INTRODUCTION

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T cell receptor (TCR)¹ -antigen binding induces gene expression, cytokine production, and cell proliferation in T lymphocytes (1). These TCR-dependent signals are mediated by tyrosine phosphorylation of various proteins including CD3, CD3, CD3, ZAP-70, Shc, Vav, and c-Cbl (2). These signaling molecules appear to be involved in the phosphorylated tyrosine-mediated protein-protein interaction and the recruitment of the other signaling molecules containing Src homology (SH) 2 domains (3). The recruitment of these signaling molecules is essential to induce various signals to the downstream events such as the activation of mitogen-activated protein kinases, Ca²⁺ influx, and transcriptional activation of various genes (1). Thus, protein tyrosine phosphorylation plays a key role during the initial phase of TCR-mediated T cell activation. However, the essential phosphorylated molecule and the precise function of each phosphorylated molecule for these signaling pathways have not yet been clarified.

Cas-L (pp105) was first identified as a 105-kDa protein that is tyrosine-phosphorylated upon 1 integrin cross-linking (4). Cas-L belongs to Cas-family along with p130^Cas and Efs/Sin (5-7). The three proteins share the structural characteristics of an N-terminal SH3 domain, followed by multiple (8 to 15) YXXP motifs, which are putative binding sites for the Crk SH2 domain, and a conserved YDYVHL sequence that possibly binds to the Src SH2 domain. (5-7). Cas-L was shown to associate with the C-terminal domain of focal adhesion kinase (FAK), a cytoplasmic tyrosine kinase that is localized to focal adhesions (5, 8). Recently, we reported that the conserved YDYVHL sequence of Cas-L was phosphorylated by FAK following 1 integrin cross-linking and that an Src family tyrosine kinase is recruited to the phosphorylated YDYVHL sequence and causes further tyrosine phosphorylation of Cas-L (9). We also reported that tyrosine-phosphorylated Cas-L binds to Crk, Nck, and SHPTP2 following 1 integrin cross-linking (5). These findings suggest that Cas-L plays an important role in the 1 integrin-mediated signaling pathway.

Although the significance of Cas-L in 1 integrin-mediated signaling has been well documented, little is known about its ability in other signaling pathways. Since Cas-L is predominantly expressed in lymphocytes (5), it is conceivable that Cas-L may participate in lymphocyte-specific signaling pathways. Recently, Kanda et al. (10) reported TCR-dependent tyrosine phosphorylation of a 105-kDa Cas-related protein. However, the precise molecular mechanism for signal transduction though Cas-L remains unclear. In this study, we show that Cas-L is tyrosine-phosphorylated and forms complexes with Crk and C3G following CD3 cross-linking. We further demonstrate that FAK is not likely involved in the CD3-dependent Cas-L phosphorylation.

	EXPERIMENTAL PROCEDURES

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Cell Culture-- A human T lymphoblastoid cell line, H9, a human breast cancer cell line, T-47D, and a monkey kidney-derived cell line, COS-1, were obtained from American Type Culture Collection (ATCC, Rockville, MD) and grown in Dulbecco's modified Eagle's medium or RPMI 1640 supplemented with heat-inactivated fetal calf serum, 2 mM L-glutamine, and gentamycin (50 µg/ml) (complete medium). JMC-T5, a Jurkat strain carrying SV40 large T antigen (a gift from Dr. Hamid Band, Brigham and Women's Hospital, Boston, MA) was maintained in the complete medium containing 1 mg/ml geneticin (Life Technologies, Inc.). Human peripheral T lymphocytes were isolated from normal healthy donors as described elsewhere (11). Briefly, mononuclear cells were separated by Ficoll-Paque gradient centrifugation from leukopheresed products followed by a positive selection with sheep erythrocytes. Contaminating monocytes were depleted by adherence to plastic plates.

