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J. Biol. Chem., Vol. 276, Issue 49, 45654-45661, December 7, 2001

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T Cell Regulation of p62^dok (Dok1) Association with Crk-L^*

Maria Paola Martelli, Jonathan Boomer, Ming Bu, and Barbara E. Bierer^§

From the Laboratory of Lymphocyte Biology, NHLBI, National Institutes of Health, Bethesda, Maryland 20892

Received for publication, June 21, 2001, and in revised form, September 10, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

In addition to engagement of the T cell receptor-CD3 complex, T lymphocytes can be activated by a variety of cell surface molecules including the ~50-kDa surface receptor CD2. While the majority of biochemical signaling elements are triggered by either CD2 or TcR-CD3 receptors, a small number of proteins are engaged by only one receptor. Recently, p62^dok (Dok1), a member of the Dok family of adapter molecules, has been reported to be activated by CD2 and not by CD3 engagement. Here we have examined the role of p62^dok in CD2-dependent signaling in Jurkat T cells. As previously reported, we find that ligation of the CD2 molecule by mitogenic pairs of anti-CD2 mAbs led to phosphorylation of p62^dok. While CD2-induced p62^dok tyrosine phosphorylation was independent of both the p36/38 membrane adapter protein linker of activated T cells (LAT) and the ZAP70/Syk family of kinases, it was dependent upon the Src family of kinases including Lck and Fyn. We find further that CD2 engagement induced the association of tyrosine-phosphorylated p62^dok to Crk-L. The CD2-dependent association of p62^dok to Crk-L was independent of expression of the ZAP70/Syk family of kinases. Of note, while T cell receptor-CD3 engagement did not induce either p62^dok phosphorylation or Crk-L association in Jurkat T cells, it did inhibit CD2-dependent p62^dok-Crk-L complexes; this TcR-CD3-mediated regulation was dependent upon ZAP70 kinase activity. Our data suggest that phosphorylation of p62^dok and its interaction with other signaling proteins may depend upon integrated signals emanating from the CD2 receptor, utilizing a ZAP70/LAT-independent pathway, and the TcR-CD3 receptor, which is ZAP70/Syk-dependent.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

T cell antigen receptor (TCR)¹ engagement by specific antigen embedded within MHC proteins or by anti-CD3 mAb triggers a complex network of biochemical signal transduction events that direct the functional program of the T cell (1). Early T cell biochemical signaling involves Src (Lck and Fyn)-related tyrosine kinase activity leading to ZAP70 kinase activity (2-5). Engagement of TcR-CD3 complexes leads to the recruitment of adaptor molecules and enzymes (protein tyrosine and serine/threonine kinases and phosphatases) in a multicomponent signaling complex in specialized membrane domains termed lipid rafts (6, 7). Signal transduction culminates in transcriptional activation leading to cytokine production and cellular proliferation.

The canonical early biochemical events that follow TcR-CD3 engagement may be mimicked by ligation, via mitogenic combinations of mAbs, of the CD2 molecule, a 50-55-kDa glycoprotein expressed on the surface of essentially all human T and natural killer cells (8-10). Pairs of anti-CD2 mAbs induce activation of the Src (Lck/Fyn) kinases, ZAP70 kinase, the adaptor molecules LAT and Cbl, and phosphatidylinositol 3-kinase, inter alia, leading to transcriptional activation of nuclear factor of activated T cells (NFAT) and cytokine (e.g. interleukin-2) production (11-20). Indeed, the remarkable similarity between these signaling pathways led to the observation that the CD2 pathway required expression of  chain family members of the TcR-CD3 complex for activity (12, 21-27). While CD2 is a coreceptor that functions to enhance T cell adhesion (23, 28-30), relatively few intracellular signaling events have been characterized that differentiate CD2 signaling from TcR-CD3 engagement (31, 32).

In order to identify CD2-dependent (apparently TcR-CD3-independent) signaling events, we characterized early biochemical signaling in the model Jurkat T cell line deficient in expression of the LAT adaptor molecule, a molecule previously shown to be required for NFAT and interleukin-2 transcriptional activation through both the CD2 and TcR-CD3 receptors and to be necessary for normal T cell development. We demonstrate preferential, CD2-dependent tyrosine phosphorylation of a 62-kDa protein identified as p62^dok. CD2-mediated p62^dok tyrosine phosphorylation was also independent of expression of the ZAP70/Syk kinases but, as previously shown (33), dependent upon Src kinase activity. We demonstrate further that CD2-mediated signal transduction results in the association of p62^dok with Crk-L but that this association is negatively regulated by CD3 engagement, the latter utilizing a signal transduction pathway that requires expression of ZAP70 kinase.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cells and Reagents-- Jurkat cell lines were cultured in RPMI 1640 (Life Technologies, Inc.) supplemented with 10% heat-inactivated fetal calf serum, 2 mM L-glutamine, 10 mM Hepes, 100 units/ml penicillin, 100 µg/ml streptomycin (Life Technologies), and 50 µM 2-mercaptoethanol (Bio-Rad), termed complete RPMI medium. The Jurkat T cell leukemia cell line (clone J77) was a gift of K. Smith (Cornell University, New York, NY). The ZAP70/Syk-deficient Jurkat clone, P116, and the ZAP70-reconstituted clone, P116-ZAP70 (34), were the kind gift of Robert Abraham (Duke University, Durham, NC). The LAT-deficient Jurkat cells, ANJ3, and the LAT-reconstituted clone, ANJ3-LATwt (35), were the gift of L. E. Samelson (NCI, National Institutes of Health, Bethesda, MD). The JCaM1.6 cells reconstituted with wild type Lck (JCaM1.6-Lck) (36) were the generous gift of David B. Straus (University of Chicago). The Lck kinase was expressed under the control of a tetracycline-repressible TetR-VP16 fusion protein in the JCaM1.6 transfectant (36) and was grown in complete RPMI medium containing 1 mg/ml Geneticin (Life Technologies, Inc.) and 250 µg/ml hygromycin B (Calbiochem). Treatment with tetracycline (Calbiochem) (1 µg/ml) for at least 4 days was used to down-regulate Lck expression. Neither cell viability nor CD3, CD2, or CD28 cell surface receptor expression was affected by tetracycline treatment (data not shown) (19).

