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Regulated Association Between the Tyrosine Kinase Emt/Itk/Tsk and Phospholipase-C1 in Human T Lymphocytes¹

Immunology, Inflammation, and Pulmonary Drug Discovery, Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, NJ 08543

	Abstract

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The Emt/Itk/Tsk tyrosine kinase is involved in intracellular signaling events induced by several lymphocyte surface receptors. Modulation of TCR/CD3-induced phospholipase-C1 (PLC1) activity by the tyrosine kinase Emt/Itk/Tsk has been demonstrated based on studies of Itk-deficient murine T lymphocytes. Here we report a TCR/CD3-regulated association between Emt and PLC1 in both normal and leukemic T cells. In addition, this association was enhanced following independent ligation of the CD2, CD4, or CD28 costimulatory molecules, but not of CD5 or CD6 surface receptors, correlating to the induced tyrosine phosphorylation of Emt. Before Ab-induced T cell activation, we found that the Emt-SH3 domain was crucial for the constitutive Emt/PLC1 association; however, upon TCR/CD3 engagement, the Emt-SH2 domain was more efficient in mediating the enhanced Emt/PLC1 interaction. Furthermore, the PLC1-SH3 domain, but not the two PLC1-SH2 domains, contributed to formation of the protein complex. Thus, we describe a regulated interaction between Emt and PLC1, and based on our studies with individual Emt and PLC1 SH2/SH3 domains, we propose a mechanism for this association.

	Introduction

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Several signaling pathways are activated following TCR/CD3 stimulation, and their components are directly regulated by protein tyrosine kinases (PTK).³ Emt/Itk/Tsk is a member of the Tec family of PTKs expressed in T, NK, and mast cells (1, 2) that is related to Bruton’s tyrosine kinase, which in human B lymphocytes has been implicated in X-linked agammaglobulinemia (reviewed in Ref. 3). Itk-deficient mice show a lower number of peripheral CD4⁺ T cells, which exhibit reduced TCR/CD3-induced IL-2 production, Ca²⁺ influx and apoptosis, diminished antiviral CTL immune activity, and other decreased responses mediated by some transgenic TCRs, while maintaining normal B cell immune responses (4, 5, 6, 7). Structurally, Emt/Itk/Tsk comprises a tyrosine kinase (TK) domain, an Src homology-2 (SH2) domain, an Src homology-3 (SH3) domain, a proline-rich (PR) motif within the Tec homology domain, and a pleckstrin homology (PH) domain (1, 2, 8). Emt/Itk/Tsk is devoid of the amino-terminal myristylation signal and the negative autoregulatory phosphorylation site present in all members of the Src family of PTKs, indicating a distinct mechanism of activity regulation (8). The intramolecular association between the SH3 and PR domains in Tec family members maintains its down-regulated state of kinase activity. However, upon TCR/CD3 engagement, Src PTKs phosphorylate Tec PTKs in the activation loop of the TK domain, leading to autophosphorylation in the SH3 domain, thereby disrupting the SH3/PR interaction and resulting in up-regulation of tyrosine kinase activity (9).

A number of Emt/Itk/Tsk interacting proteins have been discovered recently. The PH domain has been shown to interact with different protein kinase C (PKC) isoforms (10) and inositol phospholipids (11). The PR motif associates with the SH3 domains of different Src PTKs and Grb2 (9, 12, 13). Experiments performed with fusion proteins containing the Emt/Itk/Tsk SH3 domain demonstrated its association with CD28, Wiscott-Aldrich syndrome protein, hnRNP-K, Fyn, and c-Cbl (9, 13). However, the functional consequences of these SH3 interactions have not been elucidated. In addition, fusion proteins containing the SH2 domain of Emt/Itk/Tsk coprecipitate with phosphatidylinositol 3-kinase (PI3-K) after CD28 stimulation in a tyrosine phosphorylation-dependent manner (14).

Tyrosine phosphorylation and subsequent activation of the phospholipase-C isoforms (PLC1 and PLC2) were observed after cellular activation through different surface receptors including TCR/CD3, B cell Ag receptor (BCR), CD2, CD20, CD28, integrins, and several cytokine receptors (15). These receptors do not themselves possess PTK activity, but they activate a wide variety of nonreceptor PTKs, such as members of Src, Syk, and Jak families. The PLC1 associates with members of Src family PTKs and is phosphorylated by several PTK, including Src, Fyn, Lck, Lyn and Hck (15, 16). Two different consequences of PLC1 tyrosine phosphorylation have been described. First, tyrosine phosphorylation of PLC1 activates its phospholipase activity (17, 18), leading to enhanced phosphoinositide cleavage, intracellular calcium elevation, PKC activation, and subsequent downstream signaling (18). Second, tyrosine phosphorylation of PLC1 creates binding sites for recruiting regulatory proteins containing SH2 domains (15, 19).

