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The Journal of Immunology, 1999, 162: 1966-1973.
Copyright © 1999 by The American Association of Immunologists

Identification of Tyrosine Phosphorylation Sites in the CD28 Cytoplasmic Domain and Their Role in the Costimulation of Jurkat T Cells1

Ali Sadra, Tomas Cinek, Jerry L. Arellano, Jia Shi, Kenneth E. Truitt2 and John B. Imboden3

Department of Medicine, Rosalind Russell Research Laboratory, San Francisco General Hospital, and University of California, San Francisco, CA 94143


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The cytoplasmic domain of CD28 contains four tyrosine residues. Because signal transduction by CD28 appears to involve its tyrosine phosphorylation, we determined sites of CD28 tyrosine phosphorylation using mutants of mouse CD28 that retained tyrosine at one position, with the remaining three positions mutated to phenylalanine. When expressed in Jurkat cells and stimulated by mAb, only the mutants with tyrosine at position 170 or 188 were tyrosine phosphorylated. Phosphorylation of Tyr170 recruits phosphatidylinositol 3-kinase to CD28. Tyr188 has not been associated with any specific signaling event, but we found that ligation of CD28 by the natural ligand B7.2 also induced phosphorylation of Tyr188, suggesting that this event is of physiological importance. Consistent with that possibility, mutation of Tyr188 to phenylalanine severely impaired the ability of mouse CD28 to deliver a costimulus for the expression of CD69 and the production of IL-2. The functional consequences of the mutation of Tyr188 were unique; mutation of the other three tyrosines, individually or in combination, did not impair costimulation. Therefore, of the four CD28 tyrosine residues only Tyr188 is required for signaling in Jurkat cells, suggesting that its phosphorylation is a key event in the costimulation of T cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Costimulatory molecules deliver signals that promote the responses of T lymphocytes to Ag 1 . The major costimulatory molecule appears to be CD28, a member of the Ig superfamily that is expressed as a homodimer on the surface of virtually all CD4+ T cells and the majority of CD8+ T cells 2 . The interaction of CD28 with B7-1 (CD80) and B7.2 (CD86), its ligands on APCs, enhances a number of TCR-mediated responses, including the production of lymphokines 3, 4 , and can rescue cells from TCR-mediated apoptosis 5 . CD28-/- mice exhibit mild immunodeficiency and T cells from these mice have markedly impaired responses to stimulation in vitro 6 . In animal models, disruption of CD28/B7 interactions prolongs allograft and xenograft survival and can prevent or even reverse autoimmunity, whereas enhancement of CD28/B7 interactions can promote antitumor responses against established malignancies 7 .

The mechanisms of signal transduction by CD28 are not completely understood. CD28 has a cytoplasmic domain of ~40 amino acids that is highly conserved across species and that contains four tyrosine residues 2 . Several lines of evidence suggest that phosphorylation of one or more of these cytoplasmic tyrosine is important for the delivery of the CD28 costimulus. First, crosslinking of CD28 by mAbs induces its tyrosine phosphorylation in Jurkat cells and T cell hybridoma cells 8, 9 . Second, mutation of all four cytoplasmic tyrosine residues of mouse CD28 (mCD28)4 significantly impairs its ability to costimulate the production of IL-2 10, 11 . Finally, although phosphorylation of Tyr170 (murine sequence) has not been demonstrated directly, there is circumstantial evidence that its phosphorylation recruits phosphatidylinositol 3-kinase (PI3K) to CD28 8, 12, 13, 14, 15, 16, 17 . There are conflicting data as to whether the costimulation of IL-2 production by CD28 requires Tyr170 and the recruitment of PI3K 10, 11, 16, 17, 18, 19, 20, 21 .