Surface expression of both CD3 and CD2 on all cell lines was routinely evaluated by direct immunofluorescence, as previously described (19, 20), using a Coulter Epics flow cytometer (Beckman Coulter Inc.). CD3 and CD2 expression were equivalent between wild type Jurkat, P116, and P116-ZAP70 and between ANJ3 and ANJ3-LATwt (data not shown). CD3 and CD2 cell surface expression remained constant with time.

The purified rabbit anti-p62^dok antiserum was the generous gift of N. Carpino and R. Kobayashi (Cold Spring Harbor, New York, NY). The anti-CD2 mAbs T11₂ and T11₃ were the kind gift of E. Reinherz (Dana-Farber Cancer Institute, Boston, MA); the murine anti-human CD3 mAb OKT3 was purchased from the American Type Culture Collection (Manassas, VA) and used as purified ascites fluid. The rabbit polyclonal anti-PI3K (p85 subunit) antiserum, the anti-phosphotyrosine mAb 4G10, and the murine anti-Crk-L mAb were purchased from Upstate Biotechnology, Inc. (Lake Placid, NY); the rabbit polyclonal anti-Crk-L and anti-Cbl antisera, the anti-phosphotyrosine (PY99) mAb directly conjugated to agarose beads, and unconjugated protein A-agarose beads were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

Cell Stimulation and Immunoprecipitation-- For stimulation, Jurkat cells (1 × 10⁷/test) were washed in RPMI 1640 (without additives), resuspended in serum-free RPMI 1640 medium, and incubated for 30 min at 37 °C. Cells were then either left unstimulated, stimulated with OKT3 (1 µg), or stimulated with T11₂ plus T11₃ purified ascites (1:100 dilution) at 37 °C for the indicated times followed by rapid centrifugation and resuspension of the pellet in 1 ml of ice-cold lysis buffer (1% Brij 97, 150 mM NaCl, 25 mM Tris, pH 7.5, 1 mM EDTA, 1 mM Na₃VO₄, 10 µg/ml leupeptin, 10 µg/ml aprotinin, and 1 mM phenylmethylsulfonyl fluoride). After incubation on ice for 20 min, cell lysates were clarified by centrifugation at 14,000 × g for 10 min at 4 °C. When indicated, 30 µl of postnuclear lysate from each sample was set aside prior to immunoprecipitation.

For immunoprecipitations, 1 µg of specific antibody and 20 µl of protein A-agarose beads were added together to the clarified cell lysates and incubated at 4 °C for 2 h. Clarified cell lysates from the same conditions of stimulation incubated with 20 µl protein A-agarose beads alone served as the control for carryover of stimulating mAbs, as indicated. For immunoprecipitation of phosphotyrosine-containing proteins, 20 µl of anti-Tyr(P) (PY99) directly conjugated to agarose beads were used. The beads were then washed three times with washing buffer (0.1% Brij 97, 150 mM NaCl, 25 mM Tris pH 7.5, 1 mM Na₃VO₄). Proteins were boiled in SDS sample buffer, separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to polyvinylidene difluoride (Millipore Corp., Bedford, MA), and probed with primary antibodies followed by horseradish peroxidase-conjugated secondary antibodies. Polypeptides recognized in the Western blot were detected using the enhanced chemiluminescence (ECL) methods according to the manufacturer's instructions (Amersham Pharmacia Biotech). Each result shown is representative of between two and six independent experiments; representative blots are shown.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