In Itk-deficient murine T lymphocytes, TCR/CD3- and CD28-induced IL-2 secretion is defective in part due to impaired Ca²⁺ signaling, reduced inositol 1,4,5-trisphosphate (IP₃) generation and diminished tyrosine phosphorylation of PLC1 (4, 5, 6, 7). In the present work we report a regulated interaction between the Tec family kinase member Emt/Itk/Tsk and PLC1 in human T lymphocytes. This interaction was enhanced following engagement of the TCR/CD3 and the CD2, CD4, and CD28 costimulatory molecules, but not the CD5 or CD6 surface receptors. We also show that the Emt-SH3 and SH2 domains as well as the PLC1-SH3 domain are all involved in this regulated association.

	Materials and Methods

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Antibodies and GST fusion proteins

Anti-CD3 mAb G19-4, anti-CD5 mAb 10.2, anti-CD6 mAb G3-6, anti-CD2 mAb 9.6, anti-CD4 mAb G17-2, anti-CD28 mAb 9.3, and the anti-mouse CD40 mAb 40 4.8E1 were described previously (20, 21, 22). Secondary Abs (HRP-conjugated anti-rabbit IgG and HRP-conjugated anti-mouse IgG) were purchased from BioSource International (Camarillo, CA). Rabbit antiserum to Emt, mouse anti-Emt mAb, rabbit antiserum to human Sos1, (hSos1), rabbit antiserum to human Sos2 (hSos2), mouse mAb to human PLC1 (clone B-6-4), and resins linked to GST-PLC1-SH3, GST- PLC1-N-SH2, and GST-PLC1-C-SH2 were purchased from Upstate Biotechnology (Lake Placid, NY). The rabbit antiserum to human PLC1 was described previously (23). The GST-Emt fusion proteins were produced as follows. The Emt full-length cDNA was cloned by PCR from a Jurkat cDNA library, using 5'-GCGGCGGAATTCCATGAACAACTTTATCCTCCTGGAA-3' as forward primer and 5'-GCGGCGGACTCGAGCCTAAAGTCCTGATTCTGCAAT-3' as reverse primer, and cloned into the bacterial expression vector pGEX-4T-3 (Pharmacia Biotech, Piscataway, NJ) using the EcoRI and XhoI restriction sites. The plasmid construct encoding the entire Emt PTK served as a template for other constructs generated by PCR. The Emt cDNA, PTR32, encoding residues 1–342 (including PH, Tec homology, PR, SH3, and SH2 domains) was cloned using as forward primer (5'-GCGGCGGAATTCCATGAACAACTTTATCCTCCTGGAA-3') and as reverse primer (5'-GCGGCGGACTCGAGCCTACCTCCCAAAACAAACTGGATA-3'). The cDNA encoding the Emt SH3 domain (residues 177–230) was cloned using 5'-GCGGCGGAATTCCATTGCCTTATATGACTACCAA-3' as forward primer and 5'-GCGGCGGACTCGAGCCTACCACTCATAGGTTTCCAGATT-3' as reverse primer. The cDNA encoding the Emt SH2 domain (residues 240–342) was cloned using 5'-GCGGCGGAATTCCTACAATAAGAGTATCAGCCGA-3' as forward primer and 5'-GCGGCGGACTCGAGCCTACCTCCCAAAACAAACTGGATA-3' as reverse primer. The cDNA encoding the Emt SH3SH2 domains (residues 177–342) was cloned using 5'-GCGGCGGAATTCCATTGCCTTATATGACTACCAA-3' as forward primer and 5'-GCGGCGGACTCGAGCCTACCTCCCAAAACAAACTGGATA-3' as reverse primer. Constructs were introduced into the expression vector pGEX-4T-3 using the EcoRI and XhoI restriction sites and transformed into DH5 Escherichia coli cells. GST protein production and purification were performed as specified by the manufacturer.

