Regulation of Cytotoxic T Lymphocyte-Associated Molecule-4 by Src Kinases

Ellen Chuang, Kyung-Mi Lee, Michael D. Robbins, James M. Duerr, Maria-Luisa Alegre, John E. Hambor, Mark J. Neveu, Jeffrey A. Bluestone and Craig B. Thompson
J Immunol February 1, 1999, 162 (3) 1270-1277;

Abstract

Cytotoxic T lymphocyte-associated molecule-4 (CTLA-4) is a cell surface receptor expressed on activated T cells that can inhibit T cell responses induced by activation of the TCR and CD28. Studies with phosphorylated peptides based on the CTLA-4 intracellular domain have suggested that tyrosine phosphorylation of CTLA-4 may regulate its interactions with cytoplasmic proteins that could determine its intracellular trafficking and/or signal transduction. However, the kinase(s) that phosphorylate CTLA-4 remain uncharacterized. In this report, we show that CTLA-4 can associate with the Src kinases Fyn and Lck and that transfection of Fyn or Lck, but not the unrelated kinase ZAP70, can induce tyrosine phosphorylation of CTLA-4 on residues Y201 and Y218. A similar pattern of tyrosine phosphorylation was found in pervanadate-treated Jurkat T cells stably expressing CTLA-4. Phosphorylation of CTLA-4 Y201 in Jurkat cells correlated with cell surface accumulation of CTLA-4. CTLA-4 phosphorylation induced the association of CTLA-4 with the tyrosine phosphatase SHP-2, but not with phosphatidylinositol 3-kinase. In contrast, Lck-induced phosphorylation of CD28 resulted in the recruitment of phosphatidylinositol 3-kinase, but not SHP-2. These findings suggest that phosphorylation of CD28 and CTLA-4 by Lck activates distinct intracellular signaling pathways. The association of CTLA-4 with Src kinases and with SHP-2 results in the formation of a CTLA-4 complex with the potential to regulate T cell activation.

Activation of the T cell is initiated by interactions between the TCR on T cells with peptide bound to MHC on APCs. However, interactions between accessory molecules on T cells with their ligands on APCs can play critical roles in determining the T cell response that occurs following TCR stimulation. CD28 and cytotoxic T lymphocyte-associated molecule-4 (CTLA-4)4 are structurally related receptors on T cells that bind either of two related ligands, B7-1 and B7-2, on APCs. Whereas CD28 can provide important costimulatory signals for T cell activation, the majority of evidence suggests that CTLA-4 functions to down-regulate T cell function. A negative role for CTLA-4 in regulating T cell activation has been shown in murine models of tumor immunity (1, 2) and autoimmune disease (3, 4, 5) and is best demonstrated by the phenotype of CTLA-4-deficient mice, which exhibit severe lymphoproliferative disease and early death at 3–4 wk of age (6, 7).

CTLA-4-mediated inhibition of T cell activation may be accomplished by competition with CD28 for binding to B7 ligands. CTLA-4 may also interfere with TCR and/or CD28-initiated intracellular signaling pathways. In support of this, ligation of CTLA-4 on the surface of T cells has been shown to inhibit TCR/CD28-mediated IL-2 production and to delay cell cycle progression (8, 9), and biochemical studies have demonstrated that CTLA-4 ligation inhibits TCR- and CD28-mediated ERK and JNK activation (10). In addition, T cells from CTLA-4-deficient mice exhibit hyperphosphorylation of the CD3ζ chain as well as increased activity of the TCR-associated kinases Fyn, Lck, and ZAP70 (11), suggesting that CTLA-4 may also regulate early events associated with TCR activation.

The cytoplasmic domain of CTLA-4 contains 36 amino acid residues, with tyrosines present at positions 201 and 218. It has been suggested that phosphorylation of Y201 may regulate interactions between CTLA-4 and potential cytoplasmic signaling molecules. The sequence surrounding Y201 forms a consensus binding motif for the SH2 domains of the p85-regulatory subunit of phosphatidylinositol 3-kinase (PI(3)K) (12). Lipid kinase activity has been reported to coprecipitate with CTLA-4 in T cells (13), and phosphorylated peptides containing the Y201 motif have been demonstrated to bind to PI(3)K with high affinity (13, 14). CTLA-4 has also been shown to coimmunoprecipitate with the SH2 domain-containing tyrosine phosphatase SHP-2, and peptide binding studies have suggested that this association is also mediated by the phosphorylated Y201 motif of CTLA-4 (11).

