The involvement of the proto-oncogene p120 c-Cbl and ZAP-70 in CD2-mediated T cell activation
Huamao Lin1,3,
Maria Paola Martelli2 and
Barbara E. Bierer1,2
1 Department of Pediatric Oncology, Dana-Farber Cancer Institute, Boston, MA 02115, Department of Pediatrics, Harvard Medical School, Boston, MA 02115, USA
2 Laboratory of Lymphocyte Biology, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA
Correspondence to:
B. E. Bierer, National Institutes of Health, 10 Center Drive, Building 10, Bethesda, MD 20892, USA
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Abstract
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The CD2 co-receptor expressed on the surface of T lymphocytes is able to stimulate T cell activation, proliferation and cytokine production in the absence of direct engagement of the antigen-specific TCR. Engagement of human CD2 by mitogenic pairs of anti-CD2 mAb induces tyrosine phosphorylation of a number of intracellular proteins including a 120 kDa phosphoprotein that we identify as the proto-oncogene c-Cbl. Rapidly tyrosine phosphorylated following stimulation of a number of cell surface receptors, c-Cbl is an adaptor protein that has been shown to associate with a complex of intracellular signaling molecules, and to mediate both positive and negative regulatory effects. Here we show that, like TCRCD3 stimulation, stimulation of CD2 enhanced the association of c-Cbl with both Crk(L) and the p85 subunit of phosphatidylinositol-3 kinase. Overexpression of wild-type c-Cbl protein inhibited both CD2and CD3-induced NF-AT transcriptional activity, suggesting that CD2 signaling is also negatively regulated by c-Cbl. The inhibitory effect of c-Cbl depended upon its N-terminal phosphotyrosine-binding domain, the domain that has been shown to be required for inhibition of the Syk/ZAP-70 family kinases. In Syk Jurkat T cells stably expressing wild-type ZAP-70, CD2 stimulation induced only a minimal increase in ZAP-70 tyrosine phosphorylation. Nevertheless, ZAP-70 kinase was required for CD2-mediated NF-AT transcriptional activity. Thus, CD2-mediated NF-AT transcriptional activity appears to depend upon ZAP-70/Syk kinases and to be negatively regulated by c-Cbl.
Keywords: Crk, NF-AT, phosphatidylinositol-3 kinase, signal transduction, Syk, T lymphocytes, ZAP-70
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Introduction
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The engagement of the antigen-specific TCRCD3 complex with cognate peptide embedded in MHC molecules initiates the process of T cell activation (1,2). Both T cell adhesion and signaling via the TCRCD3 complex may be modulated by the interaction of a number of T cell co-receptors with their ligand(s) expressed on the surface of antigen-presenting cells. The aggregate signal will ultimately depend upon the balance of positive and negative regulatory intracellular elements, integrating receptorco-receptor input and the cytokine milieu to dictate the outcome of T cell engagement.
Human CD2 is a 4450 kDa glycoprotein expressed on the majority of human thymocytes, T lymphocytes and NK cells. T cell stimulation with certain combinations of anti-CD2 mAb leads to IL-2 production and T cell proliferation in the absence of direct ligation of the TCRCD3 complex (3). CD2 is a co-receptor that has been shown to be able to modulate TCRCD3-mediated cell activation, to enhance T cell responsiveness to cytokines including IL-12 and to reverse anergy in certain human T cell clones (49). The molecular consequences of CD2 receptor engagement have not been completely defined.
Stimulation initiated via either the TCRCD3 complex or the CD2 receptor has been shown to induce tyrosine phosphorylation of a number of intracellular proteins, some shared and some apparently unique. We and others have previously observed the tyrosine phosphorylation of a protein of mol. wt ~120 kDa; a protein of similar mol. wt has been identified as the proto-oncogene c-Cbl following anti-CD3 ligation (10,11). The predicted product of the c-Cbl cDNA contains a phosphotyrosine-binding (PTB) domain (12,13), a RING finger domain, proline-rich sequences thought to mediate interaction with SH3 domain-bearing proteins and multiple tyrosine residues that, when phosphorylated, are capable of binding to SH2 domain-bearing proteins (for review, see 13). Stimulation of a number of surface receptors has been shown to induce the tyrosine phosphorylation of c-Cbl and to lead to its association, either directly or in multimolecular complexes, with a number of intracellular mediators (11,1317). More recently, c-Cbl has been shown to target its substrates to ubiquitination and degradation (18,19). The functional roles of c-Cbl are complex, and involve both positive and negative effects; assignment of any particular function to c-Cbl and not to its associated proteins has been difficult.