Cell lines and cell culture

The human leukemic T cell line Jurkat was cultured in RPMI 1640 containing 10% heat-inactivated FCS, 100 U/ml penicillin, and 100 µg/ml streptomycin. For PHA T lymphoblast generation, human peripheral blood cells were obtained from healthy volunteers, and mononuclear cell suspensions were prepared by Ficoll-Hypaque density gradient centrifugation. T lymphocytes were isolated by 2-aminoethylisothiouronium bromide-treated SRBC rosetting. The SRBC were lysed according to standard procedures. The remaining cell preparations contained >98% T lymphocytes (referred to as purified fresh T cells) as assessed by flow cytometric analysis after staining with an anti-CD3 mAb (Becton Dickinson, Mountain View, CA). After cell isolations, T lymphocytes were cultured in RPMI 1640 (Life Technologies, Grand Island, NJ), containing 10% FCS and 1 µg/ml PHA for 7 days.

Cell stimulation, immunoprecipitation, and immunoblotting analysis

Purified fresh T cells, PHA expanded T lymphoblasts, or Jurkat cells were washed and incubated at 4°C. For TCR/CD3, CD2, CD4, CD5, CD6, or CD28 stimulation, specific mAbs were added at 10 µg/ml for 2 min at 4°C, and cells were washed to remove unbound mAb and incubated with sheep anti-mouse IgG as cross-linker (50 µg/ml) at 37°C for the indicated time periods. After stimulation, cells were lysed in 1 ml of lysis buffer containing 50 mM Tris (pH 7.5), 1% Nonidet P-40, 150 mM NaCl, 2 mM EGTA, 1 mM NaF, 1 mM sodium orthovanadate, plus Complete Protease Inhibitor Mixture (Roche Molecular Biochemicals, Indianapolis, IN). Samples were centrifuged at 14,000 rpm for 2 min (to remove nuclei and other insoluble material), and lysates were precleared twice with GammaBind protein G-Sepharose beads (Pharmacia Biotech) for 60 min at 4°C and subjected to immunoprecipitation with mAbs to Emt, PLC1, hSos1, and hSos2 or equivalent amounts of GST alone or GST fusion proteins (5 µg/sample). Beads were washed once with 10% lysis buffer in PBS and twice with PBS alone. After washing, the immunoprecipitates were resuspended in 20 µl of Laemmli sample buffer (Bio-Rad, Hercules CA), boiled for 5 min, analyzed by SDS-PAGE on 8% or gradient 4–20% gels under reducing conditions, and subsequently transferred to polyvinylidene difluoride membranes (Immobilon-P, Millipore, Bedford, MA). For protein detection, membranes were blocked in 3% BSA and treated with the indicated Abs as a primary reagent and with anti-mouse or anti-rabbit Ab linked to HRP (BioSource International) as the secondary reagent. For Far Western analysis, blots were incubated with GST-Emt-SH2 or -SH3 fusion proteins (10 µg/ml) and 1/1000 anti-GST rabbit antiserum. For phosphotyrosine analysis, blots were incubated with anti-phosphotyrosine mAb 4G10 (Upstate Biotechnology) at 0.1 µg/ml plus anti-mouse-HRP. The binding of HRP was detected by ECL (Amersham, Aylesbury, U.K.) and exposure to x-ray film. Where indicated, blot stripping was conducted by membrane incubation in 62.5 mM Tris-HCl (pH 6.8), 2% SDS, and 50 mM 2-ME at room temperature for 60 min.

	Results

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The TCR/CD3-regulated Emt/PLC1 association in human T cells

In an effort to better understand the role of Emt tyrosine kinase in the TCR/CD3 signal transduction pathway, we sought to identify Emt-associated proteins. The observation that TCR/CD3- and CD28-induced IL-2 production is defective in Itk-deficient murine T lymphocytes, in part due to reduced tyrosine phosphorylation of PLC1, diminished IP₃ generation, and impaired Ca²⁺ influx (4, 5, 6, 7), indicated a functional association between the Emt/Itk and PLC1 signaling molecules. Furthermore, overexpression of Bruton’s tyrosine kinase exhibits its participation in PLC2 activation in B cells, suggesting that Tec kinases, in general, play a particularly important role in producing the sustained level of IP₃ required for a calcium influx (24, 25). To test for the possibility of a physical association between Emt and PLC1, immunoprecipitation experiments were performed using the leukemic human T cell line Jurkat and specific Abs to Emt and PLC1. We observed a constitutive association between Emt and PLC1, which was up-regulated upon TCR/CD3 engagement (Fig. 1A, top). Reciprocally, constitutive and TCR/CD3-enhanced association of Emt/PLC1 was also detected in PLC1 immunoprecipitates from Jurkat T cells (Fig. 1A, bottom).