In addition to PI(3)K and SHP-2, CTLA-4 Y201 has been demonstrated to associate with the medium chain of the clathrin-associated adaptor complex AP-2 (14, 15, 16, 17). This interaction mediates internalization of CTLA-4 and provides a mechanism for regulating CTLA-4 cell surface expression. In contrast to the association with SHP-2 and PI(3)K, the association of CTLA-4 with AP-2 does not require tyrosine phosphorylation. In vitro studies with peptides corresponding to the cytoplasmic domain of CTLA-4 have suggested that tyrosine phosphorylation of Y201 inhibits the association of CTLA-4 with AP-2 (14, 15, 17). Although CTLA-4 tyrosine phosphorylation has been difficult to demonstrate in vivo, a low level of phosphorylation was reported to be detected in primary T cells treated with pervanadate (15). Taken together, these studies suggest that CTLA-4 Y201 may play a critical role in the regulation of CTLA-4-mediated signal transduction by first preventing the internalization of CTLA-4 from the plasma membrane and then creating a binding site for SH2 domain-containing molecules in the CTLA-4 present on the cell surface.

In the present study, we have addressed the role of tyrosine phosphorylation in regulating CTLA-4 intracellular trafficking and signal transduction. We have found that CTLA-4 can be phosphorylated by the tyrosine kinases Lck and Fyn, but not by ZAP70. Tyrosine phosphorylation was found to regulate CTLA-4 cell surface expression. CTLA-4 was found to associate with Lck and Fyn, and Lck coexpression induced the association of CTLA-4 with SHP-2. The formation of a CTLA-4 signaling complex which includes Src kinases and SHP-2 could provide a molecular basis for the regulation of T cell activation by CTLA-4.

Materials and Methods

Abs and other reagents

The anti-CD3 145-2C11 and anti-CTLA-4 4F10 mAbs have been described previously (18). Phycoerythrin (PE)-conjugated anti-CTLA-4 and hamster IgG anti-trinitrophenol were purchased from PharMingen (San Diego, CA). Mouse monoclonal anti-phosphotyrosine (4G10), rabbit polyclonal anti-PI(3)K, and rabbit polyclonal anti-Lck Abs were purchased from Upstate Biotechnology (Lake Placid, NY). Anti-SHP-2 antiserum (19) was kindly provided by Dr. G.-S. Feng (Indiana University Medical Center, Indianapolis, IN). Goat polyclonal anti-CTLA-4 Ab to the CTLA-4 extracellular domain used for Western blotting and goat polyclonal anti-CD28 Abs to the murine CD28 extracellular and intracellular domains were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit polyclonal anti-Fyn Ab was purchased from Transduction Laboratories (Lexington, KY).

The rabbit polyclonal Abs recognizing phospho-Y201 CTLA-4 or phospho-Y218 CTLA-4 were generated using as immunogens either KLH-PLTTGVpYVKMPPT or KLH-EKQFQPpYFIPIN.

Cell lines, cell culture, and transfections

The human T cell leukemia cell line J32 Jurkat (20) was kindly provided by Dr. C. June (Naval Medical Center, Bethesda, MD). Plasmids were introduced by electroporation of 20 μg of plasmid DNA using the Bio-Rad (Hercules, CA) Gene Pulser set at 250 V and 960 μF capacitance. Transfectants were selected by growth in 0.3 mg/ml G418. Single-cell subclones were obtained by the limiting dilution method, and CTLA-4 surface and intracellular expression was determined by flow cytometry. Transient transfections of the human embryonic kidney cell line HEK293 were performed according to standard calcium phosphate methods using 2.5–5 μg of CTLA-4 plasmid DNA and 2.5 μg of Lck plasmid DNA unless otherwise indicated, and cells used for experiments were harvested at 36–40 h after transfection. Cell lines were maintained in either RPMI (Jurkat) or DMEM (HEK 293) media supplemented with 10% FCS, 2–4 mM glutamine, 50 mM HEPES (pH 7.5), 100 U/ml penicillin, and 100 μg/ml streptomycin. Pervanadate treatment of cells was performed by combining 10 mM or 1 mM Na3VO4 with 10 mM H2O2 in PBS for 20 min. Cells were treated for 15 min at 37°C. The OVA-reactive T cell clone used in this experiment has been described previously (21) and was kindly provided by Dr. T. Gajewski (University of Chicago, Chicago, IL). Cells (5 × 106) were stimulated with irradiated splenocytes, 2 μM OVA, and rIL-2 (10 U/ml) for 2 days. Cells were passed over a Ficoll density gradient before lysis for immunoprecipitations.