In this study, we used a series of mutants of mCD28 expressed in Jurkat cells to determine phosphorylation sites. As expected, we found that stimulation of mCD28 induced the phosphorylation of Tyr170. We also identified a second site of phosphorylation, Tyr188. We observed phosphorylation of Tyr188 after mAb crosslinking or engagement by the natural ligand, B7.2. Mutation of Tyr188 to Phe severely impaired the ability of mCD28 to deliver a costimulus in Jurkat cells, suggesting that phosphorylation of Tyr188 plays a critical role in signaling through CD28.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Abs

Murine mAb 9.3 (IgG2a) to human CD28 (hCD28) and hamster mAb 37.51 specific for mCD28 were kind gifts of Drs. Jeffrey Ledbetter (Bristol-Meyers Squibb Pharmaceutical Research Institute, Seattle, WA) and James Allison (University of California, Berkeley, CA), respectively. Mouse mAb G46-2.6 (IgG1) to human class I-MHC Ags and mouse mAb 34-2-12 (IgG2a) against mouse class I-MHC Ag H-2Dd were obtained from PharMingen (San Diego, CA) and were dialyzed overnight against PBS to remove their sodiumazide preservative. The horseradish peroxidase- (HRP-) conjugated antiphosphotyrosine mAb 4G10-HRP was purchased from Upstate Biotechnology (Lake Placid, NY). Because CD28 is also phosphorylated on serine and threonine 22 , we confirmed the specificity of 4G10-HRP by immunoblotting in the presence of saturating levels of phosphothreonine, phosphoserine, and phosphotyrosine solutions. Only phosphotyrosine blocked the 4G10-HRP (data not shown). Rabbit antisera to the p85{alpha} subunit of PI3K (p85{alpha} Z-8), goat antisera to hCD28 (CD28 N-20) and mCD28 (CD28 M-20), and HRP-conjugated anti-goat Ig and anti-mouse Ig reagents were from Santa Cruz Biotechnology (Santa Cruz, CA).

Cells

Jurkat E6-1 cells and Rat2 embryonic fibroblast cells (American Type Culture Collection, Manassas, VA) were maintained in "complete" RPMI medium composed of RPMI 1640 supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin, and 10% heat-inactivated FCS (Life Technologies, Gaithersburg, MD). Jurkat clones expressing wild-type (WT) mCD28, and the ALL F, F170, Y170, Y185, Y188, and Y197 mutants have been described 10, 21 . The F185, F188, and F197 mCD28 mutants were made by PCR-mediated site-directed mutagenesis, subcloned into the expression vector pBSR{alpha}EN (a gift of Drs. Andrey S. Shaw and Michael W. Olszowy, Washington University School of Medicine, St. Louis, MO), and used to stably transfect Jurkat cells as described 21 . Clones that expressed mCD28 at levels comparable to the WT mCD28-expressing Jurkat cells were selected for study. The transfected Jurkat cells were passaged in complete RPMI medium supplemented with 2 mg/ml G418 (Life Technologies). Rat2 cells that expressed mouse B7.2 were produced by electroporation of Rat2 rat fibroblast cells with a pcDNA1.1 (Invitrogen, Carlsbad, CA) vector containing the mouse B7.2 cDNA (from Dr. Peter Linsley, Bristol-Meyers Squibb Pharmaceutical Research Institute) followed by selection in complete RPMI medium supplemented with 1 mg/ml G418 and analysis by flow cytometry. For studies of mouse splenocytes, a single cell suspension, prepared from the spleens of 12-wk-old female BALB/c mice, was cultured in complete RPMI medium with 5 µg/ml Con A (Sigma, St. Louis, MO) and 50 U/ml mouse IL-2 (Genzyme Diagnostics, Cambridge, MA) for 72 h before use.