View larger version (48K): [in this window] [in a new window]   FIGURE 1. Regulated Emt/PLC1 association in Jurkat T Cells. A, Jurkat T-cells (2 x 107 cells/lane) were left unstimulated or were stimulated with anti-TCR/CD3 as indicated in Materials and Methods, then lysed in 1% Nonidet P-40-containing lysis buffer and subjected to immunoprecipitation with control IgG or Abs to Emt or PLC1. B, Jurkat T cells (2 x 107) were left unstimulated or were stimulated with anti-TCR/CD3 over the course of 30 min, lysed in 1% Nonidet P-40-containing lysis buffer, and subjected to immunoprecipitation with anti-Emt Abs or control IgG at the indicated time points. Immunoprecipitated proteins were subjected to SDS-PAGE on 4–20% gradient gels and immunoblotted with antiserum to PLC1 (upper panels). Blot stripping was conducted as indicated in Materials and Methods, and the same membranes were reprobed with anti-Emt mAb (bottom panels) or anti-phosphotyrosine (B, middle panel). Stripping and subsequent reprobing with anti-Emt mAb (B, bottom panel) were conducted as a control for protein loading. Data are representative of five independent experiments.

We also performed coprecipitation experiments using PBL obtained from healthy donors (freshly purified T lymphocytes or PHA T cell lymphoblasts). Enhanced Emt/PLC1 association upon TCR/CD3 engagement was observed in both fresh T cells and PHA T lymphoblasts (Figs. 2, A and B). However, we were not able to detect constitutive Emt/PLC1 coimmunoprecipitation in freshly purified T cells (Fig. 2A). Overall, the Emt/PLC1 association was stoichiometrically reduced in normal PBL-derived T cells compared with leukemic Jurkat cells. Taken together, these results indicate that the association between Emt and PLC1 is regulated by TCR/CD3 in both normal and leukemic human T cells.

View larger version (55K): [in this window] [in a new window]   FIGURE 2. Regulated Emt/PLC1 association in normal T lymphocytes. Fresh purified T cells (A; 5 x 107 cells/lane) and PHA-expanded T lymphoblasts (B; 3 x 107 cells/lane) were left unstimulated or were stimulated with anti-TCR/CD3 as indicated in Fig. 1, then lysed in 1% Nonidet P-40-containing lysis buffer and subjected to immunoprecipitation with control IgG or Abs to Emt or PLC1. Immunoprecipitated proteins were subjected to SDS-PAGE on 4–20% gradient gels and immunoblotted with antiserum to PLC1 (upper panels). Blot stripping was conducted as indicated in Materials and Methods, and the same membranes were reprobed with anti-Emt mAb (bottom panels). Data are representative of three independent experiments.

Emt/PLC1 association is enhanced by ligation of CD2, CD4, or CD28: correlation with tyrosine phosphorylation of Emt

We next investigated the effect of engagement of different surface receptors on the regulation of the Emt/PLC1 complex. Since TCR/CD3 activation resulted in enhancement of the Emt/PLC1 association, we analyzed whether costimulatory receptors, such as CD2, CD4, CD6, CD28, or the down-regulating receptor CD5 (26), modulated the Emt/PLC1 association. As shown in Fig. 3B (top), CD2, CD4, CD28, as well as TCR/CD3 engagement significantly augmented formation of the Emt/PLC1 complex in Jurkat cells. Similar results were obtained when using PHA-expanded T cell lymphoblasts (data not shown). In contrast, engagement of CD5 or CD6 did not enhance this protein-protein association. Analysis of Jurkat cells by FACS revealed high expression levels of TCR/CD3, CD2, CD4, CD5, and CD6. However, CD28 expression was approximately 10-fold lower compared with the other Ags (Fig. 3A); thus, the results of these experiments followed bona fide ligation of the individual receptors. Interestingly, the TCR/CD3-, CD2-, CD4-, and CD28-induced enhancement of the Emt/PLC1 association (Fig. 3B, top) correlated with the induction of Emt tyrosine phosphorylation specifically regulated by these surface receptors (Fig. 3B, middle). Stimulation of Jurkat cells through CD2, CD4, CD28, or TCR/CD3 up-regulated, although to different extents, the phosphotyrosine content of Emt. In contrast, CD5 and CD6 ligation had no effect on the tyrosine phosphorylation status of Emt. It is important to note that in Jurkat cells, both CD28-enhanced Emt/PLC1 complex formation and CD28-induced augmentation of Emt tyrosine phosphorylation were less potent than other costimulatory molecules, correlating with their lower surface expression. Similar levels of Emt were immunoprecipitated from each condition, as shown by reprobing of the membrane with anti-Emt mAb (Fig. 3B, bottom). Taken together, these results demonstrate that several surface receptors potently induced both increased tyrosine phosphorylation of Emt and enhancement of the Emt/PLC1 association.