Antibodies and Glutathione S-Transferase Fusion Proteins-- The affinity-purified anti-human Cas-L rabbit antibody was raised in rabbits immunized with the glutathione S-transferase (GST) fusion protein of Cas-L containing Cas-L residues 419 to 524 (14). Anti-Crk, anti-p130^Cas, and anti-FAK monoclonal antibodies (mAbs) were purchased from Transduction Laboratories (Lexington, KY). Rabbit antibodies against C3G, Lck, and ZAP70 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-phosphotyrosine mAb (anti-P-Tyr, 4G10) and anti-c-Myc tag mAb (9E10) were purchased from Upstate Biotechnology Inc. (Lake Placid, NY) and Oncogene Science Inc. (Manhasset, NY), respectively. Anti-CD3 (OKT3) and anti-CD29 (4B4) mAbs have been described previously (11). GST-Crk SH2 was provided by Dr. Bruce J. Mayer (Children's Hospital, Boston, MA). GST-Shc SH2 was obtained from Dr. Gotz Baumann (Sandoz Pharma LTD., Basel, Switzerland). Rat p130^Cas cDNA was obtained from Dr. Hisamaru Hirai (University of Tokyo, Tokyo, Japan).

Transfections-- Plasmid DNA was transfected into COS-1 cells by the DEAE-dextran method (12). Ten µg of plasmid DNA was transfected into JMC-T5 cells (20-30 million) in 300 µl of serum-free RPMI 1640 by electroporation at 250 V and 960 microfarad using the Gene Pulser (Bio-Rad, Hercules, CA).

Cell Stimulation and Preparation of Cell Extracts-- Cells were washed three times with Iscove's serum-free medium (Sigma), and incubated with 500 µl of Iscove's media containing 10 µg/ml anti-CD3 or anti-CD29 mAb on ice for 15 min. After a wash with cold medium, cells were incubated with 200 µl of media containing 10 µg/ml anti-mouse immunoglobulin (Ig) at 37 °C for appropriate durations of time. The reactions were stopped by the addition of ice-cold Iscove's medium containing 5 mM EDTA, 10 mM sodium fluoride, 10 mM pyrophosphate, and 0.4 mM sodium vanadate. After centrifugation, cells were lysed in a lysis buffer (1% Nonidet P-40, 140 mM NaCl, 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 10 mM iodoacetamide, 1 µg/ml pepstatin A, 10 mM sodium fluoride, 10 mM sodium pyrophosphate, 0.4 mM sodium vanadate, and 50 mM Tris-HCl, pH 8.0) for 15 min on ice. For stimulation with the extracellular matrix, cells were incubated in plates coated with poly-L-lysine (PLL, Sigma) or human plasma fibronectin (FN, Life Technologies, Inc.), and then solubilized in the lysis buffer. The lysate was centrifuged at 14,000 rpm for 10 min, and the supernatant was used for precipitations with antibodies or GST fusion proteins.

Protein Precipitation-- For immunoprecipitations, cell lysates were incubated with indicated antibodies for 2 h at 4 °C and then with protein A-Sepharose (Pharmacia LKB, Upssala, Sweden) for an additional 1 h. In the case of GST fusion proteins, cell lysates were incubated with glutathione-Sepharose (Pharmacia LKB) conjugated with GST fusion proteins for 1 h at 4 °C. The beads were washed five times with the washing buffer (1% Nonidet P-40, 140 mM NaCl, 2.5 mM EDTA, and 50 mM Tris-HCl, pH 8.0). Bound proteins were eluted by boiling in Laemmli sample buffer for 5 min.

Western Blotting-- Proteins were fractionated by SDS-polyacrylamide gel electrophoresis under reducing conditions and then electrophoretically transferred to nitrocellulose membranes. Following incubation with PBS containing 5% nonfat dry milk or 3% bovine serum albumin, the filters were blotted with indicated primary antibodies. The primary antibodies were detected with horseradish peroxidase-conjugated anti-mouse or anti-rabbit Ig antibodies (Amersham Corp.) and chemiluminescence reagents (NEN Life Science Products). Alternatively, the membranes were incubated with ¹²⁵I-labeled anti-P-Tyr.