Cell activation, immunoprecipitation, and immunoblotting

For mAb crosslinking experiments, Jurkat cells were washed twice with ice-cold complete RPMI medium and resuspended at 1 x 108 cells/ml. The samples were warmed to room temperature for 5 min, then to 37°C for 5 min. The primary mAb was added at 10 µg/ml. In crosslinking experiments, the secondary crosslinking Ab was added at 40 µg/ml after 2 min. For stimulation of Jurkat cells by Rat2 cells, 1.5 x 108 Jurkat cells were pelleted with 2.5 x 107 Rat2 cells by a 10-s cenrifugation at 1700 x g in a microfuge and then incubated at 37°C. At the end of the incubation periods, mAb-stimulated Jurkat cells and the Jurkat cell/Rat2 cell mixture were pelleted in a microfuge, lysed with Nonidet P-40 lysis buffer (150 mM NaCl, 10 mM Tris-HCl (pH 7.5), 1% Nonidet P-40; 0.5 mM EDTA, 20 mM NaF, 1 mM Na3VO4, 1 mM PMSF, 1 µg/ml leupeptin, 10 µg/ml aprotinin, 0.5 µg/ml pepstatin, and 2 µg/ml antipain), and left on ice for 15 min. For samples receiving no primary mAb stimulation, appropriate mAbs were added at this point. The lysates were centrifuged at 21,000 x g at 4°C to remove insoluble material, and 20 µl of packed Ultralink protein A/G beads (Pierce, Rockford, IL) were added to the resulting supernatants. After incubation for 1 h on a rotor at 4°C, the bead-captured immune complexes were washed five times with the Nonidet P-40 lysis buffer. The immune complexes were solubilized in either reducing (containing freshly added 1% 2-ME) or nonreducing (containing freshly made 5 mM sodium iodoacetamide) Laemmli sample buffer. The samples were heated to 95°C for 10 min and then run on 10% SDS polyacrylamide gels. The separated proteins were transferred to polyvinylidene fluoride membranes using a Hoefer Scientific Instruments (San Francisco, CA) semidry transfer apparatus. After an initial air drying and rewetting in methanol and water, the membranes were blocked for 30 min at 42°C in BSA PBS blocking buffer (PBS, 0.1% Tween 20, and 5% BSA; Sigma). After immunoblot analysis with antiphosphotyrosine, the membranes were stripped with 2% SDS at 42°C for 30 min. The membranes were reblocked with PBS blocking buffer containing PBS, 0.1% Tween 20, and 10% nonfat milk, and reprobed with anti-p85 PI3K antiserum. After a second stripping and blocking, the membranes were probed with anti-CD28 antiserum. Immunoblot signals were detected with enhanced chemiluminescence using Renaissance Western Plus Reagent from NEN Life Sciences (Boston, MA). Each lane corresponds to immunoprecipitates from 1.5 x 108 cells.

IL-2 assay and measurements

IL-2 levels were measured in supernatants obtained from 50,000 Jurkat cells incubated in 200 µl of complete RPMI medium for 16 h at 37°C in room air supplemented with 5% CO2. Cells were stimulated with 1 ng/ml PMA plus 0.5 µM ionomycin and, as indicated, 5 µg/ml of either anti-human MHC class I, anti-hCD28, or anti-mCD28. IL-2 was quantified using a human IL-2 ELISA kit from Immunotech (Westbrook, ME). Optical density readings of the samples were performed on a SpectraMax 250 reader from Molecular Devices (Sunnyvale, CA).

Costimulation of CD69 expression

For measurement of CD69 induction, 105 Jurkat cells were stimulated with 5 ng/ml PMA or with 5 ng/ml PMA plus 5 µg/ml of either anti-hCD28, anti-mCD28, or control hamster IgG anti-trinitrophenol (TNP) for 2 h at 37°C in room air supplemented with 5% CO2. The cells were then washed with ice-cold staining wash buffer (PBS with 0.1% sodium azide and 3% FCS). The washed cells were stained with 1 µg of anti-hCD69 FITC-conjugated Ab. An isotype-matched, nonreactive FITC-conjugated Ab (anti-hCD19) was used as a negative control. The stained cells were then extensively washed with ice-cold staining wash buffer, and their surface staining was analyzed and profiled by a FACSort program and a Becton Dickinson FACScan instrument (Becton Dickinson, Sparks, MD).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Stimulation of CD28 induces its tyrosine phosphorylation

As reported previously, stimulation of CD28 by mAbs induced its tyrosine phosphorylation in human Jurkat cells and hCD28-transfected murine T hybridoma cells (Fig. 1Go) 8, 9 . Similarly, we find that mAb-mediated crosslinking triggered the tyrosine phosphorylation of mCD28 in mouse splenic T cells and of mCD28 when it is expressed in Jurkat cells after gene transfer (Figs. 1Go and 2Go). To establish that the anti-CD28 immunoprecipitated phosphotyrosine band was indeed CD28, the samples were electrophoresed under both reducing and nonreducing conditions. Consistent with the expression of CD28 as a covalently linked homodimer 2 , the band corresponding to CD28 migrated at a molecular mass of 75–90 kDa under nonreducing conditions and 35–45 kDa under reducing conditions (Fig. 1GoB). As shown, the tyrosine phosphorylated candidate band comigrated with CD28 under both reducing and nonreducing conditions confirming its identity as CD28.