p62^dok Was Tyrosine-phosphorylated upon CD2 but Not CD3 Stimulation in Jurkat T Cells Independently of either LAT Protein or ZAP70/Syk Kinase Expression-- We have previously shown that CD2, like TcR-CD3, required both the LAT adapter protein and ZAP70/Syk kinase expression for NFAT transcriptional activation (19, 20). In order to characterize early signaling moieties recruited by CD2 and not by TcR-CD3 engagement, we studied Jurkat T cells that were deficient in expression of the adapter protein LAT or ZAP70/Syk kinases, molecules known to be required for competent signal transduction leading to NFAT and interleukin-2 transcription (35-37). LAT-deficient mutants of the Jurkat T-cell line (ANJ3) were compared with the LAT-reconstituted clone, ANJ3-LATwt. Cells were left unstimulated or stimulated via mitogenic pairs of anti-CD2 mAbs or anti-CD3 mAb directed against the TCR-CD3 complex; anti-phosphotyrosine antisera were used to probe postnuclear lysates (Fig. 1A). A prominent phosphoprotein of 62-64 kDa was observed in the LAT-deficient cells, diminished upon reexpression of the LAT adapter protein, and enhanced by CD2 engagement. Comparably treated cells were subjected to anti-phosphotyrosine immunoprecipitation followed by Western blot analysis using an anti-p62^dok specific antibody (Fig. 1B) to demonstrate that the 62-64-kDa protein was detected by p62^dok antisera. Reciprocal immunoprecipitation experiments confirmed that p62^dok was indeed tyrosine-phosphorylated upon CD2 stimulation independently of LAT kinase expression (Fig. 1C, upper panel). Reprobing the stripped membrane with anti-p62^dok antisera confirmed equivalent loading of the immunoprecipitated protein in all lanes (Fig. 1C, lower panel). This demonstration of CD2-induced p62^dok tyrosine phosphorylation in Jurkat T cells is consistent with an earlier observation by Hubert et al. (31), who identified a p62 RasGAP-associated protein phosphorylated by CD2 engagement, an observation recently confirmed by others (33, 38-40). Here, we extend these findings to demonstrate that the induction of p62^dok tyrosine phosphorylation was independent of LAT expression; indeed, the basal and stimulated tyrosine phosphorylation of p62^dok was enhanced in the absence of LAT expression, suggesting reciprocal regulation of the protein by an as yet uncharacterized pathway.

View larger version (39K): [in this window] [in a new window]   Fig. 1.   CD2-mediated p62dok tyrosine phosphorylation is independent of LAT protein expression. A, the LAT-deficient Jurkat T cell line ANJ3 and ANJ3 reconstituted with wild-type LAT (ANJ3-LATwt) (106 cells) were either left unstimulated or stimulated at 37 °C for 5 min using anti-CD3 or pairs of anti-CD2 mAbs and lysed in 100 µl of 1% Brij 97 lysis buffer. 30 µl of PNL (0.3 × 106 cell equivalents) were separated by SDS-PAGE and immunoblotted with the anti-phosphotyrosine mAb 4G10. Tyrosine phosphorylation of a 62-64-kDa protein is shown by the arrow. B and C, either ANJ3 or ANJ3-LATwt cells (107 cells/lane) were stimulated as indicated in A. PNL were immunoprecipitated using either 20 µl of anti-phosphotyrosine (PY99) mAb-agarose-conjugated antibody (B) or 1 µg of rabbit anti-p62dok antibody (C), separated by SDS-PAGE, and immunoblotted with 4G10 (upper panels). The same membranes were stripped and reblotted with a rabbit anti-p62dok-specific antibody (lower panels). The heavy chain of the precipitating antibody (C, lower panel) is indicated by the asterisk.

Given that LAT phosphorylation and function depends upon the ZAP70/Syk kinase family in T cells, we wished to determine whether CD2-dependent p62^dok tyrosine phosphorylation would be dependent upon ZAP70 kinase activity. Antiphosphotyrosine immunoprecipitates from resting or stimulated wild type Jurkat (J77) cells or the ZAP70/Syk-deficient Jurkat mutant (P116) cells were analyzed using an anti-p62^dok specific antibody (Fig. 2A). p62^dok was basally tyrosine-phosphorylated in Jurkat T cells. Upon CD2 engagement, but not upon CD3 ligation, the amount of tyrosine-phosphorylated p62^dok precipitated was increased, and the appearance of a slower migrating species was observed. The same pattern, but even greater tyrosine phosphorylation following CD2 ligation, was observed in the ZAP70/Syk kinase-deficient P116 T cells. Reciprocal immunoprecipitation confirmed that CD2-dependent p62^dok tyrosine phosphorylation was independent of ZAP70/Syk kinase expression (Fig. 2B).

View larger version (20K): [in this window] [in a new window]   Fig. 2.   The CD2-mediated p62dok tyrosine phosphorylation is independent of ZAP70/Syk kinase expression. A, either wild type Jurkat T cells (J77) or the ZAP70/Syk-deficient Jurkat cells, P116 (107 cells/lane), were left unstimulated or stimulated with either the mitogenic pair of anti-CD2 mAbs T112 and T113 or anti-CD3 mAb OKT3 at 37 °C for 5 min. PNL were prepared as described under "Experimental Procedures," subjected to immunoprecipitation with 20 µl of agarose-conjugated anti-phosphotyrosine (PY99) mAb, separated by SDS-PAGE, and immunoblotted with a rabbit anti-p62dok-specific antibody. B, P116 cells (107 cells/lane) were stimulated as indicated in A. PNL were then subjected to immunoprecipitation with 1 µg of rabbit anti-p62dok antibody, separated by SDS-PAGE, and immunoblotted with the anti-phosphotyrosine antibody 4G10 (upper panel). The membrane was then stripped and reblotted with anti-p62dok antibody (lower panel). The heavy chain of either the stimulating (upper panel) or the precipitating antibody (lower panel) is indicated by an asterisk.