View larger version (52K): [in this window] [in a new window]   FIGURE 3. Regulation of Emt/PLC1 interaction and tyrosine phosphorylation of Emt following independent ligation of CD2, CD4, or CD28 costimulatory receptors. A, Jurkat T cells were stained with an isotype-matched control mAb (thin solid line) or the anti-CD3 mAb G19-4, anti-CD2 mAb 9.6, anti-CD4 mAb G17-2, anti-CD28 mAb 9.3, anti-CD5 mAb 10.2, or anti-CD6 mAb G3-6 (thick solid lines) plus goat anti-mouse FITC and analyzed by FACScan. B, Surface receptors were cross-linked as indicated with specific mAbs (20 µg/ml) on Jurkat cells (2 x 107/lane). After receptor ligation at 37°C for 10 min, the cells were lysed in 1% Nonidet P-40 lysis buffer and subjected to immunoprecipitation with control IgG or anti-Emt Abs. Immunoprecipitated proteins were analyzed by SDS-PAGE on 4–20% gradient gels and were immunoblotted with antiserum to PLC1 (upper panel). Blot stripping was performed as specified in Materials and Methods, and the membrane was reprobed with anti-phosphotyrosine mAb (middle panel) or anti-Emt mAb (bottom panel). Data are representative of three independent experiments.

Protein domains involved in the Emt/PLC1 association

To address the mechanism of the observed protein-protein interaction, we investigated the involvement of individual Emt domains in the formation of the Emt/PLC1 complex. For this purpose, several GST-Emt fusion proteins were generated (Fig. 4A), each including a single domain or different domain combinations. Coprecipitation experiments were conducted using equivalent amounts of GST-Emt fusion proteins added to lysates of PHA expanded T cell lymphoblasts or Jurkat T cells (data not shown). As shown in Fig. 4B, the GST-PTR32, GST-SH3, and GST-SH3SH2, but not the GST-SH2, fusion proteins coimmunoprecipitated PLC1 before TCR/CD3 activation. This constitutive association was moderately augmented upon TCR/CD3 engagement when using the GST-PTR32 and GST-SH3SH2 fusion proteins and was strongly up-regulated with the GST-SH2 fusion protein. Interestingly, the opposite result was observed in the case of the GST-SH3 fusion protein (compare amount of PLC1 coprecipitated before and after TCR/CD3 stimulation). These results suggest that both the Emt-SH3 and -SH2 domains are involved in the PLC1 association.

View larger version (48K): [in this window] [in a new window]   FIGURE 4. Emt/PLC1 interaction analysis with GST fusion proteins. A, Schematic of the human Emt protein and individual GST-fusion proteins constructed. B, Normal PHA T cell lymphoblasts (3 x 107/lane) were left unstimulated or were TCR/CD3 stimulated (8 min at 37°C), then lysed in 1% Nonidet P40-containing lysis buffer and incubated with equivalent amounts of GST or the indicated GST-Emt fusion proteins. Reactants and immunoprecipitates were subjected to SDS-PAGE on 4–20% gradient gels. Immunoblotting was performed with antiserum to PLC1. C, Far Western analysis. Jurkat T cells were stimulated with anti-TCR/CD3, and PLC1 immunoprecipitates were taken at the indicated time points. Reactants and immunoprecipitates were subjected to SDS-PAGE on 4–20% gradient gels. Immunoblotting was performed with 10 µg/ml GST-Emt-SH2 fusion protein (top), anti-phosphotyrosine 4G10 mAb (middle), or anti-PLC1 antiserum (bottom). Data are representative of two independent experiments.