The involvement of c-Cbl in T cell signaling has been extensively investigated. The SH3 domain of c-Cbl has been shown to interact with the Src family PTK, the p85 subunit of phosphatidylinositol (PI)-3 kinase, Grb2 and Crk, while phosphorylated c-Cbl protein is able to interact, via its SH2 domains, with PI-3 kinase, Vav, Crk and Fyn after stimulation through the TCRCD3 complex (10,11,15,16,20). c-Cbl has also been shown to associate directly with ZAP-70/Syk protein kinases after TCR stimulation, an association mediated by the phosphotyrosine(s) of ZAP-70/Syk binding to the PTB domain of c-Cbl (12,21,22). c-Cbl appears to regulate ZAP-70/Syk activity and has been shown to be a negative regulator of TCRCD3-dependent T cell activation (21,23,24).
While the involvement of c-Cbl in TCRCD3-mediated signaling is well documented, the role of c-Cbl in CD2 signaling is less clear. In this report, the involvement of c-Cbl in CD2 signaling was investigated. We demonstrated that, like stimulation via the TCRCD3 receptor, stimulation using mitogenic pairs of anti-CD2 mAb induced the tyrosine phosphorylation of c-Cbl as well as the association of Crk(L) and PI-3 kinases with c-Cbl. Overexpression of wild-type c-Cbl protein inhibited CD2-induced NF-AT-dependent transcriptional activity, suggesting that c-Cbl is also a negative regulator of CD2 signaling. CD2-dependent c-Cbl-mediated inhibition required an intact N-terminal c-Cbl PTB domain, the domain required for binding to the ZAP-70/Syk family of protein tyrosine kinases (PTK). Our data suggested a potential role for ZAP-70/Syk kinases in CD2-mediated T cell activation. We show that, although only marginally tyrosine phosphorylated following CD2 stimulation of (Syk) Jurkat T cells, ZAP-70 kinase is required for CD2-mediated NF-AT transcriptional activity. Taken together, our results suggest that the induction of NF-AT-dependent transcription by CD2 is negatively regulated by c-Cbl and requires the ZAP-70/Syk tyrosine family kinases.
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Methods
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Cell culture and reagents
The T cell leukemia cell line Jurkat clone J77 was a gift of Kendall Smith (Cornell University, New York, NY). The ZAP-70/Syk negative Jurkat cell line P116 and the ZAP-70-reconstituted P116 clone (P116-ZAP70) were the kind gift of Robert Abraham (Duke University, Durham, NC). Jurkat cells transformed with SV40 large tumor (T) antigen (J-TAg) (the gift of Gerald Crabtree, Stanford University, Palo Alto, CA) were used in transient transfection experiments, as indicated. All lymphocytes were grown in RPMI 1640 (Mediatech, Herndon, VA) supplemented with 10% heat-inactivated FBS (Sigma, St Louis, MO), penicillin 100 U/ml (Gibco, Grand Rapids, NY), streptomycin 100 µg/ml (Gibco), 2 mM glutamine (Gibco), 10 mM HEPES (pH 7.3) and 50 µM 2-mercaptoethanol (Sigma), termed 10% FCS media.
The anti-CD2 mAb T112 and T113 were the kind gift of Ellis Reinherz (Dana Farber Cancer Institute, Boston, MA); the anti-phosphotyrosine mAb 4G10 was a gift of Tom Roberts (Dana Farber Cancer Institute); the anti-CD3
mAb OKT3 (ATTC, Rockville, MD) was used as purified ascites fluid; the rabbit polyclonal anti-c-Cbl antibody (C-15), the anti-c-Cbl antibody directly conjugated to agarose beads, the rabbit polyclonal anti-Crk-L (C-20) antibody and the anti-ZAP-70 mAb directly conjugated to agarose beads were all purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The anti-Crk mAb (C12620, Clone 22) and the anti-ZAP-70 mAb (Z24820, Clone 29) were obtained from Transduction Laboratories (San Diego, CA); the anti-PI-3 kinase (p85 subunit) antibody was purchased form Upstate Biotechnology (Lake Placid, NY). Phorbol myristate acetate (PMA) and ionomycin were purchased from Calbiochem (La Jolla, CA).
Immunofluorescent analysis of CD3 and CD2 surface expression
Surface expression of both CD3 and CD2 was evaluated by direct immunofluorescence: cells were incubated on ice for 20 min with phycoerythrin-conjugated anti-CD3 mAb, clone UCHT1 (Beckman Coulter, Fullerton, CA) and FITC-conjugated anti-CD2 mAb, clone RPA-2.10 (BD PharMingen, San Diego, CA), or appropriate phycoerythrinand/or FITCconjugated IgG isotype control antibody (Beckman Coulter). Cells then were washed and analyzed using a Coulter Epics flow cytometer (Beckman Coulter).
cDNA constructs
The wild-type c-Cbl construct, the Cbl G306E mutant in which the glycine at amino acid 306 in the PTB domain was replaced by glutamine and the Cbl 70Z mutant (25) in which 17 amino acids (EQYELYCEMGSTFQLCK) were deleted in the c-Cbl RING finger region were kindly provided by Hamid Band (Brigham and Women's Hospital, Boston, MA). Wild-type and mutated Cbl constructs were subcloned into the pEF-BOS vector (26) and used for transfection into Jurkat cells.