	RESULTS

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Development of Cas-L Specific Antibody-- In previous studies, Cas-L was analyzed by conventional anti-p130^Cas mAb that cross-reacts with Cas-L (5, 10). To distinguish Cas-L from p130^Cas, anti-Cas-L-specific antibody was generated by the immunization of a GST fusion protein of the Cas-L residues 419-524, which shares the lowest homology with p130^Cas or Efs/Sin (5). Cellular lysates were analyzed by immunoblotting with this antibody to determine its reactivity. As shown in Fig. 1, c-myc-epitope-tagged Cas-L from COS cells and the endogenous Cas-L from H9 cells were detected by this anti-Cas-L antibody. Two species of 110- and 105-kDa were equally expressed in COS transfectant cells, whereas a 110-kDa form of Cas-L was less expressed in H9 cells. However, c-myc-tagged p130^Cas expressed in COS cells and the endogenous p130^Cas protein from T-47D cells were not detected by this antibody. In contrast, anti-p130^Cas mAb reacted with both p130^Cas and Cas-L proteins. Therefore, this anti-Cas-L antibody that we developed specifically recognizes Cas-L but not p130^Cas.

View larger version (28K): [in this window] [in a new window]   Fig. 1.   Characterization of Cas-L-specific antibody. A, cell lysates from COS-1 untransfected () and transfected with c-myc-tagged Cas-L or p130Cas plasmid vector, or from H9 and T-47D human cell line cells, were immunoprecipitated with an anti-c-Myc tag mAb (9E10) or anti-Cas mAb, respectively. Immunoprecipitates were separated by SDS-polyacrylamide gel electrophoresis and immunoblotted with anti-Cas-L antibody or anti-Cas mAb.

Tyrosine Phosphorylation of Cas-L upon CD3 Cross-linking-- We next attempted to determine whether Cas-L was tyrosine-phosphorylated upon CD3 cross-linking using the Cas-specific Ab. The CD3 molecule on the H9 cell surface was cross-linked with anti-CD3 mAb and rabbit anti-mouse IgG antibody for appropriate durations of time. Cellular lysates were analyzed by immunoprecipitation with specific antibodies and immunoblotting with anti-P-Tyr. As shown in Fig. 2A, Cas-L was tyrosine-phosphorylated 1 min and 5 min after CD3 cross-linking and then dephosphorylated 20 min after CD3 cross-linking. Similar kinetics were also observed in p59^Fyn and p56^Lck, which were phosphorylated 1 min after CD3 cross-linking. On the other hand, FAK was not significantly phosphorylated upon CD3 cross-linking. In contrast to CD3 stimulation, both FAK and Cas-L were tyrosine-phosphorylated following 1 integrin cross-linking (Fig. 2B). It should be noted that Cas-L tyrosine phosphorylation following 1 integrin cross-linking was slower (5 min) than that following CD3 cross-linking. The Cas-L tyrosine phosphorylation was still observed 20 min after 1 integrin cross-linking.

View larger version (47K): [in this window] [in a new window]   Fig. 2.   Cas-L is tyrosine-phosphorylated after the CD3 cross-linking in T cells. H9 cells stimulated with OKT3 (CD3) (A) or 4B4 (CD29) (B) or peripheral T lymphocytes stimulated with OKT3 (C), for the indicated times, were lysed and then immunoprecipitated with indicated antibodies. The immunoprecipitates were analyzed by Western blotting with the same antibodies, followed by 125I-labeled anti-P-Tyr (Anti-pTyr). The levels of tyrosine phosphorylation and amounts of proteins were determined by a densitometer. They were relative to the level of tyrosine phosphorylation in the unstimulated status as shown. Each level of tyrosine phosphorylation was normalized to the amount of the protein immunoprecipitated. Data are representative of three independent experiments.