Reciprocally, we also investigated whether individual PLC1 domains were involved in Emt association by using GST-PLC1 fusion proteins containing only its SH3 domain, N-terminal SH2 domain, or C-terminal SH2 domain. As shown in Fig. 5, an important component of the Emt/PLC1 association was mediated by the PLC1-SH3 domain in Jurkat cells, since only limited amounts of Emt were coprecipitated using either PLC1 N- or C-terminal SH2 domains. Clearly, the effect of TCR/CD3 ligation was observed regarding the association between the GST-PLC1-SH3 fusion protein and Emt, with a minor component contributed by the two PLC1 SH2 domains.

View larger version (25K): [in this window] [in a new window]   FIGURE 5. Emt/PLC1 interaction analysis with GST-PLC1 fusion proteins. Jurkat cells (2 x 107/lane) were left unstimulated or were TCR/CD3 stimulated (8 min at 37°C), then lysed in 1% Nonidet P-40-containing lysis buffer and incubated with equivalent amounts of GST, the indicated GST-PLC1 fusion proteins, or anti-Emt Abs. Reactants and immunoprecipitates were subjected to SDS-PAGE on 4–20% gradient gels. Immunoblotting was performed with anti-Emt mAb. Data are representative of two independent experiments.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The observation that in Itk-deficient T lymphocytes there is diminished TCR/CD3-induced tyrosine phosphorylation of PLC1 resulting in reduced calcium signals and IP₃ generation prompted us to investigate the putative association between Emt and PLC1. Coprecipitation experiments in both Jurkat and PBL-derived normal T cells (freshly isolated or PHA expanded), showed a TCR/CD3-regulated physical interaction between these signaling molecules. However, several differences were noticed. First, there was a constitutive Emt/PLC1 association in the leukemic T cell line Jurkat that was less apparent in normal T cells, although in both cell types a TCR/CD3-induced increase in the Emt/PLC1 association was observed. Second, although we detected in fresh T cells and PHA T lymphoblasts enhanced Emt/PLC1 association upon TCR/CD3 engagement, the degree of interaction in normal PBL-derived T cells was reduced compared with that in leukemic Jurkat cells. Since the cellular activation state seemed to be important for this association, the differences regarding the constitutive Emt/PLC1 interaction found in transformed vs normal cell types may be attributed to differential activation/differentiation states between these cell types.

To address in more detail the mechanism for Emt/PLC1 association, we investigated the involvement of individual Emt or PLC1 SH2/SH3 domains in this process. Coprecipitation experiments using GST fusion proteins containing individual or combinations of Emt or PLC1 SH2/SH3 domains demonstrated the involvement of both Emt and PLC1 SH3 domains in the constitutive association with PLC1 or Emt, respectively. However, following TCR/CD3 stimulation, the Emt-SH2 domain became involved. Importantly, the partial contribution of these domains was different in resting and TCR/CD3-activated T cells, indicating a different mechanism. In this regard, the constitutive Emt/PLC1 association seems likely to be due primarily to the Emt SH3 domain and some contribution of the PLC1 SH3 domain. Nevertheless, upon TCR/CD3 stimulation the Emt-SH3-mediated contribution decreased, and both the Emt-SH2- and PLC1-SH3-mediated interactions were significantly enhanced. This observation correlated with the TCR/CD3-induced tyrosine phosphorylation levels of PLC1. Further, we analyzed the involvement of both the Emt-SH2 and Emt-SH3 domains in mediating direct binding to PLC1. Interestingly, the Emt-SH2 domain was capable of interacting directly and specifically with TCR/CD3-induced tyrosine-phosphorylated PLC1, since we were able to detect the association using the Emt-SH2 fusion protein either by immunoaffinity reactions or by direct immunoblotting.

However, the Emt-SH3-mediated association with PLC1 was most likely indirect, since we observed binding in immunoaffinity reactions but did not detect direct binding by Far Western analysis. The effect of cross-linking the TCR/CD3 was that less PLC1 was associated with GST-Emt-SH3. This may have been due to the formation of protein complexes that sequester PXXP motifs available for binding. In addition, we identified both hSos1 and hSos2 GEFs as putative mediating proteins that contain several proline-rich motifs (37), and these were found in direct association with PLC1 (J. K. Scholler et al., submitted). Perhaps the hSos proteins serve as a docking site for both Emt-SH3 and PLC1-SH3 domains with its multiple proline-rich motifs. Indeed, TCR/CD3 ligation also led to reduced interaction between immunoprecipitated hSos1 and Emt, possibly due to sequestered PXXP motifs on hSos1 after cell activation. Alternatively, phosphorylation of tyrosine residues within an SH3 domain may disrupt interaction with proline-rich sequences, as has been shown for the Tec family kinases (9).