Cell stimulation and immunoprecipitations
Jurkat cells (1x107) were resuspended in 0.5 ml Buffer A [RPMI 1640 supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin, 2 mM glutamine and 10 mM HEPES (pH 7.3)], incubated with 0.51 µl of each of the anti-CD2 mAb T112 and T113 as ascites on ice for 15 min, as indicated, and then transferred to 37°C for 5 min, unless otherwise stated. The cells were lysed by addition of 0.5 ml cold lysis buffer B [2% Brij 97, 150 mM NaCl, 25 mM Tris (pH 7.5), 2 mM EDTA, 2 mM Na3VO4, 20 µg/ml leupeptin, 20 µg/ml aprotinin and 2 mM PMSF] on ice for 20 min. Lysates were clarified by centrifugation at 15,000 g for 10 min at 4°C.
In immunoprecipitation experiments, 20 µl of a 1:1 ratio of Protein A:Protein G slurry and 12 µg of indicated antibodies were added to the clarified cell lysates and incubated at 4°C for 2 h. For ZAP-70 or c-Cbl immunoprecipitations, 25 µl of the specific antibody directly conjugated to agarose beads were used. The beads were then washed 3 times with washing buffer C [0.1% Brij 97, 150 mM NaCl, 25 mM Tris (pH 7.5) and 1 mM Na3VO4]. Immunoprecipitated proteins were separated by SDSPAGE, transferred to PVDF membranes (Millipore, Bedford, MA) and probed with primary antibodies followed by horseradish peroxidase-conjugated secondary antibodies. Proteins were detected using the ECL method according to manufacturer's instructions (Amersham, Uppsala, Sweden). Quantification of band density was determined by densitometry using the Imagequant software (Molecular Dynamics, Sunnyvale, CA).
Transient transfection and luciferase assays
Jurkat cells J-TAg (1x107 cells) were incubated with 20 µg of vector, wild-type c-Cbl or mutated Cbl plasmid DNA as indicated, each together with 5 µg of the luciferase reporter plasmid p3xNF-AT-luc (27), carrying the luciferase gene driven by three tandem repeats of the distal NF-AT sequences derived from the IL-2 promoter, for 15 min at room temperature. Alternatively, Jurkat cells were incubated with 10 µg of the luciferase reporter plasmid p3xNF-AT-luc and 1 µg of pRL-TK vector (Promega, Madison, WI), which provides constitutive expression of Renilla luciferase. Cells were then electroporated at 250V, 800 µF, low
(Gibco/BRL). After electroporation, the cells were transferred to 10% FCS media and incubated at 37°C for 1224 h. Transfected cells (1x106 cells/test) were either left unstimulated or stimulated for 6 h with a 1:1000 dilution of each of the anti-CD2 mAb T112 and T113 ascites, unless otherwise stated, with either soluble (1µg/ml) or plate-bound (10 µg/ml) anti-CD3 mAb OKT3, or with PMA (10 ng/ml) plus ionomycin (2 µM). Cells were washed with PBS and samples were prepared using the Enhanced Luciferase Kit (Analytic Luminescent Laboratory, San Diego, CA) or, alternatively, the Dual-Luciferase Reporter Assay System (Promega), according to the manufacturer's instructions. The relative luciferase units are presented as the percentage of the maximal stimulation induced by PMA plus ionomycin for each transfection condition. As shown, the activity of firefly luciferase was normalized to that of Renilla luciferase to correct for transfection efficiency.
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Results
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CD2 stimulation induced the tyrosine phosphorylation of c-Cbl
Stimulation by mitogenic pairs of anti-CD2 mAb has been shown to induce T cell proliferation and IL-2 production (3). Early T cell activation is in part mediated by activation of PTK that phosphorylate a number of other intracellular proteins. Anti-CD2 mAb, like anti-CD3 mAb, induced a time-dependent tyrosine phosphorylation in Jurkat T cells as detected by anti-phosphotyrosine antibody (Fig. 1A
). Basal tyrosine phosphorylation of a number of intracellular proteins was observed in Jurkat cells; treatment of the cells with mitogenic pairs of anti-CD2 mAb enhanced the tyrosine phosphorylation of a number of proteins, including proteins of mol. wt ~3436, 5560 and 120 kDa (Fig. 1A
, lanes 26). Increased phosphorylation of the ~120 kDa protein was detected as early as 30 s following stimulation and began to diminish 15 min after stimulation.