CD3-dependent Recruitment of Crk and C3G to Tyrosine-phosphorylated Cas-L-- Based on the finding of CD3-dependent tyrosine phosphorylation of Cas-L, we next attempted to define binding molecules to tyrosine-phosphorylated Cas-L. Since Cas-L contains multiple possible binding sites for the Crk SH2 domain, we determined the Cas-L-Crk association following CD3 cross-linking. For this purpose, H9 cells were lysed 1 min after CD3 cross-linking. Then, cellular lysates were immunoprecipitated with anti-Crk mAb and analyzed by immunoblotting with anti-Cas-L-specific antibody. As shown in Fig. 3A, CD3 cross-linking increased Cas-L coprecipitation with Crk. The co-immunoprecipitated species by anti-Crk antibody migrated slower than those by anti-Cas-L antibody. This result suggests that a large form of Cas-L could significantly bind to Crk. In addition to a 105-110-kDa tyrosine-phosphorylated Cas-L band, a 120-kDa tyrosine-phosphorylated molecule was immunoprecipitated by anti-Crk antibody upon CD3 cross-linking (Fig. 3A). Since this 120-kDa protein was not observed after immunodepletion with anti-Cbl antibody (data not shown), this 120-kDa species could be Cbl (10, 13). We also performed a reverse experiment of Crk coprecipitation with Cas-L. As shown in Fig. 3B, Crk was co-immunoprecipitated with Cas-L 1 min following CD3 cross-linking, whereas Crk was not coprecipitated 20 min after CD3 stimulation. The immunoprecipitated Cas-L was remarkably tyrosine-phosphorylated 1 min after CD3 cross-linking, whereas Cas-L phosphorylation was diminished 20 min after cross-linking, indicating that this association of Cas-L and Crk corresponds to tyrosine phosphorylation of Cas-L. Furthermore, as shown in Fig. 3C, Cas-L was precipitated by the GST-Crk SH2 domain, whereas Cas-L was not precipitated by the GST or GST-Shc SH2 proteins. Taken together, these results indicate that Cas-L associates with Crk via the Crk SH2 domain following CD3 cross-linking in a tyrosine phosphorylation-dependent manner.

View larger version (22K): [in this window] [in a new window]   Fig. 3.   Cas-L binds to Crk via the Crk SH2 domain. H9 cells were stimulated with (+) or without () OKT3 for 1 min (A and C) or the indicated times (B). A, cell lysates were immunoprecipitated with control antibody, anti-Crk mAb, or anti-Cas-L antibody and then analyzed by Western blotting with anti-Cas-L antibody followed by anti-Crk mAb. The immunoblots were reprobed with 125I-labeled anti-P-Tyr (Anti-pTyr). B, cell lysates were immunoprecipitated with control or anti-Cas-L antibody. The immunoprecipitates were immunoblotted with anti-Crk mAb and anti-Cas-L Ab, followed by 125I-labeled anti-P-Tyr. C, cell lysates were incubated with GST-, GST-Shc SH2-, and GST-Crk SH2-conjugated beads. The precipitates were analyzed by immunoblotting with anti-p130Cas mAb. The amounts of proteins coimmunoprecipitated were quantitated by a densitometer and normalized to the amounts of proteins immunoprecipitated with primary antibodies. Data are representative of two independent experiments.

View larger version (50K): [in this window] [in a new window]   Fig. 4.   Cas-L forms complexes with C3G following CD3-stimulation in T cells. Immunoprecipitates from CD3-stimulated H9 cell lysates using anti-Cas-L, anti-C3G, or control antibody were analyzed by Western blotting with anti-Cas-L or anti-C3G Ab, followed by 125I-labeled anti-P-Tyr (Anti-pTyr). The amounts of coimmunoprecipitated Cas-L proteins were analyzed as described in Fig. 3. Data are representative of two independent experiments.

The SH3 Domain-independent Phosphorylation of Cas-L upon CD3 Cross-linking-- Cas-L is tyrosine-phosphorylated and binds to Crk upon both 1 integrin (5) and CD3 stimulation. We next investigated the mechanism by which Cas-L is tyrosine-phosphorylated upon such stimulation. We recently found that FAK initiates tyrosine phosphorylation of Cas-L following integrin stimulation (9). However, FAK is not tyrosine-phosphorylated upon CD3 cross-linking (Fig. 2A), suggesting that Cas-L may be tyrosine-phosphorylated upon CD3 cross-linking in a FAK-independent manner. To elucidate this hypothesis, we studied CD3-dependent tyrosine phosphorylation of a Cas-L mutant, c-myc-tagged Cas-LSH3, which lacks the SH3 domain and does not bind to FAK (data not shown). This mutant was expressed in JMC-T5, a Jurkat cell strain that is available for transient transfection analysis. After CD3 cross-linking, Cas-LSH3 was immunoprecipitated with anti-c-Myc mAb and analyzed by immunoblotting with anti-P-Tyr. As shown in Fig. 5, Cas-LSH3 was tyrosine-phosphorylated upon CD3 cross-linking, whereas Cas-LSH3 was not phosphorylated upon FN stimulation. The CS1 region of FN also failed to induce Cas-LSH3 tyrosine phosphorylation (data not shown). In contrast, wild-type Cas-L was tyrosine-phosphorylated following the ligation of CD3 or 1 integrin. These results indicate that Cas-L tyrosine phosphorylation upon CD3 cross-linking is caused by a different mechanism from 1 integrin-dependent Cas-L tyrosine phosphorylation.