To date, there is no evidence of regulation of hSos enzymatic activity following receptor stimulation (38). However, the functions of Emt, PLC1, and hSos, although distinct, may be brought into direct contact for augmenting molecular density in the signaling complexes and possibly bridging Ras activation in proximity to PKC/calcium activation. Finally, the PLC1-SH3 domain may also contribute to the protein complexes directly through binding to the proline-rich sequences present on Emt (1, 8) or through the proline-rich sequences of other proteins (i.e., hSos1 and hSos2) found in association with Emt.

Membrane targeting by the PH domain is believed to render Tec family kinases activated, since they become more accessible to membrane-anchored Src family PTKs. The regulation of Tec family PTKs depends on at least two events, membrane anchoring to phospholipids produced by PI3-K and tyrosine phosphorylation by Src family kinases (14, 39). Engagement of surface receptors, such as CD2, CD4, or CD28, activates both Src family PTKs and PI3-K (14, 40, 41, 42, 43, 44). Consistently, we have demonstrated up-regulation of Emt tyrosine phosphorylation after ligation of TCR/CD3, CD2, CD4, or CD28 on T cells, correlating to enhancement of the Emt/PLC1 association. Previous reports have demonstrated that PLC1 becomes tyrosine phosphorylated and activated after TCR/CD3, CD2, CD4, or CD28 stimulation (17, 23, 43, 45), thus producing the sustained elevation of cytoplasmic free calcium necessary for the downstream activation of transcription factors and gene expression resulting in T cell activation. Emt/Itk/Tsk is not upstream of either TCR -chain or ZAP-70 tyrosine phosphorylation; however, it is required for optimal PLC1 activation after TCR/CD3 engagement (7). In addition, the defect in calcium influx across the plasma membrane in murine Itk-deficient T cells is probably due to the reduced tyrosine phosphorylation and activation of PLC1 in these cells (4, 6, 7). Perhaps the formation of the Emt/PLC1 complex facilitates PLC1 recruitment and tyrosine phosphorylation, thereby leading to activation of downstream effector functions.

Taken together, our results provide the first evidence for a physical interaction between Emt and PLC1 in human T cells. Further, the data suggest that Emt may be involved in PLC1 tyrosine phosphorylation/activation following TCR/CD3 and costimulatory molecule engagement, resulting in an enhanced signaling module necessary for downstream signaling. The formation of numerous heteromeric complexes containing Emt, PLC1, and/or hSos proteins may drive the extent to which signals are delivered through costimulation of T lymphocytes.

	Acknowledgments

 
We thank Dr. Deryk Loo for critical reading of the manuscript, Gena Whitney and Kathy O’Day for their valuable technical advice and helpful discussions, and Derek Hewgill for technical assistance. We also especially thank Dr. Alejandro Aruffo, the members of the Signal Transduction laboratory, and the Immunology, Inflammation, and Pulmonary Drug Discovery Department at Bristol-Myers Squibb for their continuous support during this project.

	Footnotes

 
¹ This work was supported by the Bristol-Myers Squibb Pharmaceutical Research Institute and Grant FP 08986319 from the Spanish Ministry of Education and Culture (to J.J.P.-V.).

² Address correspondence and reprint requests to Dr. Juan J. Perez-Villar, Immunology, Inflammation, and Pulmonary Drug Discovery, Bristol-Myers Squibb Pharmaceutical Research Institute, P.O. Box 4000, Princeton, NJ 08543. E-mail address:

³ Abbreviations used in this paper: PTK, protein tyrosine kinase; TK, tyrosine kinase; PR, proline rich; BCR, B cell Ag receptor; SH2, Src homology 2 domain; SH3, Src homology 3 domain; PH, pleckstrin homology domain; PKC, protein kinase C; PI3-K, phosphatidylinositol 3-kinase; PLC1, phospholipase C1; IP₃, inositol 1,4,5-trisphosphate; hSos, human Sos.

Received for publication July 13, 1999. Accepted for publication September 28, 1999.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

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