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  		FIGURE 1. MAb crosslinking induces the tyrosine phosphorylation of CD28. A, Tyrosine phosphorylation of CD28 in mouse splenocytes. Splenocytes from female BALB/c mice were cultured in complete RPMI medium supplemented with 5 µg/ml Con A and 50 U/ml mIL-2. After 72 h, the cells were washed and then incubated in medium alone (-) or stimulated with crosslinked mAbs to mouse MHC class I or mCD28 for 5 min at 37°C as described in the Materials and Methods. Immunoprecipitates of mouse MHC class I and mCD28 were analyzed by immunoblotting with antiphosphotyrosine, anti-mCD28, and anti-PI3K p85. B, Tyrosine phosphorylation of hCD28 in Jurkat cells. Jurkat cells were incubated in medium alone (-) or stimulated with crosslinked mAbs to human MHC class I or hCD28. Immunoprecipitates of MHC class I and hCD28 were analyzed by immunoblotting for phosphotyrosine, hCD28, and PI3K p85 under reducing and nonreducing conditions.

 


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  		FIGURE 2. Time course of the tyrosine phosphorylation of mCD28 in Jurkat cells. Jurkat cells expressing WT mCD28 were stimulated with the mCD28 mAb, 37.51, for the time points indicated. A 5-min engagement of MHC class I receptor was performed as control. Immunoprecipitates of mCD28 and MHC class I were were analyzed by immunoblotting for phosphotyrosine, mCD28, and PI3K p85.

 
The phosphorylation of mCD28 in Jurkat cells occurred within 1 min, was maximal at ~5 min, and persisted for at least 30 min (Fig. 2Go). A low level of tyrosine phosphorylation of the mCD28, as well as of the endogenous hCD28, was occasionally seen in unstimulated Jurkat cells, but there was a consistent, substantial increase in phosphorylation in response to the anti-CD28 mAbs. As reported, perturbation of hCD28 and mCD28 led to the recruitment of PI3K, as evidenced by the coimmunoprecipitation of PI3K p85 with CD28 after crosslinking by mAb (Figs. 1Go and 2Go; Refs. 8, 12–15, and 17).

Tyr170 and Tyr188 of mCD28 are sites of phosphorylation

The ability of Jurkat cells to support the tyrosine phosphorylation of mCD28 allowed us to examine a series of mCD28 mutants with phenylalanine to tyrosine mutations (shown schematically in Fig. 3Go) to determine specific sites of tyrosine phosphorylation. We selected stable Jurkat transfectants expressing comparable cell surface levels of WT mCD28 and the mCD28 mutants for analysis and used a mAb specific for mCD28 to selectively crosslink the transfected mCD28. In parallel experiments, the endogenous hCD28 was stimulated by a mAb specific for hCD28 (Fig. 4GoA).



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  		FIGURE 3. Schematic presentation of the cytoplasmic domains of mCD28 mutants used in these studies. The tyrosine (Y) residues of mCD28 cytoplasmic domain were mutated to phenylalanine (F) as shown. All four tyrosine residues were mutated to phenylalanine in the ALL F mutant. The "Y" series of mutants preserved one tyrosine residue with the remaining three tyrosine mutated to phenylalanine. The "F" series of mutants had single phenylalanine for tyrosine substitutions. Each mutant and the WT mCD28 were stably expressed at comparable levels in Jurkat cells.