Plasmids

The mammalian expression plasmids encoding murine CTLA-4 and murine CD28 were constructed as previously described (16). Point mutations were generated using the Chameleon mutagenesis system (Stratagene, La Jolla, CA), and mutations were confirmed by DNA sequencing. The CTLA-4Δtail plasmid was constructed using mutagenesis to create an XhoI site distal to the transmembrane region and subcloning into the pcDNA3 plasmid (Invitrogen, San Diego, CA). The CTLA-4Δtail plasmid encodes the CTLA-4 amino acids 1–189. The expression plasmids encoding ZAP70 and Fyn (22) were generously provided by Dr. L. Samelson (National Institute of Child Health and Human Development, Bethesda, MD). The pEF plasmid encoding wild-type (WT) Lck was constructed by cloning the murine Lck cDNA into a derivative of the pEF-BOS expression vector (23) and was kindly provided by Dr. D. Straus (University of Chicago).

Immunoprecipitations and Western blotting

HEK 293 cells (5–10 × 106) or Jurkat T cells (30–40 × 106) were lysed in buffer containing 150 mM NaCl, 50 mM HEPES (pH 7.5), 1 mM EDTA, 1% Nonidet P-40, 10% glycerol, 10 mM NaF, 1 mM Na3VO4, 1 mM PMSF, 8 μg/ml aprotinin, and 2 μg/ml leupeptin. For experiments using the T cell clone, 25 × 106 cells were used for each immunoprecipitation. Lysates were precleared by incubating with protein G-Sepharose for 1 h, and immunoprecipitations were performed by the addition of 5 μg of anti-CTLA-4 4F10 Ab or control hamster Ig and protein G-Sepharose for 3–16 h at 4°C. The precipitates were separated by SDS-PAGE, transferred onto nitrocellulose, and Western blotted with the indicated Abs. Results were visualized using enhanced chemiluminescence (Amersham, Arlington Heights, IL). Where indicated, the blots were stripped with 1% SDS and 100 mM 2-ME in PBS for 30 min at 55°C and reprobed using the indicated Abs.

For the in vitro phosphorylation assay, 1 μg of bacterially expressed glutathione S-transferase (GST) or GST-CTLA-4c, a fusion protein containing the cytoplasmic domain of CTLA-4 fused to GST, was incubated with 0.5 μg of baculovirus-expressed Lck in PBS with 24 mM MgCl2 and 10 μM ATP for 20 min at 37°C. The reaction was electrophoresed using SDS-PAGE and tyrosine phosphorylation detected by Western blotting with an anti-phosphotyrosine Ab.

Flow cytometry

Cells were washed with PBS containing 1% BSA and 0.1% sodium azide and incubated with PE-conjugated anti-CTLA-4 or control hamster Ig for 30 min at 4°C or fixed with 1% paraformaldehyde and permeabilized with 0.3% saponin before incubation with the Abs for intracellular staining. Cells were analyzed on a FACSort flow cytometer (Becton Dickinson Immunocytometry Systems, Mountain View, CA) using Cellquest software.

Results

Cell surface expression and tyrosine phosphorylation of CTLA-4 in pervanadate-treated Jurkat T cells

Phosphorylation of Y201 has been proposed to negatively regulate the interaction between the CTLA-4 internalization motif and clathrin-association adaptor complex proteins (14, 15, 17). To analyze the role of tyrosine-phosphorylated Y201 in CTLA-4 cell surface expression, Jurkat T cells were stably transfected with full length (WT) CTLA-4 or a CTLA-4 mutant in which a valine substitution is present at position 201 (CTLA-4 Y201V). The effect of the tyrosine phosphatase inhibitor pervanadate on CTLA-4 cell surface expression was assessed. As shown in Fig. 1⇓A, the majority of WT CTLA-4 protein was detected intracellularly, whereas significant amounts of the CTLA-4 Y201V mutant protein accumulated on the cell surface. This is consistent with our previous findings that the CTLA-4 Y201V mutant protein interacts poorly with AP-2 and is internalized less rapidly than the WT molecules (16). As shown in Fig. 1⇓B, treatment of the cells with pervanadate resulted in a dose-dependent enhancement of WT CTLA-4, but not CTLA-4 Y201V cell surface expression. The increase in cell surface expression correlated with pervanadate-induced tyrosine phosphorylation of WT CTLA-4 (Fig. 1⇓C). In contrast, tyrosine phosphorylation of the CTLA-4 Y201V mutant was significantly reduced, and pervanadate treatment had little effect on its cell surface expression. These results are consistent with a role for phosphorylation of Y201 in the regulation of CTLA-4 intracellular trafficking. In the unphosphorylated state, Y201 of WT CTLA-4 interacts with AP-2, and CTLA-4 is rapidly internalized from the cell surface. Upon its phosphorylation, the affinity of Y201 for AP-2 is diminished, resulting in the accumulation of CTLA-4 on the cell surface. In the CTLA-4 Y201V mutant, the AP-2 binding site has been mutated, and its cell surface expression is not regulated by clathrin-mediated endocytosis or by tyrosine phosphorylation. As a result, CTLA-4 Y201V is constitutively expressed at high levels on the cell surface.