View larger version (37K): [in this window] [in a new window]   Fig. 5.   The SH3-deletion mutant of Cas-L, as well as wild-type Cas-L, is tyrosine-phosphorylated after CD3-cross-linking in Jurkat T cells. Control plasmid, c-myc-tagged wild-type Cas-L, or c-myc-tagged-SH3 Cas-L were transfected into JMC-T5, a Jurkat strain carrying SV40 large T antigen, by electroporation as described under "Experimental Procedures." After 2 days of culturing, live cells (20 × 106) were stimulated with (+) or without () OKT3 plus anti-mouse IgG antibody for 2 min, or with poly-L-lysine (PLL) or fibronectin (FN) for 1 h, and then lysed. The cell lysates were immunoprecipitated with anti-c-Myc mAb (9E10), and analyzed by Western blotting with 9E10, followed by 125I-labeled anti-P-Tyr (Anti-pTyr). The levels of tyrosine phosphorylation were expressed as a ratio to that of the unstimulated status, normalized to the amounts of proteins immunoprecipitated. Data are representative of three independent experiments.

	DISCUSSION

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It has been reported that Cas family proteins, p130^Cas, Cas-L/HEF-1, and Efs/Sin are tyrosine-phosphorylated upon various stimuli, including integrin-mediated cell adhesion, growth factors, chemokines, and cross-linking of the B cell receptor (4, 5, 16-19). Tyrosine-phosphorylated Cas family proteins bind to signaling molecules containing SH2 domains (5, 20, 21). However, the precise nature of signaling pathways through Cas family proteins is poorly understood. Especially, since Cas-L is preferentially expressed in lymphocytes, it is conceivable that Cas-L may play an important role in lymphocyte-specific function. In this study, we have demonstrated that Cas-L is tyrosine-phosphorylated following CD3 cross-linking and forms complexes with Crk and C3G. The tyrosine phosphorylation of Cas-L and its association with Crk and C3G are rapid, but transient, events following CD3 cross-linking. These results demonstrated a novel signaling pathway through Cas-L following TCR stimulation.

Antigen-TCR binding induces various signals in T cells (1, 2). Our study raised the question as to what signal Cas-L is involved in. We showed that tyrosine-phosphorylated Cas-L binds to Crk via the Crk SH2 domain following CD3 cross-linking. Crk is an adapter protein composed of one SH2 domain and one or two SH3 domains, and binds to various proteins including C3G, Dock180, Sos, and c-Abl via the Crk SH3 domain (15, 22-24). This suggests that Cas-L can recruit these signaling molecules in a tyrosine phosphorylation-dependent manner via Crk. One of the major Crk SH3 domain-binding proteins, C3G, is a guanine nucleotide exchange factor that activates a small GTPase, Rap1A/K-rev1 (15). K-rev1 was first reported to be a protein that reverts the K-Ras transformed phenotype of NIH-3T3 cells (25). Rap1A has been shown to inhibit the binding of Ras to Raf-1 and Ras-dependent Raf-1 activation (26). These findings indicate that K-rev1/Rap1A competitively inhibits Ras-signaling pathway. Therefore, the recruitment of C3G to Cas-L may be involved in the regulation of Ras-mediated signaling pathways. The other putative signal downstream of C3G is the JNK/SAPK pathway. Tanaka et al. (27) recently reported that overexpression of Crk induces activation of JNK/SAPK and that coexpression of C3G further enhances the JNK activity. Overexpression of Crk has been shown also to induce tyrosine phosphorylation of Cas-L and p130^Cas, as well as the recruitment of C3G to the phosphorylated Cas proteins (5, 6). Our study demonstrated that Cas-L is one of the major Crk-binding proteins upon CD3 cross-linking. On the other hand, it has been reported that JNK is activated upon TCR stimulation and that JNK activation plays a significant role in T cell activation (28). These findings suggest that Cas-L is involved in TCR-mediated JNK activation through the recruitment of C3G. Ras/Raf-1 and JNK pathways have been shown to serve an important function in T cell activation (28, 29). Cas-L may regulate T cell responses through such pathways.