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Fig. 1. Stimulation with mitogenic pairs of anti-CD2 mAb increased the tyrosine phosphorylation of c-Cbl. (A) Jurkat cells were either left unstimulated (lane 1), stimulated with the anti-CD2 mAb T112 and T113 (lanes 26) or stimulated by anti-CD3 mAb OKT3 (lane 7) at 37°C for the indicated periods of time before lysis. Whole-cell lysates were separated by SDSPAGE and were visualized by Western blot using the anti-phosphotyrosine mAb 4G10. The appearance of a dominant band at ~120 kDa is indicated by the arrow. (B) Jurkat T cells were either left unstimulated () or stimulated with the anti-CD2 mAb T112 and T113 at 37°C for 5 min. (+) before lysis. c-Cbl was then immunoprecipitated from the cell lysates. Tyrosine phosphorylated c-Cbl was detected by Western blot analysis using the anti-phosphotyrosine mAb 4G10 (upper panel). The membrane was stripped and reprobed using anti-c-Cbl antisera as described in Methods (lower panel).
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CD3 stimulation has been shown previously to induce the tyrosine phosphorylation of a 120 kDa protein that was later identified by immunoblotting as the proto-oncogene c-Cbl (10,28). To determine whether CD2 stimulation also increased the tyrosine phosphorylation of c-Cbl, the c-Cbl polypeptide was immunoprecipitated from lysates of either unstimulated or CD2-stimulated Jurkat cells. Immunoprecipitated proteins were resolved by SDSPAGE, transferred to PVDF membranes and detected using anti-phosphotyrosine antibody. Western blot analysis demonstrated that, while phosphorylated c-Cbl was detected in resting cells, the amount of phosphorylated protein quantitatively increased following CD2 stimulation (Fig. 1B
, upper panel). The membrane was stripped and reprobed with anti-c-Cbl antisera to demonstrate that equivalent amounts of c-Cbl polypeptide had been precipitated from each cell population (Fig. 1B
, lower panel).
Stimulation with anti-CD2 mAb increased the association of c-Cbl both with the p85 subunit of PI-3 kinase and with Crk
It has been suggested that a potential function of c-Cbl is to act as an adaptor protein, mediating the association of multiple signaling molecules. Two proteins that have been demonstrated to associate with c-Cbl after anti-CD3 mAb stimulation are the p85 subunit of PI-3 kinase and Crk (14,29). To investigate whether c-Cbl associated with the p85 subunit of PI-3 kinase and Crk after CD2 engagement, lysates from Jurkat cells stimulated with either anti-CD3 mAb or mitogenic pairs of anti-CD2 mAb were prepared; c-Cbl was immunoprecipitated from the cell lysates and the membranes were probed with anti-Crk antibody (Fig. 2A
). Following CD2 stimulation, an increased amount of Crk was detected in c-Cbl immunoprecipitates (Fig. 2A
, lane 2). As expected, stimulation of the cells with anti-CD3 mAb also induced the association of c-Cbl with Crk proteins (Fig. 2A
, lane 3) migrating at ~40 kDa. Based on migration, we were unable to discriminate precipitation of CrkII from Crk(L). The anti-Crk mAb (Transduction Laboratories) used in the above experiments recognizes the homologous CrkI and CrkII isoforms of the Crk family; whether it recognizes Crk(L) is not known. To analyze whether Crk(L) expressed in T cells also co-precipitated with c-Cbl after CD2 ligation, c-Cbl immunoprecipitations followed by immunoblotting with a specific anti-Crk(L) antibody were performed (Fig. 2B
). Cross-linking CD2 (Fig. 2B
, lane 3), like CD3 (Fig. 2B
, lane 2), on the surface of Jurkat T cells increased the amount of Crk(L) adaptor protein (lower panel) that co-precipitated with c-Cbl (middle panel). In addition we demonstrated the CD2-inducible association of c-Cbl with the p85 subunit of PI-3 kinase (Fig. 2B
, middle panel). Similar amounts of c-Cbl protein were immunoprecipitated in each lane, as confirmed by stripping and reprobing the membrane with anti-c-Cbl specific antibody (Fig. 2B
, middle panel). In reciprocal experiments, increased amounts of tyrosine phosphorylated c-Cbl and of p85 PI-3 kinase were demonstrated in Crk(L) immunoprecipitates following either CD2 or CD3 stimulation of Jurkat T cells (Fig. 2C
).

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Fig. 2. Stimulation with mitogenic pairs of anti-CD2 mAb increased the association of c-Cbl with Crk proteins and PI-3 kinase. (A) Jurkat T cells were either left unstimulated (lane 1), stimulated with the anti-CD2 mAb T112 and T113 (lane 2) or the anti-CD3 mAb OKT3 at 37°C for 5 min (lane 3) before lysis. c-Cbl was then immunoprecipitated from the cell lysates. c-Cbl (upper panel) and Crk proteins (lower panel) were visualized by Western blot using the anti-c-Cbl or anti-Crk antibodies respectively, as described in Methods. (B) Jurkat T cells were treated as in (A). c-Cbl was then immunoprecipitated from the cell lysates using an anti-c-Cbl antibody directly conjugated to agarose beads; precipitated proteins were separated by SDSPAGE, transferred to PVDF and probed with the anti-phosphotyrosine-specific antibody 4G10. The CD3and CD2-inducible tyrosine phosphorylation of a 120 kDa protein is shown (upper panel). The same membrane was stripped and reblotted with anti-c-Cbl and anti-p85 PI-3 kinase antibodies (middle panel) and anti-Crk(L) antibody (lower panel). (C) Jurkat T cells were treated as in (A). Crk(L) was immunoprecipitated from the cell lysates. The membrane was blotted with the anti-phosphotyrosine-specific antibody 4G10 demonstrating co-precipitation of a 120 kDa phosphoprotein (a). The membrane was stripped and reprobed with anti-c-Cbl antibody (b). While it is possible that a number of ~120 kDa proteins are tyrosine phosphorylated and thus detected in (a), a dominant ~120 kDa protein is detected by anti-c-Cbl antibody (b). Western analyses using anti-p85 PI-3 kinase (c) and anti-Crk(L) (d) antibodies were also performed on the same membrane.