FIGURE 1.
FIGURE 1.

Pervanadate treatment increases CTLA-4 cell surface expression and induces CTLA-4 tyrosine phosphorylation in Jurkat T cell transfectants. A, Human Jurkat T cells were transfected with expression plasmids encoding either WT murine CTLA-4 or a mutant containing a valine substitution at Y201 (CTLA-4 Y201V). Single-cell subclones were obtained and stained with anti-CTLA-4-PE to visualize cell surface CTLA-4 or permeabilized with saponin and stained with anti-CTLA-4-PE to visualize intracellular CTLA-4. Cells were analyzed by flow cytometry, and CTLA-4 fluorescence is indicated by the unshaded histograms. The shaded histograms represent results for cells transfected with the parental vector alone and stained with anti-CTLA-4-PE as above. CTLA-4 expression of one representative subclone of each construct is shown. B, The CTLA-4-expressing Jurkat T cells from A were treated with either 0.1 mM or 1 mM pervanadate for 15 min and stained with anti-CTLA-4-PE for cell surface CTLA-4 expression. The results are representative of experiments performed on two independent subclones each of CTLA-4 WT and CTLA-4 Y201V transfectants. C, Jurkat T cells expressing CTLA-4 WT, CTLA-4 Y201V, or vector control-transfected cells were treated or not treated with 0.5 mM pervanadate and lysed in lysis buffer. Lysates were equalized for total protein content and CTLA-4 was immunoprecipitated by the addition of the anti-CTLA-4 Ab 4F10. Immunoprecipitates were separated by SDS-PAGE and phosphoproteins detected by Western blotting using the anti-phosphotyrosine Ab 4G10. The location of CTLA-4 is indicated. ∗, nonspecific tyrosine-phosphorylated proteins that are also present in the control transfectants.

Interestingly, a low level of tyrosine phosphorylation of the CTLA-4 Y201V mutant remained detectable following pervanadate treatment, suggesting that the other tyrosine, Y218, becomes phosphorylated as well.

CTLA-4 phosphorylation by Src kinases

The above findings suggested that tyrosine phosphorylation of CTLA-4 can be a mechanism for regulating its cell surface expression. To identify tyrosine kinases that potentially phosphorylate CTLA-4 in vivo, CTLA-4 was transiently expressed in HEK293 cells together with candidate tyrosine kinases, and the presence of CTLA-4 tyrosine phosphorylation was determined. As candidate kinases, the Syk family kinase ZAP70 and the Src family kinases Lck and Fyn were chosen because of their activity at sites of MHC/TCR interactions. These are sites to where CTLA-4 may be colocalized through interactions with B7 molecules. As shown in Fig. 2⇓A, coexpression of CTLA-4 with Lck or Fyn in HEK293 cells resulted in the tyrosine phosphorylation of CTLA-4, whereas coexpression of ZAP70 did not. Since optimal activation of ZAP70 requires its phosphorylation by Src kinases, Fyn and ZAP70 were coexpressed together with CTLA-4. However, this did not lead to phosphorylation of CTLA-4 above the levels achieved by expression of Fyn alone, despite the presence of ZAP70 activity as assessed by the detection of increased tyrosine phosphorylation of proteins with molecular masses of 70 to 100 kDa in cells that express both ZAP70 and Fyn (Fig. 2⇓B). Furthermore, the lack of an additive effect of ZAP70 in Fyn-expressing cells was not due to saturation of CTLA-4 tyrosine phosphorylation, since increasing the quantity of transfected DNA encoding Fyn alone resulted in the detection of increased CTLA-4 phosphorylation (Fig. 2⇓A). A similar increase was seen when the quantity of Lck cDNA transfected into cells was increased (data not shown). The above findings indicate that Fyn and Lck, but not ZAP70, induce the tyrosine phosphorylation of CTLA-4. To determine whether CTLA-4 is a direct substrate for Src kinases, a fusion protein containing the cytoplasmic domain of CTLA-4 fused to GST (GST-CTLA-4c) was combined with recombinant Lck protein in an in vitro phosphorylation assay. As shown in Fig. 3⇓, incubation of Lck with GST proteins results in the tyrosine phosphorylation of GST-CTLA-4c, but not GST. The results demonstrate that Lck can directly phosphorylate CTLA-4 and that phosphorylation occurs within the cytoplasmic domain of CTLA-4.