 


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  		FIGURE 4. Stimulation of mCD28 leads to its phosphorylation on Tyr170 and Tyr188. A, MAb-induced tyrosine phosphorylation of the Y170 and Y188 mutants. Jurkat cells stably transfected with WT mCD28, the ALL F mCD28 mutant, and mCD28 mutants with only single tyrosine residues were incubated in medium alone (-) or were stimulated by crosslinked mAbs to human MHC class I, hCD28, or mCD28. Immunoprecipitates of human MHC class I, hCD28, or mCD28 were analyzed by immunoblotting for phosphotyrosine, CD28 (using antisera to hCD28 and mCD28), and PI3K p85. Crosslinked mAb to mCD28 induced the tyrosine phosphorylation of the Y170 and Y188 mutants, but not Y185 or Y197. Only Y170 recruited PI3K p85. Crosslinking the endogenous hCD28 on each cell line induced its tyrosine phosphorylation and recruited PI3K p85. B, The effect of single mutations to phenylalanine at position 170 or 188. The F170 and F188 mCD28-expressing transfected-Jurkat T cells were incubated in medium alone (-) or were stimulated by anti-mCD28 mAb before immunoprecipitation of mCD28 and immunoblot analysis.

 
As expected, mutation of all four cytoplasmic tyrosine of mCD28 (ALL F mutant) abrogated its tyrosine phosphorylation after mAb stimulation (Fig. 4GoA). Immunoblot analysis with anti-CD28 confirmed the presence of the ALL F mutant in the immunoprecipitates, and parallel studies of crosslinked hCD28 demonstrated that the ALL F-expressing Jurkat clone supported the tyrosine phosphorylation of hCD28. Preclearing experiments have failed to detect heterodimer formation between hCD28 and mCD28 in Jurkat cells that express WT mCD28 21 . The absence of tyrosine-phosphorylated CD28 in the ALL F immunoprecipitates provides further evidence against the presence of confounding heterodimers in this system.

We then examined mutants that preserved a single cytoplasmic tyrosine residue with the remaining three mutated to phenylalanine. Of these, the Y170 and Y188 mutants exhibited a low level of basal tyrosine phosphorylation that increased substantially after mAb-mediated stimulation (Fig. 4GoA). In contrast, we did not detect phosphorylation of the Y185 and Y197 mutants in either unstimulated or stimulated cells, even though mAb crosslinking induced tyrosine phosphorylation of hCD28 in the Y185- and Y197-expressing cells. Therefore, Tyr170 and Tyr188 are sites of mCD28 phosphorylation in Jurkat cells whereas Tyr185 and Tyr197 are either not phosphorylated under these conditions or are phosphorylated at levels below the limits of detection. Consistent with the conclusion that both Tyr170 and Tyr188 are phosphorylated, single mutation to phenylalanine at either position 170 or 188 did not prevent tyrosine phosphorylation of mCD28 (F170 and F188 mutants; Fig. 4GoB). However, mutation of Tyr188 had a more impressive inhibitory effect on mCD28 phosphorylation than did mutation of Tyr170 (Fig. 4GoB). As previously reported by ourselves and others, Tyr170 is required for the recruitment of PI3K to mCD28 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 . Among the mCD28 mutants expressing single tyrosine residues, only Y170 recruited PI3K p85, and a single mutation to phenylalanine at position 170 (the F170 mutant) abrogated the association with PI3K (Fig. 4GoB), as reported 21 .

Interaction with B7.2, the natural ligand of CD28, induces tyrosine phosphorylation of WT mCD28 and the Y188 mutant form of mCD28

To determine whether CD28 is tyrosine phosphorylated after interactions with its natural ligands, we incubated WT mCD28-expressing Jurkat cells with either Rat2 cells or Rat2 cells that express high levels of mouse B7.2 (B7 Rat2) after gene transfer. Interaction with the B7.2-expressing cells induced readily detectable tyrosine phosphorylation of mCD28 within 2 min, and the phosphorylation persisted for at least 10 min (Fig. 5GoA). Recruitment of the p85 subunit of PI3K to mCD28 after similar kinetics, indicating that engagement by B7.2 triggers the phosphorylation of Tyr170. To determine whether phosphorylation of Tyr188 is also induced by binding B7.2, we incubated Y188-expressing Jurkat cells with the B7 Rat2 cells. As was the case with mAb-mediated crosslinking, incubation with B7 Rat2 cells led to the tyrosine phosphorylation of the Y188 mutant (Fig. 5Go). The kinetics of tyrosine phosphorylation for Y188 were somewhat slower than for WT mCD28 and there was no recruitment of p85 subunit of PI3K to the phosphorylated Y188 mutant.