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Overexpression of c-Cbl, requiring an intact PTB domain, inhibited both CD2and CD3-induced NF-AT transcriptional activity
c-Cbl has been shown to associate with a number of tyrosine kinases and intracellular signaling molecules, and has been shown, in the case of ZAP-70 kinase, to down-regulate kinase activity (21,23). We investigated whether overexpression of c-Cbl negatively regulated CD2-induced T cell activation as assessed by the induction of NF-AT transcriptional activity. Large T antigen-transformed Jurkat T cells were co-transfected transiently with a reporter plasmid of multimerized NF-AT sequences driving the luciferase gene and either empty vector, wild-type c-Cbl construct or the c-Cbl G306E mutant in which glycine at amino acid 306 in the PTB domain was replaced by glutamine. While overexpression of wild-type c-Cbl inhibited NF-AT transcriptional activity initiated by either anti-CD3 mAb or mitogenic pairs of anti-CD2 mAb, no inhibition was observed following overexpression of the G306E c-Cbl mutant (Fig. 3
). Indeed, we observed that transfection of the G306E c-Cbl mutant appeared to enhance NF-AT-transcriptional activity following anti-CD3-, but not anti-CD2-, dependent stimulation. These results are consistent with the hypothesis that c-Cbl functions as a negative regulator of both CD2and CD3-induced signal transduction pathways, and that the c-Cbl-mediated inhibitory effect requires an intact PTB domain, the domain that has been shown to mediate the interaction of c-Cbl with phosphorylated ZAP-70/Syk family members via a D(N/D)XpY motif (12,2124).

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Fig. 3. Overexpression of c-Cbl inhibited CD3and CD2-induced NF-AT activity. J-Tag Jurkat cells were transiently transfected with plasmids encoding the vector, wild-type c-Cbl or the Cbl PTB domain mutant G306E. Cells were co-transfected with the reporter plasmid p3xNF-AT-luc. Twelve hours after transfection, 106 cells were left unstimulated or stimulated with different dilutions of the mitogenic pair of anti-CD2 mAb T112 and T113 (A) or anti-CD3 mAb (B) as indicated. Cells were also stimulated with PMA (10 ng/ml) plus ionomycin (2 µM). The relative luciferase units are presented for each transfection condition as the percentage of maximal stimulation induced by PMA plus ionomycin.
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The c-Cbl RING finger domain was required for inhibition of CD2 signaling
c-Cbl has been shown to inhibit the activities of tyrosine kinases including the ZAP-70/Syk family members. Recently, it has been reported that the RING finger domain of c-Cbl, required for ZAP-70/Syk kinases inhibition (30), mediates protein ubiquitination (18,19,31,32). Isolated from a pre-B cell lymphoma, the Cbl mutant 70Z has previously been shown to contain an intact PTB domain, but a 17 amino acid deletion in the RING finger region. Overexpression of the Cbl 70Z mutant was shown to transform NIH-3T3 fibroblast cells and to induce T cell activation (30,33,34). We tested the effect of overexpression of Cbl 70Z on CD2-induced NF-AT transcriptional activation (Fig. 4
). In contrast to overexpression of the wild-type c-Cbl protein, overexpression of the Cbl 70Z mutant induced NF-AT transcriptional activation in the absence of any other stimulation. Interestingly, overexpression of 70Z mutant further enhanced CD2-induced NF-AT activity. These observations suggested that the PTB binding domain itself is not unique in its ability to regulate T cell activation; the RING finger domain of c-Cbl is also involved in the inhibition of CD2 signaling.

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Fig. 4. The c-Cbl RING finger mutant enhanced CD2-induced NF-AT activity. J-Tag Jurkat cells were transiently transfected with plasmids encoding the vector, wild-type c-Cbl or the 70Z Cbl mutant containing a 17 amino acid deletion in the RING finger region. Cells were co-transfected with the reporter p3xNF-AT-luc. Twelve hours after transfection, 106 cells were left unstimulated or stimulated with mitogenic pair of anti-CD2 as indicated. Cells were also stimulated with PMA (10 ng/ml) plus ionomycin (2 µM). The relative luciferase units are presented for each transfection condition as the percentage of maximal stimulation induced by PMA plus ionomycin.
