SH3 domains and
Grb3-3 were expressed as GST fusion proteins in Escherichia
coli according to standard methods. These were kind gifts from M. Moran (University of Toronto, Toronto, Canada), G. Koretzky (University
of Iowa College of Medicine, Iowa City, Iowa), and B. Tocqué
(Rhône-Poulenc Rorer, Vitry sur Seine, France). The cDNA encoding
the cytoplasmic domain of CD28 was amplified by polymerase chain
reaction, and cloned into the PGEX4-T1 vector (Amersham Pharmacia
Biotech). Nonphosphorylated GST-CD28 was expressed in the E. coli BL21(DE3) pLysS strain (Novagen). Phosphorylated GST-CD28 was
expressed E. coli TKB1 strain (Stratagene), a BL21(DE3)
derivative strain that harbors a plasmid-encoded, inducible tyrosine
kinase gene. Bacterial cultures were grown to log phase, induced by 0.4 mM isopropyl-1-thio--D-galactopyranoside, and incubated for 3-7 h at 28 °C. GST-CD28 was
tyrosine-phosphorylated by subsequently incubating the TKB1 cultures in
tryptophan-free media at 37 °C to induce the tyrosine kinase,
according to the supplier's instructions. The bacteria were lysed in
PLC
lysis buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1.5 mM MgCl2, 1 mM EGTA, 10 µg/ml
leupeptin, 10 µg/ml aprotinin, 1 mM phenylmethylsulfonyl
fluoride), and purified on glutathione-Sepharose beads (Amersham
Pharmacia Biotech). The amount of GST fusion protein was estimated by
comparison with bovine serum albumin standards on Coomassie-stained
SDS-PAGE gels. 10 µg of each GST fusion protein was used for each
coprecipitation.
Plasmid cDNAs--
Human CD28 cDNA (clone A53, gift from
B. Seed, Massachusetts General Hospital, Boston, MA) was cloned into
the EcoRI site of pME18-226Neo, which contains the SR
promoter (gift from Gerrard Zurawski, DNAX, Palo Alto, CA). Polymerase
chain reaction-based overlap extension mutagenesis was used to create
point mutations within the cytoplasmic domain of CD28. The plasmids
containing Myc epitope-tagged Grb2 under control of the cytomegalovirus
promoter were a gift from David Pot (Chiron Corp., Emeryville, CA) and have been described previously (48).
Transfections of Cell Lines-- 293T cells were transfected using a standard calcium phosphate method. COS cells were transfected using Lipofectamine (Life Technologies), according to the manufacturer's instructions.
Cell Stimulation, Lysis, and Coprecipitation--
VCD28 cells
were harvested and resuspended at 2-4 × 107 cells/ml
in PBS. 1 µg of mAb 9.3 was added to each stimulated sample (0.5 ml)
in 1.5-ml Eppendorf tubes and incubated at 37 °C for 5 min. The
cells were transferred to ice, and 0.5 ml of 2× PLC lysis buffer
was added. Following lysis, 1 µg of mAb 9.3 was added to unstimulated
samples. Confluent Rat2-FF3 cells were stimulated with 500 ng of
colony-stimulating factor-1/ml for 5 min at 37 °C, and lysed in 1×
PLC
lysis buffer. The lysate from one 100-mm plate was used per
condition. 293T cells were lysed directly in 1× PLC
lysis buffer in
100-mm tissue culture dishes, and
th of each lysate was used
per coprecipitation. Lysates were centrifuged at 21,000 × g for 15 min, and the supernatant was incubated with protein
A-Sepharose beads (Amersham Pharmacia Biotech) or with immobilized GST
fusion proteins on glutathione beads for 2-4 h at 4 °C. The beads
were washed three times with cold lysis buffer and boiled in the
presence of SDS sample buffer containing
-mercaptoethanol (Laemmli
buffer). The protein complexes were resolved by SDS-PAGE and
transferred to polyvinylidene difluoride membranes (Immobilon). CD28
was immunoblotted using anti-CD28 serum in PBS with 0.05% Tween and
5% skim milk powder, followed by incubation with horseradish peroxidase-conjugated donkey anti-rabbit antibody or protein A (The
Jackson Laboratory). Protein bands were detected by Renaissance enhanced luminol reagent (NEN Life Science Products).