FIGURE 2.
FIGURE 2.

Lck and Fyn, but not ZAP70, induce tyrosine phosphorylation of CTLA-4 in the cell line HEK293. A, HEK293 cells were transiently transfected with a CTLA-4 expression plasmid encoding CTLA-4 together with plasmids encoding either ZAP70, Fyn, ZAP70 plus Fyn, or Lck using the indicated amounts of DNA (micrograms). Cells were harvested 36 h following transfection. CTLA-4 was immunoprecipitated using the anti-CTLA-4 Ab 4F10. Immunoprecipitates were separated by SDS-PAGE, and Western blotting was performed using the anti-phosphotyrosine Ab 4G10. B, Cell lysates obtained in A were analyzed directly for the presence of tyrosine-phosphorylated proteins.

FIGURE 3.
FIGURE 3.

Lck induces tyrosine phosphorylation of the cytoplasmic domain of CTLA-4 in vitro. GST or a GST fusion protein containing the cytoplasmic domain of CTLA-4 (GST-CTLA-4c) was combined with Lck in the presence of 10 μM ATP. Reactions were incubated for 20 min at 37°C, electrophoresed on SDS-PAGE, and Western blotted with Abs to phosphotyrosine or to GST.

Phosphorylation of CTLA-4 occurs on both Y201 and Y218

The cytoplasmic domain of CTLA-4 contains two tyrosines. To determine which tyrosine was phosphorylated by Src kinases, mutagenesis of Y201 or Y218 was performed, and the effect of these mutations on CTLA-4 phosphorylation by Lck was analyzed. As shown in Fig. 4⇓, substitution of phenylalanine for Y201 significantly diminished the amount of phosphorylation detected. Substitution of phenylalanine for Y218 also diminished the observed levels of tyrosine phosphorylation. These findings suggest that both tyrosines are phosphorylated by Lck. To directly confirm the presence of phosphorylated Y201 or Y218, Abs recognizing these epitopes were generated. Western blotting using these phosphospecific Abs confirmed that in the WT CTLA-4 molecule, both Y201 and Y218 were phosphorylated by Lck. The specificity of these Abs is confirmed by the specific detection of mutant CTLA-4 with these Abs. These findings demonstrate that both Y201 and Y218 are substrates for the tyrosine kinase activity of Lck. Due to the different affinities of the anti-phosphotyrosine Abs for their epitopes, however, the relative contribution of the individual phosphorylated tyrosine residues to the total tyrosine phosphorylation detected in the WT CTLA-4 molecule cannot be determined.

FIGURE 4.
FIGURE 4.

Phosphorylation of CTLA-4 by Lck occurs on both Y201 and Y218. HEK293 Cells were transiently transfected with plasmids encoding WT CTLA-4 (CTLA-4 WT), a CTLA-4 mutant lacking the CTLA-4 cytoplasmic domain (CTLA-4 Δtail), or CTLA-4 mutants containing a phenylalanine substitution at either Y201 (CTLA-4 Y201F) or Y218 (CTLA-4 Y218F). Cells cotransfected with the plasmid encoding Lck are indicated. The cells were processed as described in Fig. 2⇑, and Western blotting was performed using either the anti-phosphotyrosine Ab 4G10 (top), an Ab specific for CTLA-4 phosphorylated on Y201 (second panel), an Ab specific for CTLA-4 phosphorylated on Y218 (third panel), or an anti-CTLA-4 Ab (bottom panel). The same blot was successively stripped and reprobed with the indicated Abs.

Association of CTLA-4 with Lck

The above studies demonstrated that Src kinases can induce the phosphorylation of CTLA-4. To determine whether CTLA-4 could also associate with Src kinases, the Western blot shown in Fig. 4⇑ was reprobed with an anti-Lck Ab (Fig. 5⇓A). Lck was found to coimmunoprecipitate with CTLA-4. Mutation of either Y201 or Y218 did not reduce the amount of Lck detected, suggesting that these residues were not required for binding to Lck. To confirm the association of CTLA-4 with Lck found in HEK293 cells, CTLA-4 was immunoprecipitated from Jurkat cells that were stably transfected with either WT CTLA-4 or the CTLA-4 Y201V mutant. Western blotting using an anti-Lck Ab indicated that Lck coimmunoprecipitated with CTLA-4 (Fig. 5⇓B), and the amount of Lck precipitated correlated with the amount of CTLA-4 present (Fig. 5⇓B, top and bottom left panels). Finally, to determine whether CTLA-4 and Lck associations could be observed in nontransfected T cells, an activated T cell clone expressing CTLA-4 was investigated for CTLA-4/Lck interactions. As shown in Fig. 5⇓C, CTLA-4 immunoprecipitates were found to contain Lck.