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ZAP-70/Syk was involved in CD2-induced T cell activation
As mentioned above, c-Cbl has been shown to inhibit ZAP-70/Syk kinase activity; c-Cbl-dependent inhibition of ZAP-70/Syk activity correlated with inhibition of CD3-mediated T cell activation. The involvement of ZAP-70/Syk family kinases in CD2-induced signal transduction, however, has been controversial. Our data demonstrating the involvement of the PTB domain of c-Cbl, the domain reported to bind ZAP-70, in inhibition of CD2-mediated NF-AT transcriptional activity suggested a potential role for ZAP-70/Syk kinases in CD2-mediated T cell activation. Thus, we investigated whether CD2-stimulation induced the tyrosine phosphorylation of ZAP-70 in Jurkat cells. Jurkat cells were either left unstimulated or stimulated with mitogenic pairs of anti-CD2 mAb or with anti-CD3 mAb. Duplicate samples of cell lysates were prepared and subjected to immunoprecipitation using agarose conjugated anti-ZAP-70 antibody. Immunoprecipitated proteins were separated by SDSPAGE, transferred to PVDF membranes and subjected to Western blot analysis using either an anti-phosphotyrosine-specific mAb (Fig. 5A
, upper panel) or an anti-ZAP-70-specific antibody, the latter to demonstrate that similar amounts of protein were precipitated in each lane (Fig. 5A
, lower panel). Upon stimulation of Jurkat T cells with mitogenic pairs of anti-CD2 mAb, only a minimal (but reproducible) increase in tyrosine phosphorylation of ZAP-70 kinase was detected (Fig. 5A
, upper panel). Tyrosine phosphorylation was quantitated by densitometry and the mean (± SEM) of two independent experiments is shown (Fig. 5B
). Of note, following either CD2 or CD3 stimulation, ZAP-70 was found to co-precipitate with two tyrosine-phosphorylated proteins of ~120 and 2123 kDa. These proteins are likely to be c-Cbl and the TCR
chain respectively, although the amount of protein was below the limits of detection following stripping and reprobing the membrane.

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Fig. 5. Stimulation via mitogenic pairs of anti-CD2 mAb induced modest tyrosine phosphorylation of ZAP-70. (A) Jurkat T cells were either left unstimulated or stimulated in duplicate with either the anti-CD2 mAb T112 and T113 or the anti-CD3 mAb OKT3 at 37°C for 5 min. Cells were then lysed in 1% Brij 97 lysis buffer; post-nuclear lysates were incubated with an anti-ZAP-70-specific antibody directly conjugated to agarose beads, as described in Methods. Immunoprecipitated proteins were resolved by SDSPAGE, transferred to PVDF membranes and immunoblotted with either the anti-phosphotyrosine-specific antibody 4G10 (upper panel) or anti-ZAP-70 antibody (lower panel). Results are representative of two independent experiments. The heavily phosphorylated 5055 kDa band is the heavy chain of the anti-ZAP-70 antibody (data not shown). (B) Quantification of ZAP-70 tyrosine phosphorylation was determined by densitometry, as described in Methods. Results are expressed as the mean ± SE of two independent experiments.
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To test the requirement for ZAP-70/Syk family kinases in CD2-mediated downstream events, we used the variant Jurkat T cell line P116, deficient in expression of both ZAP-70 and Syk kinases (35 and data not shown). Neither CD2 nor CD3 ligation was able to stimulate NF-AT transcriptional activity in Jurkat P116 cells (Fig. 6A
). Furthermore, co-ligation of both CD3 and CD2 receptors did not bypass the requirement for ZAP-70/Syk kinase expression for induction of NF-AT transcriptional activity. Importantly, reconstitution of P116 with the wild-type ZAP-70 protein (P116-ZAP70) restored NF-AT transcriptional activity upon either CD3 or CD2 cross-linking. CD2and CD3-stimulated NF-AT-driven transcription were comparable between the parental Jurkat and the ZAP-70-reconstituted P116 cells, and ZAP-70 protein was expressed at levels approximately equivalent to the parental (Syk) Jurkat cell line (36 and data not shown). Of note, the additive effect of CD3 and CD2 co-stimulation on NF-AT transcriptional activation was also restored. Surface expression of CD2 and CD3 molecules did not differ significantly between these two cell lines, as assessed by immunofluorescent cytometry (Fig. 6B
). Our data demonstrate that ZAP-70 kinase expression is required not only for CD3but also for CD2-dependent NF-AT transcriptional activation.