CD2-mediated p62^dok Tyrosine Phosphorylation was Dependent on Lck Expression in Jurkat T Cells-- The identity of the upstream tyrosine kinase required for CD2-dependent tyrosine phosphorylation of p62^dok remains unclear. Previous reports have concluded that the Src-related Lck kinase is able to mediate p62^dok tyrosine phosphorylation (33); murine p62^dok was tyrosine-phosphorylated in human CD2-expressing cells derived from Fyn^+/+ mice (38). To explore the specific role of Lck in CD2-mediated p62^dok tyrosine phosphorylation, we used the CD2-positive Lck-reconstituted clone of JCaM1.6, the Lck-deficient Jurkat cell line in which Lck kinase was expressed under the control of a tetracycline-repressible TetR-VP16 fusion protein. Prolonged (~8 days) tetracycline (1 µg/ml) treatment of these cells induced complete inhibition of Lck expression, as evaluated by Western blot (data not shown) (19). Untreated or tetracycline-treated cells were either left unstimulated or stimulated via either CD2 or CD3 receptors for the indicated periods of time, and the pattern of tyrosine-phosphorylated proteins was analyzed. As expected, both basal and receptor-mediated protein tyrosine phosphorylation were severely diminished by inhibition of Lck expression. In particular, the CD2-induced tyrosine phosphorylation of a 62-64-kDa protein (Fig. 3A), recognized as p62^dok by immunoprecipitation studies (Fig. 3B), was affected. In these Jurkat T cells, Fyn kinase is expressed at low levels. The residual phosphorylation of p62^dok and of other proteins (e.g. p97 and p120) observed upon CD2 and not upon CD3 stimulation in the tetracycline-treated cells (Fig. 3, longer exposure) may be due to CD2-dependent Fyn activation. Taken together, these data demonstrate that, in Jurkat T cells, CD2-mediated p62^dok tyrosine phosphorylation was regulated, at least in part, by the Src-kinase family member Lck.

View larger version (32K): [in this window] [in a new window]   Fig. 3.   Lck kinase expression is required for p62dok tyrosine phosphorylation upon CD2 stimulation in Jurkat T cells. A, the Lck-deficient JCaM 1.6 cells reconstituted with wild type Lck (JC1-Lckwt) were either left untreated (Ø) or treated with tetracycline (Tet) 1 µg/ml for 8 days. Either untreated or Tet-treated cells (106 cells/test) were then either left unstimulated or stimulated via either CD2 or CD3 for the indicated periods of time. Cells were lysed in Brij 97 1% lysis buffer, and PNL from equivalent cell numbers (0.3 × 106) were separated by SDS-PAGE and immunoblotted with 4G10. Tyrosine phosphorylation of a 62-64-kDa protein is shown by the arrow. A longer exposure of the same gel is shown at the right. B, Lck-reconstituted JCaM 1.6 cells were treated as in A and then either left unstimulated or stimulated via either CD3 or CD2 for the indicated time periods. PNL were subjected to immunoprecipitation with 20 µl of agarose-conjugated anti-phosphotyrosine (PY99) mAb, separated by SDS-PAGE, and immunoblotted with a rabbit anti-p62dok-specific antibody.

Tyrosine-phosphorylated p62^dok Coprecipitated with Crk-L upon CD2 Stimulation in T Cells-- Hyperphosphorylated p62^dok was recently shown to coprecipitate with Crk-L in Bcr/Abl-transformed cells (41, 42); whether p62^dok associated with Crk-L in the absence of the Bcr/Abl kinase or in the absence of transformation was not examined. We have recently shown that, like CD3 stimulation, CD2 stimulation of Jurkat T cells induced the association of Crk-L with the proto-oncogene c-Cbl and the p85 subunit of PI3K in a multicomponent complex (20). Here we questioned whether the CD2-stimulated tyrosine-phosphorylated p62^dok protein in Jurkat T cells was found to associate with Crk-L, Cbl, or the p85 subunit of PI3K. For this purpose, wild type Jurkat T cells were either left unstimulated or stimulated for 5 min via either TcR-CD3 or CD2, after which Crk-L protein was immunoprecipitated from cell lysates using a rabbit anti-Crk-L-specific antibody. As previously described (20), the phosphoproteins p120 c-Cbl and the p85 subunit of PI3K were found to coprecipitate with Crk-L upon either TcR-CD3 or CD2 stimulation (Fig. 4A, upper panel). Upon CD2 stimulation but not upon TcR-CD3 engagement, a phosphoprotein of ~62-64-kDa was found to coprecipitate with Crk-L (Fig. 4A, upper panel). The 62-64-kDa phosphoprotein was identified as the docking protein p62^dok by reblotting the stripped membrane with a rabbit anti-p62^dok-specific antibody (Fig. 4A, lower panel). Since the identification of p62^dok was performed on stripped membranes, further analysis was performed. Immunoprecipitations using a mouse anti-Crk-L-specific antibody followed by protein A-agarose or with protein A-agarose alone (to control for carryover of the stimulating antibody) from clarified postnuclear lysates of unstimulated or CD3- or CD2-stimulated Jurkat T cells were blotted directly with a rabbit anti-p62^dok-specific antibody (Fig. 4B, upper panel). Again, p62^dok protein was detected in Crk-L immunoprecipitates specifically from CD2-stimulated cells (Fig. 4B, upper panel). Reblotting the stripped membrane with an anti-phosphotyrosine antibody demonstrated that the Crk-L-associated p62^dok was tyrosine-phosphorylated (Fig. 4B, middle panel). Quantitatively similar amounts of Crk-L protein were immunoprecipitated in each lane (Fig. 4B, lower panel). Kinetic analysis demonstrated that, following CD2 stimulation, p62^dok associated with Crk-L transiently, peaking at early (2-5 min) time points and undetectable 15 min after stimulation (data not shown).