Biotinylation of Cell Surface Proteins-- Before stimulation, cells were washed in PBS and resuspended in 10 mM sodium borate, 150 mM NaCl, pH 8.8. The biotinylation reaction was initiated by the addition of 5 µl of 10 mg/ml sulfosuccinimidyl-6-(biotinamido)hexanoate (Pierce) in Me2SO. After 15 min, the biotinylation reaction was quenched by the addition of 10 µl of 1 M NH4Cl. The cells were washed three times with PBS plus 10 mM Tris-Cl, 1 mM EDTA before stimulation and lysed as described above. The protein blots were probed with avidin-horseradish peroxidase (Amersham Pharmacia Biotech) diluted in 1% skim milk powder in PBS, 0.05% Tween. To verify that the cells were uniformly labeled, an aliquot of cells was incubated with avidin-Spectral Red (Southern Biotechnology Associates Inc.) and analyzed by flow cytometry.
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RESULTS |
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CD28 Is an SH3-binding Protein--
SH3 and WW domains are
distinct polypeptide structures that function to form multimeric
protein complexes as a result of their capacity to bind to proline-rich
sequences. The structure of SH3 domains consists of a -barrel of two
three-stranded, antiparallel
-sheets, which presents an array of
conserved hydrophobic side chains appropriately spaced for interaction
with polyproline helices (49). WW domains are composed of three
anti-parallel
-sheets rich in aromatic amino acids that form a
hydrophobic ligand pocket (50). The ligand specificity of SH3 and WW
domains may in some instances be overlapping (51). The cytoplasmic
domain of CD28 contains two polyproline sequences and therefore may
form complexes with SH3- and WW-containing proteins.
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The Interaction between Grb2 and CD28 Is Inducible and Is Mediated by the SH3 Domains of Grb2-- We next examined which of the Grb2 domains were capable of binding to CD28 using recombinant GST fusion proteins. VCD28 lysates were prepared from resting or mAb 9.3-stimulated cells and incubated with equivalent amounts of immobilized GST, GST-N-terminal SH3 (GST-SH3N), GST-SH2, or GST-C-terminal SH3 (GST-SH3C) fusion proteins. The protein complexes were resolved by SDS-PAGE and immunoblotted with CD28-specific antibodies. CD28 from unstimulated cells did not complex to any of the Grb2-derived constructs (Fig. 2A, lanes 1, 3, and 5). GST-SH3C demonstrated strong and inducible binding to CD28, while neither the GST-SH3N nor the GST-SH2 domain coprecipitated detectable amounts of CD28 (Fig. 2A, compare lane 6 to lanes 2 and 4). In other experiments, weak binding of the N-terminal SH3 domain, but not the SH2 domain, of Grb2 to CD28 was detected (data not shown). The isolated C-terminal SH3 domain was less effective than the full-length Grb2 molecule in coprecipitating CD28 (data not shown). The capacity of the Grb2-SH2 fusion protein to efficiently bind to tyrosine-phosphorylated proteins was tested in Fig. 2A, lower part. The Flt3 receptor tyrosine kinase binds to Grb2 at tyrosine 958 within the carboxyl tail in a manner similar to epidermal growth factor receptor or Met (53). In contrast to CD28, the activated Flt3 receptor was coprecipitated with the SH2 domain of Grb2, while there was no detectable interaction with either of the Grb2-SH3 domains (Fig. 2A, compare lane 13 to lanes 11-15). Sos was detected in complex with both the N- and C-terminal SH3 domains but not with the SH2 domain of Grb2 (Fig. 2C, lanes 1-6).
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Grb2 Binds to Diproline Motifs in the Cytoplasmic Domain of CD28-- Structural studies of the Grb2-SH3 domains bound to their ligands show that the two prolines in the PXXP motif represent contact residues with the SH3 hydrophobic binding groove. Mutation of either of these prolines results in significant attenuation of the binding interaction (54). We changed the first proline of each PXXP motif present in the cytoplasmic tail of CD28 to alanine by mutagenesis at codons 178 (P178A) and 190 (P190A) (Fig. 3A). The wild type and mutant forms of CD28 were expressed in COS cells at similar levels as demonstrated by Western blotting (Fig. 3B, lanes 1, 3, and 5). Cells were stimulated and lysed as above. Wild type and the P178A mutant forms of CD28 from stimulated cells bound to GST-Grb2 at similar efficiency (Fig. 3B, lanes 2 and 4). Substitution within the C-terminal diproline motif (P190A) reduced the amount of CD28 that coprecipitated with Grb2 (Fig. 3B, lane 6). These data suggest that the C-terminal diproline motif is the primary binding site for the SH3 domains of Grb2.