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Fig. 6. Expression of ZAP-70 kinase was required for CD2and CD3-induced NF-AT transcriptional activity. (A) Either the ZAP-70/Syk-deficient Jurkat cells P116 or the wild-type ZAP-70-reconstituted cells P116-ZAP70 were transiently co-transfected with the p3xNF-AT-luc reporter plasmid and pRL-TK vector, which provides constitutive expression of Renilla luciferase, as described in Methods. Twenty-four hours after transfection, 106 cells were either left unstimulated or stimulated with either 1 µl T112 plus T113, plate-bound anti-CD3 mAb OKT3 or a combination of anti-CD2 mAb and anti-CD3, as indicated. Cells were also stimulated with PMA (10 ng/ml) plus ionomycin (2 µM). Results are reported as activity of firefly luciferase normalized to that of Renilla luciferase (to correct for transfection efficiency) in the lysate and expressed as the percentage of maximal stimulation induced by PMA plus ionomycin (0.82 and 0.50 arbitrary units for P116 and P116-ZAP70 cells respectively). Results are representative of three independent experiments. (B) Immunofluorescent flow cytometry depicting CD2 (left) and CD3 (right) expression on the surface of Jurkat, P116 and P116-ZAP70 cells.
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Discussion
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In this report, we have investigated the role of c-Cbl and of ZAP-70 in CD2-dependent signal transduction processes. Engagement of the CD2 surface receptor by combinations of anti-CD2 mAb induced the tyrosine phosphorylation of c-Cbl and, in turn, the enhanced association of c-Cbl with the Crk(L) protein and the p85 subunit of PI-3 kinase. Like stimulation via the TCRCD3 complex, CD2-mediated stimulation of NF-AT transcriptional activity, in transient overexpression experiments, was negatively regulated by c-Cbl; this negative regulation involved the c-Cbl PTB domain and the RING finger domain. The ZAP-70/Syk family of tyrosine kinases has been shown to be substrates of c-Cbl. Here we show that stimulation via CD2, like CD3, induced the tyrosine phosphorylation of ZAP-70, a kinase whose expression was required for the induction of NF-AT transcriptional activity.
Previous work has analyzed the role of CD2 co-stimulation of TCRCD3-dependent signaling. CD2 has been shown to be co-localized with CD3 and other signaling components in T cell glycoprotein-enriched membrane domains, or rafts (37). CD2 ligation by either mAb or ligand is known to enhance cytokine production and proliferation, and multicomponent signaling complexes have also been studied (3845). More recently, CD2 ligation by a single anti-CD2 mAb has been shown to enhance the CD3-dependent tyrosine phosphorylation of c-Cbl and of Shc (46). These investigators demonstrated the involvement of the p72 Syk tyrosine kinase, a member of the ZAP-70/Syk kinases, but did not study the role of ZAP-70 in Jurkat T cells (46). The role of CD2 engagement in the absence of direct TCRCD3 engagement was the focus of the current study. The Jurkat cells used in this current study are deficient in Syk kinase expression (35 and data not shown); neither we nor others have been able to generate stable transfectants of Syk kinase in Jurkat cells (36 and data not shown). The studies contained herein, therefore, do not address the role of Syk kinase or compare ZAP-70 to Syk kinase in CD2-dependent signaling.
The CD2 and CD3 surface receptors both induce tyrosine phosphorylation of a number of intracellular proteins, and both have been shown to induce the phosphorylation and activation of the Src-related kinases Lck and Fyn, and the Tec-related kinases Itk (4751). Here we show that ZAP-70 and c-Cbl are also activated by CD2 stimulation. While the induction of phosphorylation does differ quantitatively between CD2 and CD3 signaling, these signaling intermediates do not differ qualitatively. These intermediates may be important for signaling to cytokine production and T cell proliferation, outcomes of T cell engagement shared between the two cell surface receptors. These intermediates are unlikely, however, to explain the differences between CD2 and CD3 in function, e.g. the ability of CD2 to mediate reversal of T cell anergy (9) or to regulate T cell IL-12 responsiveness (7,8).
We have shown that c-Cbl is involved CD2-dependent signaling as it is in CD3 signaling and both required the PTB domain of c-Cbl. Recently, c-Cbl has been shown to down-regulate a number of substrates, including platelet-derived growth factor receptor (52), epidermal growth factor receptor (53), colony-stimulating factor-1 receptors (54) and Syk family protein kinases (24) via ubiquitination. This effect of c-Cbl on protein turnover required its RING finger domain, a potential ubiquitin-protein ligase and was abolished in the Cbl 70Z mutant protein (18,19,31,32). With an intact PTB domain but a disrupted ubiquitin-protein ligase domain, the 70Z Cbl mutant acts as a dominant negative protein (30,33,34). Consequently, CD2-induced NF-AT transcriptional level is enhanced in cells transfected with 70Z mutant. Our data demonstrate that both the PTB domain and the RING finger domain of c-Cbl are involved in the regulation of CD2 signaling. The direct effect of c-Cbl, however, on CD2-stimulated degradation of Syk/ZAP-70 kinase remains to be determined.