FIGURE 5.
FIGURE 5.

CTLA-4 associates with Lck. (A) The Western blot shown in Fig. 4⇑ was reprobed with an Ab to Lck. B, CTLA-4 was immunoprecipitated from lysates prepared from Jurkat cells stably transfected with either CTLA-4 WT or CTLA-4 Y201V or from cells transfected with the control plasmid (Neo). Immunoprecipitates were separated by SDS-PAGE and Western blotting was performed using an anti-Lck Ab (upper left). The blot was stripped and reprobed with an Ab to CTLA-4 (lower left). The amount of lysate shown represents 1% of the amount used for immunoprecipitations (upper right). C, Cells from a T cell clone were stimulated with peptide-loaded APCs for 2 days. Cells were passed over a Ficoll gradient and incubated in lysis buffer. Immunoprecipitations were performed using either the anti-CTLA-4 Ab 4F10 or a control hamster Ig (H Ig). Western blotting was performed with either an anti-Lck Ab (top) or an anti-CTLA-4 Ab (bottom). The amount of lysate shown represents 2.5% of the amount used for immunoprecipitations. The sensitivity of the anti-CTLA-4 Ab is insufficient to detect CTLA-4 present within the lysate sample.

Association of CTLA-4 with PI(3)K and SHP-2

It has been proposed that tyrosine phosphorylation of CTLA-4 may create a binding site for SH2 domain-containing proteins. Peptide-based binding studies have suggested that the association of CTLA-4 with PI(3)K and SHP-2 occurs via the SH2 domains of these molecules with the phosphorylated Y (201)VKM motif contained within the cytoplasmic domain of CTLA-4. To determine whether the tyrosine-phosphorylated full length CTLA-4 molecule could associate with PI(3)K or SHP-2, the coprecipitation of endogenous PI(3)K or SHP-2 with CTLA-4 was analyzed. Phosphorylation of CTLA-4 by Lck did not increase binding of PI(3)K over the basal level (Fig. 6⇓A, top). However, coexpression of Lck with CTLA-4 induced binding of SHP-2 to CTLA-4 (Fig. 6⇓A, bottom). In contrast, Lck-induced phosphorylation of CD28 recruited significant amounts of PI(3)K but did not recruit SHP-2. Interestingly, a 60–69-kDa tyrosine-phosphorylated protein was found to coimmunoprecipitate with CTLA-4 under conditions of Lck coexpression (Fig. 6⇓B, left). Stripping and reprobing of this blot with an Ab to SHP-2 demonstrated that SHP-2 comigrates with this tyrosine-phosphorylated protein (Fig. 6⇓B, right). These results confirm that Lck enhances the formation of a complex between CTLA-4 and SHP-2. Furthermore, under these conditions a subset of the SHP-2 which is coassociated with CTLA-4 is tyrosine phosphorylated.

FIGURE 6.
FIGURE 6.

Association of CTLA-4 with PI(3)K and SHP-2. A, HEK293 cells were transiently transfected with plasmids encoding CD28 or CTLA-4 or with control plasmids. The Lck plasmid was cotransfected as indicated. Cells were processed as described in Fig. 2⇑ and subjected to CD28 immunoprecipitation or CTLA-4 immunoprecipitation. Western blotting was performed using an anti-PI(3)K Ab (top). The same blot was reprobed with an anti-SHP-2 Ab (bottom). B, CTLA-4 immunoprecipitates were prepared from HEK293 cells transiently transfected with CTLA-4 plasmid with or without Lck plasmid. Tyrosine-phosphorylated proteins were visualized by Western blotting using an anti-phosphotyrosine Ab (4G10) (left). The predicted positions of SHP-2, Lck, and CTLA-4 are indicated. The blot was stripped and reprobed with an Ab against SHP-2 (right).

Coimmunoprecipitation of CTLA-4 with Fyn

Both Lck and Fyn can induce the phosphorylation of CTLA-4. To determine whether, like Lck, Fyn could also associate with CTLA-4, Fyn and CTLA-4 were transiently expressed in HEK293 cells, and the presence of Fyn in CTLA-4 immunoprecipitates was determined. In some samples, ZAP70 was coexpressed to assess the effects of ZAP70 expression and kinase activity on potential CTLA-4/Fyn associations. As shown in Fig. 7⇓, Fyn coimmunoprecipitates with CTLA-4, and this association was unaffected by ZAP70 coexpression.