View larger version (53K): [in this window] [in a new window]   Fig. 4.   Tyrosine-phosphorylated p62dok coprecipitates with Crk-L upon CD2 stimulation of Jurkat T cells. A, wild type Jurkat cells (107cells/test) were left unstimulated or stimulated with either T112 plus T113 or OKT3 for 5 min. Cells were then lysed, and clarified PNL were immunoprecipitated with 1 µg of rabbit anti-Crk-L antibody, resolved by SDS-PAGE, and immunoblotted with the anti-phosphotyrosine 4G10 mAb (upper panel). The bands corresponding to tyrosine-phosphorylated c-Cbl, p85 PI-3 kinase (p85PI3K), and p62dok, confirmed by reprobing the stripped membrane with specific antisera (not shown), are shown at arrows. The same membrane was stripped and reblotted with a rabbit anti-p62dok specific antibody (lower panel). B, wild type Jurkat cells were stimulated as in A. Postnuclear lysates were immunoprecipitated with 1 µg of mouse anti-Crk-L antibody (lanes 1-3) or incubated with protein A-agarose alone (lane 4-6), resolved by SDS-PAGE, and immunoblotted with a rabbit anti-p62dok-specific antibody (upper panel). The stripped membrane was probed with the anti-phosphotyrosine 4G10 mAb (middle panel). Comparable amounts of Crk-L protein were immunoprecipitated, confirmed by blotting the stripped membrane with a rabbit anti-Crk-L-specific antibody (lower panel).

To determine whether the Crk-L-p62^dok complex coprecipitated c-Cbl and/or the p85 subunit of PI3K in a multicomponent complex, we performed a series of reciprocal immunoprecipitation experiments. While both CD2 and CD3 stimulation induced the association of both c-Cbl and p85 PI3K with Crk-L (data not shown) (20), no p62^dok protein was detected in c-Cbl or p85 PI3K immunoprecipitates (data not shown). While these negative results do not preclude an association under different experimental conditions, they leave open the possibility that 1) the p62^dok-Crk-L complex is distinct from the Crk-L-c-Cbl-p85^PI3K complex previously described or 2) the association of c-Cbl or p85 PI3K with p62^dok is too weak to withstand the conditions of immunoprecipitation.

The CD2-induced p62^dok/Crk-L Association Was Independent of LAT Protein and ZAP70/Syk Kinase Expression-- Knowing that CD2-mediated p62^dok tyrosine phosphorylation was independent of LAT protein expression (Fig. 2), we asked whether the p62^dok/Crk-L association was also independent of LAT. Crk-L immunoprecipitates from resting or CD2- or CD3-stimulated LAT-deficient Jurkat cells ANJ3 and the LAT-reconstituted clone ANJ3-LATwt were immunoblotted with an anti-phosphotyrosine-specific antibody (Fig. 5, upper panel). Independent of LAT protein expression, phosphoproteins of 120 and 85 kDa, corresponding to c-Cbl and p85^PI3K, respectively (demonstrated by reblotting the stripped membrane with specific antibody; data not shown), coprecipitated with Crk-L. A 62-64-kDa phosphoprotein, recognized as p62^dok (data not shown), was also detected in the complex upon CD2 stimulation of either the LAT-deficient or LAT-reconstituted Jurkat cells. Similar amounts of Crk-L were immunoprecipitated in each lane, as confirmed by stripping and reprobing the membrane with an anti-Crk-L specific antibody (Fig. 5, lower panel). A similar experiment was performed using the ZAP70/Syk-deficient Jurkat T cell line P116 and the ZAP70-reconstituted cells P116-ZAP70. As shown in Fig. 6, p62^dok coprecipitated with Crk-L upon CD2 stimulation independently of ZAP70/Syk kinase expression.

View larger version (50K): [in this window] [in a new window]   Fig. 5.   Tyrosine-phosphorylated p62dok coprecipitates with Crk-L upon CD2 stimulation of Jurkat T cells independently of LAT protein expression. The LAT-deficient Jurkat T cell line ANJ3 and the ANJ3 reconstituted with wild-type LAT (ANJ3-LATwt) Jurkat cells (107 cells/lane) were left either unstimulated or stimulated with either T112 + T113 or OKT3 for 5 min. Crk-L immunoprecipitation was performed as described in the legend to Fig. 4A. Crk-L immunoprecipitates were resolved by SDS-PAGE and immunoblotted with the anti-phosphotyrosine 4G10 mAb (upper panel). The stripped membrane was blotted with an anti-Crk-L-specific antibody to confirm immunoprecipitation of comparable amounts of Crk-L protein (lower panel). The bands corresponding to tyrosine-phosphorylated c-Cbl, p85PI3K, and p62dok are shown by the arrow.