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In Vivo Association between CD28 and Grb2-- In order to determine whether CD28 and Grb2 formed a protein complex in vivo, as suggested by our in vitro experiments, cellular lysates from VCD28 cells were incubated with purified Grb2-specific antiserum or with nonspecific Ig covalently linked to CNBr-coupled Sepharose beads. CNBr-coupled beads were used instead of protein A-Sepharose beads so that CD28 could not be immunoprecipitated by the stimulating antibody. Protein complexes present in Grb2 immunoprecipitates were resolved by gel electrophoresis and immunoblotted with CD28 antibodies. Grb2-specific anti-serum co-immunoprecipitated CD28 (Figs. 4, A and B, lanes 1 and 2). Ligation of CD28 increased the amount of CD28 present in Grb2 immune complexes (Fig. 4, A and B, compare lane 2 with lane 1), consistent with the results from Fig. 2.
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Phosphotyrosine-dependent and -independent Association between CD28 and Grb2-- Previous data have suggested that Grb2 may bind to CD28 via its SH2 domain in a phosphotyrosine-dependent manner (29). We have presented evidence that Grb2 binds to the C-terminal diproline motif CD28 via its SH3 domains. To assess the role of tyrosine phosphorylation in the binding of CD28 to Grb2, we expressed GST fusion proteins containing the cytoplasmic domain of CD28 in the BL21(DE3) strain of E. coli. or its derivative, BL21TK, which harbors a plasmid encoding an inducible tyrosine kinase. Recombinant GST-CD28 expressed in the BL21 bacteria (CD28BL) was not tyrosine-phosphorylated, whereas GST-CD28 protein expressed in the BL21TK strain (CD28TK) was quantitatively tyrosine-phosphorylated, as detected by phosphotyrosine-specific antibodies and a shift in electrophoretic mobility (Fig. 5D). GST does not become tyrosine-phosphorylated under these conditions (data not shown). GST-CD28BL or phosphorylated GST-CD28TK fusion proteins were used to coprecipitate transiently expressed Grb2 from 293T cells. Wild-type and mutant Grb2 constructs carrying point mutations in the SH3 or SH2 domains were Myc epitope-tagged to distinguish them from the endogenous Grb2 protein (48). Grb3-3, a Grb2 isoform with a nonfunctional SH2 domain resulting from an internal deletion within the SH2 coding sequence (55), was also used in these experiments. The unphosphorylated GST-CD28BL protein formed a complex with wild type Grb2, the mutant N-terminal SH3 form, and Grb3-3 (Fig. 5A, lanes 1, 2, and 5). These data support the observation that the association of CD28 with Grb2 can occur in the absence of tyrosine phosphorylation and does not require a functional Grb2-SH2 domain. GST-CD28BL did not coprecipitate Grb2 when either the C-terminal or both SH3 domains were mutated, which, in accordance with the data presented in Fig. 2, indicates that the C-terminal SH3 domain of Grb2 is the dominant binding domain to CD28. As shown in Fig. 5C, the coprecipitation of Sos with Grb2 was also SH3-dependent, but in contrast to CD28, the N-terminal SH3 domain rather than the C-terminal SH3 domain defines the high affinity interaction (34).
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Tyrosine-phosphorylated Shc Binds to CD28--
We have shown that
Grb2 forms a protein complex with CD28 via an SH3-proline interaction.
Grb2 may thus function to link CD28 with tyrosine-phosphorylated
proteins present in activated T cells. The SH2 domain of Grb2 can bind
to proteins that contain a common consensus binding site,
pYXNX, where pY represents phosphotyrosine (56).
Tyrosine-phosphorylated proteins present in activated T cells that bind
to the Grb2-SH2 domain include p36LAT, p62/68,
Shc, the -chain of the TCR complex, and SHP-2 (38-40, 57-59). The
multiplicity of Grb2 binding partners suggests that Grb2 may have
distinct and varied functions during T cell activation.