FIGURE 7.
FIGURE 7.

Coimmunoprecipitation of Fyn with CTLA-4. CTLA-4, Fyn, and ZAP70 were coexpressed in HEK293 cells in the indicated combinations. CTLA-4 immunoprecipitates were prepared as in Fig. 2⇑. Immunoprecipitates and lysate samples representing 5% of the total protein used for immunoprecipitations were electrophoresed using SDS-PAGE. Western blotting was performed using an antiserum to Fyn (top and middle) or an anti-CTLA-4 Ab (bottom). ∗, Ig heavy chain.

Discussion

Tyrosine phosphorylation of CTLA-4 is a potential mechanism by which CTLA-4 intracellular trafficking and CTLA-4 signal transduction can be regulated. In this study, we have identified the kinases that can phosphorylate CTLA-4 and investigated the role of CTLA-4 phosphorylation in regulating its cell surface expression and its ability to form complexes with signaling molecules. We have found that Lck and Fyn, but not ZAP70, can phosphorylate CTLA-4 on tyrosine residues. Phosphorylation of Y201 correlated with accumulation of CTLA-4 on the cell surface. Lck induced the formation of a CTLA-4 complex which included Lck and SHP-2, and a subset of the coassociated SHP-2 was tyrosine phosphorylated.

Both ZAP70 and Lck are critical elements for TCR-initiated signal transduction (24, 25, 26). Whereas ZAP70 appears to function primarily within the TCR signaling pathway, the Src kinases have been implicated in the regulation of multiple T cell activation-signaling pathways, including those initiated by costimulatory molecules, adhesion molecules, and cytokine receptors (27). Our findings that Lck both associates with and phosphorylates CTLA-4 suggest that Lck is a strong candidate to phosphorylate CTLA-4 in vivo. However, Lck may not be the only kinase that can perform this function, as Fyn appears to share an ability to associate with and phosphorylate CTLA-4.

Using mutagenesis as well as Abs that specifically detect phosphorylated Y201 or Y218, we have shown that phosphorylation of CTLA-4 by Lck occurs on both tyrosines. These findings are also consistent with a recent report in which both tyrosines of CTLA-4 were required for optimal phosphorylation by Lck in transfected COS cells (17). Furthermore, treatment of CTLA-4-expressing Jurkat T cells with pervanadate resulted in tyrosine phosphorylation of both WT CTLA-4 and a CTLA-4 Y201 mutant, suggesting that phosphorylation of Y218 also occurs in cells that have not been transfected with tyrosine kinases. Although the role of Y201 in regulating CTLA-4 internalization and signal transduction is becoming better understood, the role of Y218 in CTLA-4 biology is not known at the present; additional studies will be directed at determining the function of Y218 in CTLA-4 signal transduction.

The enzymatic and physical association of Lck with CTLA-4 could have important functional consequences for T cell activation since Lck can participate in the signal transduction pathways of the TCR and CD28, the two receptors that CTLA-4 has been proposed to regulate. In TCR signaling, phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAMs) of the CD3 complex by Lck results in the recruitment and activation of ZAP70 (24). Within the CD28 signaling pathway, Lck has been shown to be required for activation-induced tyrosine phosphorylation of CD28 (28). Phosphorylation of CD28 by Lck has been shown to recruit PI(3)K and growth factor receptor-bound protein Grb-2 to CD28 (29) and may also regulate some of the tyrosine phosphorylation events seen in response to CD28 ligation such as phosphorylation of ITK (30), Vav, and the rasGAP-associated protein p62dok (31, 32). We have found that CTLA-4 associates with Lck when CTLA-4 is transiently expressed in HEK293 cells, when CTLA-4 is stably expressed in Jurkat T cells, and when CTLA-4 is induced in a T cell clone. This association does not appear to be mediated by the Lck SH2 domain, as mutation of individual CTLA-4 tyrosines does not reduce binding. CTLA-4 contains proline-rich sequences in its cytoplasmic domain which could interact with the SH3 domain of Lck. Association of CTLA-4 with the Lck SH3 domain could facilitate phosphorylation of CTLA-4, resulting in the creation of a binding site for SHP-2 and/or other signaling molecules.