View larger version (55K): [in this window] [in a new window]   Fig. 6.   Tyrosine-phosphorylated p62dok coprecipitates with Crk-L upon CD2 stimulation of Jurkat T cells independently of ZAP70/Syk kinases expression. The ZAP70/Syk-deficient Jurkat T cell line P116 and the P116 reconstituted with wild-type ZAP70 (P116-ZAP70) Jurkat cells (107 cells/test) were left either unstimulated or stimulated with either OKT3 for 5 min or T112 + T113 for 2 and 5 min. Crk-L immunoprecipitation was performed as described above. Crk-L immunoprecipitates were then resolved by SDS-PAGE and immunoblotted with a rabbit anti-p62dok specific antibody (upper panel). The same membrane was also blotted with an anti-Crk-L-specific antibody (lower panel).

TcR-CD3 Engagement Regulated the CD2-dependent p62^dok/Crk-L Association-- While the functional role of the CD2-induced p62^dok-Crk-L complex in T cells has not yet been elucidated, it is clear that CD2 functions as a coreceptor in cellular adhesion and to modulate signal transduction events in TcR-CD3-stimulated cells. Because our data supported a model in which CD2 regulated p62^dok/Crk-L association independently of TcR-CD3 (and ZAP70) engagement, we questioned whether TCR-CD3 ligation modulated the CD2-mediated and -specific association of p62^dok with Crk-L. Wild type Jurkat T cells were either left unstimulated or stimulated for 2 and 5 min with either anti-CD3 mAb, mitogenic pairs of anti-CD2 mAbs or a combination of anti-CD3 and pairs of anti-CD2 mAbs. Clarified postnuclear lysates were subjected to anti-Crk-L-specific immunoprecipitation and blotted directly with an anti-p62^dok-specific antibody (Fig. 7, upper panel). Surprisingly, compared with CD2-stimulated cells, the amount of p62^dok that coprecipitated with Crk-L upon stimulation via both CD2 and CD3 was significantly reduced. The stripped membrane was blotted with an anti-Crk-L-specific antibody to confirm immunoprecipitation of equal amounts of protein (Fig. 7, lower panel). These data suggested that ligation of the TcR-CD3 complex modulated the quantitative association of p62^dok with Crk-L following CD2 stimulation.

View larger version (59K): [in this window] [in a new window]   Fig. 7.   Modulation of CD2-induced p62dok/Crk-L association by TCR-CD3 stimulation. Wild type Jurkat cells (107 cells/test) were left unstimulated or stimulated with either the anti-CD2 mAbs T112 + T113 or the anti-CD3 mAb OKT3 or a combination of T112 + T113 and OKT3 for 2 and 5 min. Cells were then lysed, and clarified PNL were immunoprecipitated with 1 µg of mouse anti-Crk-L antibody, resolved by SDS-PAGE, and immunoblotted with a rabbit anti-p62dok-specific antibody (upper panel). Immunoprecipitation of comparable amounts of Crk-L protein was confirmed by blotting the membrane with a rabbit anti-Crk-L-specific antibody (lower panel).

TcR-CD3 Regulation of CD2-induced p62^dok/Crk-L Association Required ZAP70/Syk Kinase Expression-- Many of the downstream signal transduction events mediated by TcR-CD3 engagement appear to require ZAP70/Syk kinase expression (2, 34). We wished to determine whether ZAP70/Syk kinase expression was required for TcR-CD3-mediated regulation of the CD2-induced p62^dok/Crk-L association. In the absence of ZAP70/Syk kinase expression but following CD2-dependent stimulation, p62^dok coprecipitated with Crk-L (Fig. 8; see Fig. 6); the amount of p62^dok coprecipitating with Crk-L was not modified by coligation of CD3 with CD2. The modulation of p62^dok/Crk-L association required expression of ZAP70/Syk kinases (Fig. 8).

View larger version (45K): [in this window] [in a new window]   Fig. 8.   ZAP70/Syk kinase expression is required for the TCR-CD3-mediated modulation of the CD2-induced p62dok/Crk-L association. Either the ZAP70/Syk-deficient Jurkat cells P116 or wild type Jurkat cells (107 cells/lane) were left unstimulated or stimulated with either T112 + T113 or OKT3 or a combination of T112 + T113 and OKT3 for 5 min. Clarified PNL were either subjected to immunoprecipitation with 1 µg of mouse anti-Crk-L antibody or incubated with protein A-agarose alone, resolved by SDS-PAGE and immunoblotted with a rabbit anti-p62dok specific antibody (upper panel). 30 µl of PNL (0.3 × 106 cell equivalents) from unstimulated cells were also electrophoresed. Immunoprecipitation of comparable amounts of Crk-L protein was confirmed by blotting the membrane with a rabbit anti-Crk-L specific antibody (lower panel). The differing amounts of p62dok coprecipitating with Crk-L upon CD2 stimulation in the ZAP70/Syk-deficient P116 cells compared with the wild type Jurkat (clone J77) cells correlates with the basal expression of p62dok in the two cell lines, as shown in the PNL controls (right lanes).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

In this study, we analyzed the signaling requirements for the induction of CD2-dependent tyrosine phosphorylation of the Ras GTPase-activating protein-associated adapter protein p62^dok and its inducible association with Crk-L. Inductive tyrosine phosphorylation of p62^dok following CD2 stimulation has been noted recently by a number of laboratories (31, 33, 38-40); here we extend these findings to demonstrate that this signaling pathway is independent of ZAP70/Syk kinase and of LAT expression and leads to the association with Crk-L but can be regulated, in a ZAP70/Syk kinase-dependent fashion, by engagement of the TcR-CD3 complex.