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DISCUSSION |
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We have demonstrated that CD28 is a binding target for a limited number of SH3-containing proteins. The isolated SH3 domains from Itk and Grb2 as well as the WW domain of YAP coprecipitated CD28 from cellular lysates. We have recently shown that the association of Itk with CD28 through the Itk-SH3 domain and the N-terminal diproline motif of CD28 results in the partial activation of the Itk kinase (60). In this report, we have presented data that demonstrate that Grb2 forms an inducible complex with CD28 via its SH3 domains binding to the C-terminal diproline motif of CD28. The preferred binding sites of Itk and Grb2 are therefore distinct and correspond to the two respective diproline motifs present in the cytoplasmic domain of CD28. Binding of Grb2 to CD28 does not require tyrosine phosphorylation; nor does tyrosine phosphorylation preclude Grb2-SH3-mediated interactions with CD28. We have shown that the nonphosphorylated CD28 cytoplasmic domain expressed as a recombinant fusion protein in prokaryotic cells bound Grb2 from cellular lysates in an SH3-specific manner and was independent of the Grb2-SH2 domain. Tyrosine 173 in the cytoplasmic tail of CD28 is imbedded in a motif that has been shown to be the common binding site for both the Grb2 and the C-terminal p85-SH2 domains, although the C-terminal p85-SH2 domain binds to this site with 100-fold greater affinity than does the Grb2-SH2 domain (29). We observed that when GST-CD28 was quantitatively phosphorylated, the isolated Grb2-SH2 domain could bind to CD28 presumably through this site. However, even under these conditions the SH2 domain was not required for binding as evidenced by the capacity of Grb3-3, an isoform of Grb2 lacking a functional SH2 domain, to bind to both the unphosphorylated and phosphorylated forms of CD28. Furthermore, we observed that mAb-mediated ligation of CD28 that was insufficient to induce tyrosine phosphorylation necessary for p85 binding was nonetheless sufficient to induce Grb2 binding. These data suggest that Grb2 can bind to CD28 in two distinct configurations depending on the degree of receptor clustering and the state of CD28-tyrosine phosphorylation. Initial receptor aggregation induces a CD28-Grb2 complex, which is mediated by proline-SH3 interactions. Under conditions where tyrosine 173 is phosphorylated, a second Grb2 binding site that requires the SH2 domain is created. However, CD28 can generate signals to induce IL-2 transcription in the absence of P85-SH2 association to tyrosine 173 (13, 23, 26). It is therefore of considerable interest to identify other regions of the CD28 cytoplasmic domain involved in protein interactions. We have demonstrated that CD28 utilizes proline-rich motifs to recruit SH3-containing proteins, providing an alternate mechanism for the initiation of signaling through CD28.
The Inducibility of Grb2-SH3 Binding to CD28 Correlates with Decreased Binding of Grb2 to Cbl-- The factors that regulate the inducible interaction between Grb2 and CD28 described in this paper are presently unknown. SH3-diproline interactions are generally considered to be constitutive. For example, cytosolic Grb2 is bound to Sos in a preformed heteromeric complex (61). The interaction of Grb2 with Sos can be modulated, however, since serine/threonine phosphorylation of Sos diminishes Grb2 binding (62), whereas engagement of the Grb2-SH2 domain by a phosphopeptide can enhance the association between Grb2 and Sos (63).
The enhanced binding of CD28 to SH3-containing proteins such as Grb2 following ligation of the receptor could be a result of phosphorylation, allosteric changes in the cytoplasmic domain, or the release of another protein that blocks the interaction between CD28 and Grb2. We observed that tyrosine phosphorylation of GST-CD28 did not enhance the binding of Grb3-3 to the cytoplasmic tail of CD28, suggesting that tyrosine phosphorylation does not alter this association. Threonine phosphorylation of CD28 following phorbol 12-myristate 13-acetate treatment (64) also did not affect the binding of Grb2 to CD28 (data not shown). There is currently no evidence for a constitutive interaction between CD28 and another molecule that could block the binding of CD28 to Grb2. We therefore support the possibility that an allosteric modification of the intracellular domain of CD28 exposes the diproline motif to SH3 domains following ligation of the receptor. We provide evidence that CD28 ligation results in the redistribution of Grb2 within intracellular protein pools. The inducible interaction between Grb2 and CD28 corresponds to a concomitant decline in the amount of Cbl coimmunoprecipitated with Grb2. Cbl has previously been shown to bind to Grb2 through SH3-dependent interaction (43). These results raise the possibility that CD28 and Cbl compete for limited access to SH3 domains of Grb2 and that these two proteins bind Grb2 in a mutually exclusive manner. Cbl functions as a suppressor of FcPotential SH2 Binding Targets of the CD28-associated Grb2-- The structural requirements for CD28-mediated costimulation are controversial. In some cellular systems, mutation of the PI3K binding site at 173 abrogates CD28-dependent IL-2 production, while in other systems it does not. The cytoplasmic tail of CD28 contains four conserved tyrosine residues, which, when all are mutated to phenylalanine (ALL F mutant), impairs signaling. Reconstitution of PI3K binding by a single add-back mutation at Tyr173 in the ALL F mutant is insufficient to reconstitute costimulation (26). Add-back of tyrosine 191 within the motif PY191APPR that mediates binding of the SH3 domains of Grb2 to CD28 is sufficient to completely reconstitute CD28-dependent IL-2 production. Furthermore, deletion of the C-terminal 10 or 17 amino acids of CD28, which disrupts or deletes this diproline motif, profoundly impairs IL-2 production, whereas a seven-amino acid deletion, which leaves this motif intact, leads to enhanced costimulation (26, 27). This region has more recently been shown to be required for the CD28-dependent tyrosine phosphorylation of the GTPase-activating protein-associated p62 protein (p62DOK) (68). We have demonstrated that a 10-amino acid C-terminal truncation mutant of CD28 no longer binds to Grb2 (Fig. 3D). Thus, a limited peptide sequence, which includes the proposed Grb2-SH3 binding site, is required for costimulation.