The other question raised from our study is how Cas-L is phosphorylated following CD3 cross-linking. We found that FAK initiates tyrosine phosphorylation of Cas-L by plural tyrosine kinases following 1 integrin-mediated cell adhesion (9). In this study, we showed that the Cas-LSH3 mutant lacking the SH3 domain responsible for Cas-L binding to FAK (30) is not tyrosine-phosphorylated upon 1 integrin cross-linking. These findings indicate that FAK plays an important role in Cas-L phosphorylation in the 1 integrin-mediated signaling pathway. However, we found that FAK is not tyrosine-phosphorylated upon CD3 cross-linking. Since FAK phosphorylation is well correlated to its activation (31), FAK is not likely to be activated upon CD3 cross-linking. In contrast to our findings, Maguire et al. (32) demonstrated that FAK is synergistically tyrosine-phosphorylated upon both CD3 and 1 integrin cross-linking in T cells and that anti-CD3 mAb (OKT3) alone also induces low levels of FAK tyrosine phosphorylation. On the other hand, Kanner et al. (33) reported that FAK is not tyrosine-phosphorylated following CD3 cross-linking, which supports our results. This paradoxical observation may be caused by the difference of the cross-linking method; liquid phase cross-linking was used in our system, whereas solid phase cross-linking was used in the Maguire et al. (32) observation. However, the Cas-LSH3 mutant that fails to bind to FAK is demonstrated to be tyrosine-phosphorylated, as well as the wild-type Cas-L, upon CD3 cross-linking. This result strongly suggests that FAK is not involved in CD3-mediated Cas-L phosphorylation.

How, then, is Cas-L phosphorylated upon CD3 cross-linking? We reported that activated p56^Lck phosphorylates Cas-L (5), and we also found that the coexpression of ZAP-70 also phosphorylates Cas-L.² As both p56^Lck and ZAP-70 are involved in TCR/CD3 signaling pathways (1, 2), these tyrosine kinases are, therefore, likely involved in the CD3-dependent phosphorylation of Cas-L. Recently, Ingham et al. (18) reported that Cas-L is observed in the membrane fraction in B lymphocytes, suggesting a putative subcellular localization of Cas-L. Taken together, we hypothesize that Cas-L is localized to the cytoplasmic membrane by an unknown mechanism and interacts with TCR/CD3 molecules or their downstream signaling molecules, and that Cas-L is tyrosine phosphorylated by p56^Lck or ZAP-70 upon cross-linking of TCR/CD3 molecules.

We demonstrated Cas-L tyrosine phosphorylation not only upon 1 integrin cross-linking but also upon CD3 cross-linking. However, Cas-L is rapidly phosphorylated but dephosphorylated quickly after CD3 cross-linking, whereas Cas-L phosphorylation upon 1 integrin cross-linking is slow but stable. Such a difference in the kinetics of Cas-L tyrosine phosphorylation suggests that Cas-L may serve as a distinct regulatory function in TCR-mediated T cell responses from those mediated by 1 integrin. Cas-L has also been demonstrated to be tyrosine-phosphorylated upon B cell receptor cross-linking (18, 19). These findings indicate that Cas-L is tyrosine-phosphorylated following the interaction of lymphocyte receptors to antigens and is involved in the lymphocyte receptor-specific signaling events.

In summary, TCR stimulation induces tyrosine phosphorylation of Cas-L by a FAK-independent manner and also the recruitment of signaling molecules, Crk and C3G, to Cas-L. These results suggest a potential role of Cas-L in TCR/CD3-mediated signal transduction that leads to cellular responses. Further biological analysis may help to elucidate the mechanisms by which Cas-L functions in lymphocytes.