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  		FIGURE 5. B7.2, a natural ligand of CD28, induces the tyrosine phosphorylation of WT mCD28 and the Y188 mutant of mCD28. Jurkat T cells expressing WT mCD28 (A) or the Y188 mutant (B) were incubated with either Rat2 fibroblasts or Rat2 cells that expressed cell surface mouse B7.2 after gene transfer (B7 Rat2 cells). The cells were pelleted by centrifugation and then incubated at 37°C for the indicated times before lysis. Immunoprecipitates of human MHC class I, mCD28, and hCD28 were analyzed by immunoblotting for phosphotyrosine, CD28 using antisera to hCD28 and mCD28 and PI3K p85.

 
Tyr188 is required for costimulation in Jurkat

Perturbation of CD28 delivers a costimulus that enhances a number of T cell responses. In Jurkat, CD28 stimulation promotes CD69 expression by PMA-treated cells and augments IL-2 production induced by the combination of ionomycin and PMA. We used these responses to assess the functional consequences of mutations of the tyrosine phosphorylation sites. Because subclones of Jurkat differ in the magnitude of their responses to activation 10, 21 , we compared the costimulatory ability of the mCD28 mutants to that of the endogenous hCD28 expressed by each subclone.

PMA induces Jurkat cells to express CD69, an early activation Ag, and stimulation of CD28 enhances this response. WT mCD28, as well as the F185 and the F197 mutants, delivered costimuli for CD69 expression that were only slightly less effective than costimulation by the endogenous hCD28 expressed by each Jurkat subclone (Fig. 6GoA). Mutation of the Tyr170 phosphorylation site to phenylalanine did not impair the ability of mCD28 to promote the expression of CD69 in PMA-treated Jurkat cells; indeed, the F170 mutant was more effective than the endogenous hCD28 in delivering this costimulus. Quite different results were obtained with mutation of the Tyr188 phosphorylation site. Mutation of Tyr188 to phenylalanine severely diminished the ability of mCD28 to promote the expression of CD69 (F188 mutant; Fig. 6GoA). The functional impairment exhibited by the F188 mutant was similar to that of the ALL F mutant, which lacks cytoplasmic tyrosine residues. When tyrosine was retained at position Y188 and the remaining three tyrosine residues were mutated to phenylalanine (Y188 mutant), mCD28 retained its ability to costimulate expression of CD69 (Fig. 6GoB). Thus, in terms of CD28 cytoplasmic tyrosine residues, Tyr188 is both necessary and sufficient for the delivery of a costimulus in this particular system.



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  		FIGURE 6. Tyr188 is required for mCD28-mediated costimulation of CD69 expression in Jurkat cells. A, Clones of Jurkat cells expressing WT mCD28 or the indicated mCD28 mutants were stimulated with PMA (5 ng/ml) alone or PMA and either 5 µg/ml hamster IgG mAb (anti-TNP) (open bars), anti-hCD28 mAb (stippled bars), or anti-mCD28 mAb (closed bars) for 2 h at 37°C. Cells were then stained with anti-CD69 or with isotype-matched control FITC-conjugated mAb (anti-CD19) at 4°C and were analyzed by flow cytometry. The data are presented as the percent change in CD69 expression induced by the combination of mAb and PMA compared with PMA alone. Three independent experiments are shown, each of which was performed in duplicate. B, The abilities of the F188, ALL F, and Y188 mutants to enhance CD69 expression were compared in a single experiment. These data are representative of at least three independent experiments for each mutant.