We have found that CTLA-4 is associated at low levels with endogenous SHP-2 and that this association was enhanced by the coexpression of Lck. Unlike the tyrosine phosphatase SHP-1, which is expressed predominantly in cells of hemopoietic origin and can negatively regulate activation of the B cell receptor (33), TCR (34, 35), and NK inhibitory receptor (36, 37), the function of the ubiquitously expressed SHP-2 in cells of the immune system has been less well characterized. In fibroblasts, activation of the PDGF receptor induces the recruitment and phosphorylation of SHP-2. Phosphorylation of SHP-2 creates a Grb-2-SOS docking site, leading to activation of the Ras pathway and enhanced cell proliferation (38, 39, 40). In T cells, both positive and negative roles for SHP-2 have been suggested. SHP-2 has been proposed to negatively regulate TCR-mediated activation in primary T cells (11). In contrast, expression of a dominant negative SHP-2 inhibited TCR activation in Jurkat cells (41), suggesting that SHP-2 may have a positive regulatory role in T cell activation. Previous reports addressing the molecular basis of CTLA-4 activation have suggested that the inhibition of TCR signal transduction by CTLA-4 may occur at the level of Ras activation (10, 11). Although SHP-2 has been closely linked to the Ras pathway, under most circumstances it is thought to activate, not inhibit this pathway. Until the role of SHP-2 in T cell signaling is better clarified, it is difficult to speculate how association of CTLA-4 with SHP-2 results in the inhibition of T cell proliferation.

Both CTLA-4 and CD28 contain consensus sequences for binding to the SH2 domains of the p85 subunit of PI(3)K, and it has previously been demonstrated that phosphorylated peptides based on either the CD28 YMNM motif or the CTLA-4 YVKM motif have similar affinities for PI(3)K (13). We have found that although phosphorylation of the CD28 molecule by Lck resulted in recruitment of significant amounts of PI(3)K, phosphorylation of the CTLA-4 molecule by Lck did not. The inability of CTLA-4 to recruit PI(3)K may be due to several factors. Although we have shown that phosphorylation of CTLA-4 by Lck occurs on both Y201 and Y218, it is possible that the level of phosphorylation on Y201 is not sufficient for significant recruitment of PI(3)K. Another possibility is that within the context of the full length CTLA-4 molecule, the phosphorylated Y201 motif may be structurally inaccessible to PI(3)K. Other mechanisms may be related to the overexpression of Lck and include the sequestration of PI(3)K by Lck (42), or a competition between PI(3)K and Lck for CTLA-4 binding. Presumably, the same mechanism is not operative when CD28 is phosphorylated by Lck. These results demonstrate a distinct difference in the recruitment of PI(3)K following the phosphorylation of CTLA-4 and CD28 by Lck and suggest that the regulation of CTLA-4 and CD28 by Lck can result in distinct intracellular effects.

Increasing biochemical evidence suggests that cell surface receptor signaling involves the formation of multimeric complexes through intermolecular interactions mediated by adaptor modules contained within individual proteins (24, 40). The positioning of the kinases and phosphatases that exist within these complexes relative to their substrates and the regulation of their enzymatic activities are likely to be important in determining the integrated effects of signals generated through activation of multiple cell surface receptors. We have found that CTLA-4 associates with Lck and SHP-2, two molecules that have been identified as important participants of signaling complexes at the TCR and/or the CD28 receptor. Complex formation with these molecules could provide the molecular basis for CTLA-4-mediated regulation of TCR and/or CD28 signaling pathways. Our findings are consistent with a dual role for the Src kinases in the regulation of CTLA-4 activation: prevention of CTLA-4 endocytosis through tyrosine phosphorylation; and induction of the formation of a CTLA-4 signaling complex at the plasma membrane.

Acknowledgments

We thank D. Straus, L. Samelson, G.-S. Feng, and T. Gajewski for reagents and E. Masteller, R. Arch, and B. Ober for critical reading of the manuscript.

Footnotes

  • ↵1 This work was supported by National Institutes of Health Grants K08CA78591 to E.C. and P01DK49799 to C.B.T. and J.A.B.

  • ↵2 These authors contributed equally to this work.

  • ↵3 Address correspondence and reprint requests to Dr. Craig B. Thompson, University of Chicago, 924 E. 57th Street, R413A, Chicago, IL 60637-5420.

  • ↵4 Abbreviations used in this paper: CTLA-4, cytotoxic T lymphocyte-associated molecule-4; PI(3)K, phosphatidylinositol 3-kinase; PE, phycoerythrin; GST, glutathione S-transferase; WT, wild type.

  • Received July 20, 1998.
  • Accepted October 9, 1998.

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The Journal of Immunology: 162 (3)
The Journal of Immunology
Vol. 162, Issue 3
1 Feb 1999
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