We have shown that the unphosphorylated form of bacterially expressed CD28 can bind to both Grb2 and to the phosphorylated form of Shc derived from activated T cell lysates. We propose that CD28-bound Grb2 links phosphorylated Shc to the CD28 cytoplasmic domain. The role of Shc remains elusive in T cell receptor signaling. Grb2-Sos binds to phosphorylated Shc at tyrosine 317 and thereby stimulates Ras activation in response to growth factor stimulation (69, 70). Cross-linking of TCR and CD4 was observed to induce phosphorylation of both the 48- and 52-kDa isoforms of Shc (57). Grb2 has been detected in the phosphorylated Shc complexes in T cells. Shc has also been observed to bind to phosphorylatedCreation of the Signaling Patch: Recruitment of CD28 into the
TCR-CD3 Complex--
The Grb2-SH2 domain can bind to the doubly
phosphorylated -immunereceptor tyrosine-based activation motif,
although this interaction is of lower affinity than that between Zap-70
and the
-chain (71). No Sos was detected in
-immunereceptor
tyrosine-based activation motif precipitates, suggesting that the
stoichiometry of this interaction is low or that Grb2 may be bound to a
protein distinct from Sos (71). Stimulation of T cell clones by
alloantigen and B7 on an APC induces a physical association between
CD28 and the phospho-
-chain (74). This result is consistent with the observation that optimal costimulation occurs when both antigen and B7
are expressed on the same APC, which would allow this complex to form
(75, 76). Therefore, CD28 may be brought into the TCR-CD3 complex
through a Grb2 bridge linking CD28 via its SH3 domains and the
-chain by way of its SH2 domain. Alternatively, this bridge may be
formed through Shc, which may bind the
-chain through its SH2 domain
(57) and which in the phosphorylated state binds the SH2 domain of Grb2
(69). This would avail CD28 to Src family kinases associated with the
TCR complex including Lck and Fyn, which may be required for activating
events such as the phosphorylation of tyrosine 173 within the PI3K
binding site (77) and/or phosphorylation of the CD28-associated kinase Itk, a step required for its full activation (78). The formation of a
multimeric protein complex composed of TCR, CD3, and CD28 within the
contact patch between the T cell and the APC raises the possibility
that integration between the TCR and CD28 signaling may occur near the
plasma membrane.
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ACKNOWLEDGEMENTS |
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We thank Jose LaRose for technical help, Dominique Couez, Gary Koretzky, Peter Linsley, Michael Moran, David Pot, Bruno Tocqué, and Gerrard Zurawski for generously sharing reagents used in this study. We thank Mina Marmor, Philippe Poussier, and Michael Julius for critical reading of this manuscript.
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FOOTNOTES |
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* This work was supported by the Medical Research Council of Canada and the Arthritis Society of Canada.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.
A senior research scientist of the Arthritis Society of
Canada. To whom correspondence should be addressed: Experimental
Therapeutics, Ontario Cancer Institute, Princess Margaret Hospital, 610 University Ave., Toronto, Ontario M5G 2M9, Canada. Tel.: 416-946-2233;
Fax: 416-946-2984; E-mail: Rottapel{at}oci.utoronto.ca.
The abbreviations used are:
TCR, T cell
receptor; IL-2, interleukin 2; PI3K, phosphatidylinositol 3-kinase; mAb, monoclonal antibody; GST, glutathione S-transferasePLC, phospholipase C
PAGE, polyacrylamide gel electrophoresisPBS, phosphate-buffered salineSH2 and SH3, Src homology 2 and 3, respectively.
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REFERENCES |
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