We have shown that CD2-dependent p62^dok tyrosine phosphorylation appeared to depend, at least in part, on the Src-related kinase Lck, confirming the reports of Nemorin and Duplay (33). In the JCaM.1 cells used here, deficient in Lck expression but expressing low levels of Fyn kinase, minimal inductive tyrosine phosphorylation could be observed following CD2 stimulation in the absence of Lck kinase, suggesting that another kinase is capable of p62^dok tyrosine phosphorylation. We suggest that this kinase may be the Src-related kinase Fyn. This suggestion would be consistent with the observation that, in murine cells, p62^dok phosphorylation appeared to be dependent upon expression of Fyn (38). While p62^dok tyrosine phosphorylation was dependent upon an upstream tyrosine kinase (e.g. Lck/Fyn), it was independent of ZAP70/Syk expression and of the adapter protein LAT (Figs. 1, 2, 5, and 6).

The biochemical events initiated by engagement of the CD2 receptor overlap extensively with those initiated by the TcR-CD3 complex, including activation of and/or recruitment of tyrosine kinases such as the Src-related kinases Lck and Fyn, ZAP70 kinase, and Tec-related kinases; of serine/threonine kinases such as protein kinase C and the MAP kinases; of phospholipid kinases such as PI3K; of phosphatases such as calcineurin; and of adapter proteins such as LAT and Cbl (11-20). In most studies reported, the differences in signaling pathways initiated by cross-linking the TcR-CD3 complex or by mitogenic pairs of anti-CD2 mAbs are quantitative, and, indeed, many biochemical events depend upon common intermediates (ZAP70, LAT). It is important to appreciate that ZAP70-dependent functions may be subserved by Syk kinase. Studies using cell lines derived from patients with genetic deficiencies in ZAP70 expression have demonstrated variable CD2-dependent signaling (43, 44); in these cell lines, Syk expression may have compensated for ZAP70 deficiency. The Jurkat P116 cell lines used in the studies reported here are deficient in expression of both ZAP70 and Syk kinases (data not shown; see Ref. 34). We were unable to detect p62^dok tyrosine phosphorylation following CD3 engagement in either ZAP70-or LAT-deficient cells, and, indeed, CD3 engagement inhibited CD2-mediated p62^dok association with Crk-L; this CD3-dependent regulation required ZAP70 kinase expression. It is important to note that, in our studies, ligation of CD3 occurred concurrently with that of CD2; other outcomes may result from stimulating one receptor in advance of the other (39). It will be interesting to determine whether, with additional experimentation and other systems analyzed, the induction of p62^dok phosphorylation remains a qualitative difference between CD2 and TcR-CD3 signaling.

The role of p62^dok has been most extensively analyzed to date in cells overexpressing the Bcr-Abl fusion protein common to almost all patients with chronic myeloid leukemia (45). The 210-kDa chimeric Bcr-Abl protein is a fusion between Bcr sequences and the Abl tyrosine kinase, the fusion conferring increased Abl tyrosine kinase activity and transforming ability (46-48). A major tyrosine-phosphorylated protein in CML cells (49-52), the RasGAP-associated p62^dok protein was first cloned from cells transformed by oncogenic Abl (53, 54). Phosphorylation of p62^dok has been shown to correlate with fibroblast transformation (55, 56) but not with acquisition of growth factor independence (41). In transformed cells, proteins associating with p62^dok have been extensively characterized, and two previous reports have noted the association of p62^dok with Crk-L. In p210^bcr-abl-expressing cells, p62^dok has been shown to be both constitutively tyrosine-phosphorylated and constitutively associated with the Src homology 2 domain of Crk-L (41, 42). In the Jurkat T cells used here, cells other than CML and not transformed by p210^bcr-abl, neither tyrosine phosphorylation of p62^dok nor association with Crk-L was detected in the absence of stimulation. Inductive p62^dok tyrosine phosphorylation and association with Crk-L in T lymphocytes was demonstrated only following CD2 stimulation. Like p210^bcr-abl-transformed cells, these events required the activity of an Src-tyrosine kinase. Inductive phosphorylation of p62^dok has also been observed following inhibitory FcRIIB signaling in B cells (57-59) as it has in response to growth factor receptor signaling (49, 52, 60, 61). In these latter systems, however, the participation of Crk-L was not observed or investigated.