 
Analysis of costimulation of IL-2 led to similar conclusions. Single mutations to phenylalanine at positions 170, 185, and 197 did not impair mCD28 function; like WT mCD28, the F170, F185, and F197 mutants delivered costimuli for IL-2 production at least as effectively as the hCD28. However, mutation of Tyr188 significantly diminished the ability of mCD28 to promote IL-2 production (Fig. 7GoA). The functional impairment of the F188 mutant was comparable to that of the ALL F mutant (Fig. 7GoB). As reported previously, the Y188 mutant was as effective as hCD28 in the costimulation of IL-2 production 10 .



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  		FIGURE 7. Mutation of Tyr188 impairs costimulation of IL-2 production in Jurkat cells. A, Jurkat cells expressing WT mCD28 or the indicated mCD28 mutants were stimulated with the combination of ionomycin and PMA only (open bars) or with that combination plus either anti-MHC class I mAb (stippled bars), anti-hCD28 mAb (filled bars), or anti-mCD28 mAb (striped bars) for 16 h. The IL-2 levels in culture supernatants were determined by ELISA. Each individual experiment was performed in duplicate. The results of three independent experiments are shown. B, IL-2 production by clones expressing F188, All F, and Y188 were analyzed in a single experiment. These data are representative of at least three independent experiments for each individual clone.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
A critical step in signal transduction by a wide range of receptors is the phosphorylation of tyrosine residues in the cytoplasmic domain of the receptor, leading to the recruitment of signaling molecules to the receptor and their subsequent activation 23 . Because signaling by CD28 appears to involve a similar mechanism 24 , we used a series of mCD28 mutants expressed in Jurkat cells to determine sites of tyrosine phosphorylation and to study the functional consequences of mutating those sites to phenylalanine. We find that stimulation of mCD28 by mAb triggers its phosphorylation on Tyr170 and Tyr188. Finding that Tyr170 is phosphorylated was not unexpected in view of the indirect evidence implicating phosphorylation of this residue in the recruitment of PI3K to CD28 8, 12, 13, 14, 15, 17 . Tyr188 has not been linked with a specific signaling molecule, but we find that engagement of CD28 by its natural ligand, B7.2, also triggers its phosphorylation on this site, underscoring the potential importance of this event. Moreover, mutation of Tyr188 severely impairs the ability of mCD28 to enhance CD69 expression or to promote IL-2 production. Therefore, Tyr188 is necessary for the delivery of a costimulus in the Jurkat model, suggesting that its phosphorylation is a critical event in signaling by CD28 during the costimulation of T cells.

In Jurkat cells, the functional consequences of the mutation of Tyr188 are unique among mCD28 tyrosine residues; mutation at any of the other three tyrosine residues does not diminish costimulation of CD69 expression or IL-2 production. Moreover, preservation of Tyr188 permits mCD28 to deliver costimuli even when the other three tyrosine residues have been mutated to phenylalanine (Figs. 6Go and 7Go) 10 . Therefore, in terms of CD28 cytoplasmic tyrosine residues, Tyr188 is both necessary and sufficient for costimulation in the Jurkat system.

The functional consequences of the mutation of Tyr188 to Phe raise the possibility that phosphorylation of Tyr188 recruits to CD28 a signaling molecule involved in costimulation. The nature of the Tyr188-based signal is currently unknown. CD28 signaling events include the recruitment and activation of PI3K 8, 12, 13, 14, 15, 17 and the delivery of a signal that promotes TCR-mediated activation of Jun N-terminal kinase 25, 26 . However, neither pathway requires the integrity of Tyr188, and neither appears to be involved in the Tyr188-based signal (Fig. 4Go and our unpublished observations). Mutation of Tyr188 to Phe does not abrogate the recruitment of PI3K to mCD28 (F188 mutant; Fig. 4GoB). We have found that truncation of the mCD28 at position 182 does not alter its ability to synergize with the TCR in the activation of Jun N-terminal kinase, even though this deletes Tyr188 (our unpublished observations).

As noted, the identification of Tyr170 as a phosphorylation site was anticipated. Tyr170 lies within a sequence motif that, when phosphorylated, is predicted to form a high affinity binding site for the SH2 domains of PI3K p85 subunit 8, 12, 13, 14, 15, 17 and also has been reported to bind the Grb2 adapter protein 27 . The association of CD28 with PI3K p85 depends upon Tyr170, and observations with synthetic peptides corresponding to this region indicate that Tyr170 must be phosphorylated to bind PI3K p85 13, 14, 15 . These findings together with the direct evidence presented here establish that ligand-induced phosphorylation of Tyr170 recruits PI3K to CD28.