It is also possible that alternative mechanisms of down-regulation by c-Cbl exist. c-Cbl has been shown to bind to Crk(L)/C3G that, in turn, is able to activate the small GTP-binding protein Rap1 (5557). In several model systems, Rap1 has been shown to inhibit Ras signaling by competing with downstream substrates (56,58,59). C3G, GTP-bound Rap1 and Raf are also detected in CD3-stimulated c-Cbl complexes (16). Overexpression of a constitutively active Rap1 mutant in Jurkat cells inhibited IL-2 transcription induced by TCRCD3 and CD28 stimulation, suggesting that the c-CblCrkC3GRap1 pathway was a negative regulator of signaling (60). Furthermore, increased Rap1 activity has been demonstrated in anergic cells (60). Anti-CD2 stimulation induced an association of c-Cbl with Crk(L); whether this association initiates the c-CblCrkC3GRap1 pathway and negative regulation of Ras signaling in anergic cells remains to be shown. It should be noted, however, that overexpression of the c-Cbl PTB domain mutant G306E and the RING finger mutant 70Z Cbl did not inhibit CD2-induced NF-AT transcriptional activation. If the sites of c-Cbl phosphorylation are conserved following stimulation of the CD2 and CD3 receptor, our findings imply that the c-Cbl association with Crk(L) is not sufficient to inhibit CD2-driven NF-AT transcription, data consistent with the report that the function of oncogenic 70Z Cbl is independent of the Crk(L) association (30).
Our data regarding the involvement of ZAP-70 in CD2-dependent signaling differs from some, but not all, previously published reports (37,6164). Others have been unable to demonstrate ZAP-70 tyrosine phosphorylation following CD2 stimulation (61). We have shown that ZAP-70 phosphorylation following CD2 stimulation is less robust than following CD3 stimulation but nevertheless detectable; the specific conditions of stimulation, the Jurkat subclone, or the sensitivity of the reagents might differ. In our opinion, the positive result was informative. Because demonstration of tyrosine phosphorylation is necessarily dependent upon the threshold of the assay, we investigated the responsiveness of a Jurkat subclone that did not express either ZAP-70 or Syk kinases. In this line, CD2 was unable to induce NF-AT transcriptional activation. Transfection of ZAP-70 restored NF-AT transcription. Our data confirms that of Koyasu et al. (64) who used Syk+ T lymphocytes from a ZAP-70-deficient patient to demonstrate that ZAP-70 was required for CD2-dependent proliferation, cytokine production and NF-AT transcription. Taken together, these data would imply that ZAP-70 is required for CD2-mediated NF-AT-dependent transcription. Reinherz and colleagues have reported an Lck Jurkat variant in which stimulation via CD2 induced IL-2 production and NF-AT transcription in the apparent absence of ZAP-70 tyrosine phosphorylation (65). It is important to note that these cells had been sorted and subcloned repeatedly; the modulation of other signaling intermediates was not analyzed nor was their dependence on ZAP-70 expression for NF-AT transcription. Multiple signaling pathways (and cells treated under very different conditions) may result in different functional outcomes.
We have repeatedly observed that Jurkat cells transfected with NF-AT-luc and control vector alone are more responsive to CD2 than to CD3 stimulation as measured by NF-AT-dependent transcriptional activity. The ability of CD2 to stimulate greater NF-AT-dependent transcription does not correlate with the observation that CD3 stimulation induces greater tyrosine phosphorylation of intracellular proteins. The protein tyrosine kinase activity in the cells is not an appropriate surrogate for downstream functional events. Indeed, in anergic cells, a number of intracellular proteins including c-Cbl become hyperphosphorylated but fail to induce IL-2 production (66).
c-Cbl has been suggested to be a negative regulator that plays a role in the induction of T cell anergy. In certain model T cell clones, CD2 ligation was able to reverse T cell anergy induced by engagement of the TCRCD3 complex in the absence of co-stimulation (9). To a first approximation, the demonstration that CD2-, like CD3-, induced signaling is negatively regulated by c-Cbl would appear to counter the hypothesis that CD2 is able reverse the anergic state. It is unlikely that the relatively modest quantitative differences in CD2versus CD3-stimulated c-Cbl phosphorylation, c-CblCrk association or NF-AT-dependent transcriptional activation would control the choice between T cell unresponsiveness and immune competence. It is possible that c-Cbl is involved in the induction of unresponsiveness but not in its reversal. CD2 has a large cytoplasmic domain that associates with CD2AP, Itk and the Src family PTK (67,68 and data not shown). It will be interesting to determine whether these proteins or other CD2-associated molecules modulate the anergic state.
 |
Abbreviations
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---|
PI |
phosphatidylinositol |
PMA |
phorbol myristate acetate |
PTB domain |
phosphotyrosine-binding domain |
PTK |
protein tyrosine kinase |
 |
Notes
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3 Present address: Department of Immunology and Hemostasis, Genetics Institute, Inc., Andover, MA 01810, USA 
The first two authors contributed equally to this work
Transmitting editor: T. Watanabe
Received 19 June 2000,
accepted 22 September 2000.
 |
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