The biological role of p62^dok in T cell activation is not yet defined. p62^dok has been shown to associate with RasGAP (33, 54) and, because of this association, postulated to regulate Ras activity (41, 54, 58, 62-64). Crk-L, via C3G and Rap1 GTPase, itself an antagonist of Ras (see Ref. 65), has also been shown to regulate Ras activity (66-72). Whether p62^dok affects Ras pathways by modulating RasGAP activity or by its association with Crk-L and, via Crk-L, C3G and Rap1, or both, is the subject of ongoing investigation. In immune and hematopoietic cells, the available data suggest that p62^dok may serve largely to down-regulate T cell responses as has been demonstrated for both p62^dok and other members of the Dok family. As mentioned above, inhibitory B cell signaling via the FcRIIB receptor induced p62^dok phosphorylation (57, 58). Analysis of primary B cells derived from mice rendered genetically deficient in p62^dok expression demonstrated that p62^dok expression was required for the negative regulation by FcRIIB of B cell proliferation and was shown to suppress mitogen-activated protein kinase activation (62). Similarly, overexpression of p56^dok2 (Dok-2) (73) has also been shown to negatively regulate mitogen-activated protein kinase activity in cell lines (74, 75). In macrophages, increased expression of p56^dok2 inhibited cell proliferation and diminished p56^dok2 expression (by stable expression of p56^dok2 antisense mRNA) accelerated proliferation (76). Since transcription of p56^dok2 has been shown to increase in response to cytokine treatment (74, 76), it has been suggested that p56^dok2 participates in a negative feedback loop in which p56^dok2, induced by cytokines, subsequently inhibits cytokine-dependent proliferation (76). In T cells, stable overexpression of p62^dok inhibited both CD2- and CD3-induced NFAT and interleukin-2 transcriptional activation (40), although the mechanism remained unexplained.

Both p62^dok and Crk-L have been shown to play a role in cell migration (42, 77-80). We suggest that one functional role for the inducible p62^dok-Crk-L complex mediated by CD2 may be to regulate cell migration, perhaps through integrin binding as has been shown in other systems (78, 81), and adhesion, the latter potentially dependent upon the CD2-binding protein CD2AP (82)/CMS (83). While the avidity of CD2 to CD58 can be up-regulated by activation (84, 85), basal adhesion of CD2 to CD58, broadly expressed on a variety of hematopoietic and other cells, has been documented (29, 86). If p62^dok is a negative regulator of T cell activation, CD2-dependent p62^dok phosphorylation may exert tonic suppression of T cell activation and promote T cell circulation in the absence of recognition of specific antigen-MHC complexes. Upon antigen-specific recognition, signaling through the TcR-CD3 receptor would serve both to down-regulate CD2-dependent p62^dok/Crk-L association and to release the T cell from inhibition.

We first pursued the analysis of CD2-dependent signaling events in cells deficient in ZAP70 and LAT expression in order to isolate signaling intermediates that were not necessarily conserved between the TcR-CD3 complex and CD2. We have not yet identified a biological consequence of p62^dok tyrosine phosphorylation in the absence of ZAP70 or LAT expression, in part because many biological outcomes (NFAT transcriptional activation, cytokine production, proliferation) depend upon integrated and coordinately regulated signals involving a number of these signaling moieties (e.g. ZAP70 and LAT) (19). Therefore, the involvement of p62^dok, if any, in these functions cannot be ascertained in cell lines deficient in these proteins. In the presence of ZAP70 or LAT, it is difficult to isolate or attribute a specific role of p62^dok distinct from other components, since quantitative signaling differences will impact upon the response observed. We are further limited in that the biological outcomes following overexpression (or genetic deletion) of a protein may not accurately reflect its in vivo role. Indeed, this concern is illuminated by the recent report that p62^dok overexpression inhibited both CD2- and CD3-induced NFAT and interleukin-2 transcriptional activation but that only CD2-induced, and not CD3-induced, calcium influx, phospholipase C1 phosphorylation, and extracellular signal-regulated kinase activation were inhibited (40). The inhibition of distal read-outs (both dependent on ZAP70 and LAT expression) remained unexplained. We are currently developing systems appropriate for this study.

	ACKNOWLEDGEMENTS

We thank Robert Abraham for providing P116 and the ZAP70-reconstituted P116 cells, Larry Samelson for providing LAT-deficient ANJ3 and wild-type LAT-reconstituted ANJ3wt cells, and David E. Straus for providing the Lck-reconstituted JCaM1.6 cells.

	FOOTNOTES

^* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Supported by a fellowship from Fondazione, "Istituto Pasteur-Fondazione Cenci-Bolognetti," University of Rome "La Sapienza," Italy.

^§ To whom correspondence should be addressed: NHLBI, Bldg. 10, Rm. 6C208, 10 Center Dr., Bethesda, MD 20892. Tel.: 301-402-6786; Fax: 301-480-1792; E-mail: biererb@nih.gov.

Published, JBC Papers in Press, September 11, 2001, DOI 10.1074/jbc.M105777200

	ABBREVIATIONS

The abbreviations used are: TCR, T cell antigen receptor; PAGE, polyacrylamide gel electrophoresis; IP, immunoprecipitation; LAT, linker of activated T cells; PI3K, phosphatidylinositol 3-kinase; mAb, monoclonal antibody; PNL, postnuclear lysates.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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