The functional significance of the recruitment of PI3K to Tyr170 depends upon the cell system studied. In several systems, Tyr170 (or its human equivalent) is required for costimulation and mutation of this site to phenylalanine abrogates CD28 costimulation 8, 28 . However, in Jurkat cells, mutation of Tyr170 to phenylalanine prevents the recruitment of PI3K to mCD28 but does not impair the ability of mCD28 to promote IL-2 production 21, 29 or to enhance CD69 expression (Fig. 6Go). Activation of PI3K inhibits the transcriptional capacity of NF of activated T cells (NF-AT) in Jurkat cells, indicating that PI3K can function as a negative regulator of TCR signaling events 30 . Wortmannin, an inhibitor of PI3K, prevents Ag-induced IL-2 production by freshly isolated T cells from DO11.10 TCR transgenic mice 31 . However, the wortmannin-sensitive step does not appear to be at the level of CD28 costimulation of IL-2 production. Wortmannin inhibits conjugate formation between T cells and APCs, an event that does not depend upon CD28 31 . When the TCR and CD28 are engaged by mAbs, wortmannin enhances IL-2 production by DO11.10 T cells, an observation consistent with the negative regulatory role for PI3K defined in Jurkat cells 31 .

Interestingly, the two phosphorylation sites identified here, Tyr170 and Tyr188, are the only cytoplasmic tyrosine residues conserved between CD28 and CTLA-4 2, 32, 33 , a related molecule that functions primarily to shut Tyr188, are the only cytoplasmic tyrosine residues conserved between CD28 and T cell activation 34 . The homologue of Tyr170 is a regulator of the cell surface expression of CTLA-4 and may recruit PI3K and the tyrosine phosphatase SYP to CTLA-4 35, 36, 37 . The functional significance of the homologue of Tyr188 is not known.

In contrast to the results obtained with Tyr170 and Tyr188, we did not detect tyrosine phosphorylation of Tyr185 or Tyr197 of mCD28 (Fig. 4Go). Tyr185 and Tyr197 could be phosphorylated at levels below the limitations of detection or under activation conditions other than those studied here. It is also possible that their phosphorylation requires the integrity of another CD28 cytoplasmic tyrosine residue. In that case, our strategy of using mutants that express single tyrosine residues would prevent us from observing phosphorylation on these sites. Tyr185 and Tyr197 are not necessary for mCD28 to promote CD69 expression in PMA-treated Jurkat cells or to augment IL-2 production in response to ionomycin plus PMA (Fig. 6Go and 7Go). Therefore, these tyrosine residues do not generate critical costimulatory signals in this system, but we cannot rule out the possibility that our experimental conditions rendered signals based on Tyr185 and Tyr197 unnecessary.

In summary, we have identified two sites of tyrosine phosphorylation of CD28: Tyr170, whose phosphorylation recruits and activates PI3K; and Tyr188, whose integrity, we find, is required for costimulation. These studies support the hypothesis that CD28 signaling involves its tyrosine phosphorylation and pinpoint the phosphorylation of Tyr188 as a critical step in the delivery of a costimulus.


   Acknowledgments
 
We thank Dr. Joel Ernst (University of California, San Francisco) for his critical reading of this manuscript.


   Footnotes
 
1 This work was supported by Grant RO1 AI26644 from the National Institutes of Health and by the Rosalind Russell Arthritis Center. Back

2 Current address: Merck Research Laboratories, 126 East Lincoln Avenue, P.O. Box 2000, RY32-645, Rahway NJ 07065. Back

3 Address correspondence and reprint requests to Dr. John Imboden, Box 0868, University of California, San Francisco, CA 94143. E-mail address: Back

4 Abbreviations used in this paper: m, mouse; h, human; PI3K, phosphatidylinositol 3-kinase; WT, wild type. Back

Received for publication July 22, 1998. Accepted for publication November 2, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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