Sam68 Association with p120GAP in CD4+ T Cells Is Dependent on CD4 Molecule Expression

Nada Jabado, Sébastien Jauliac, Annaïck Pallier, Frédéric Bernard, Alain Fischer and Claire Hivroz
J Immunol September 15, 1998, 161 (6) 2798-2803;

Abstract

p120 GTPase-activating protein (p120GAP) is a major negative regulator of p21ras activity in several cell types including T cells. Catalytic activity of this enzyme is regulated in part by its interaction with several associated tyrosine-phosphorylated proteins. Sam68 was initially described as associated with p120GAP. It has been further established that Sam68 is a substrate of src kinases in mitosis and that it is not associated with p120GAP in transformed fibroblasts. We describe herein that Sam68 associates with p120GAP and PLCγ1 in human mature T cells and in a T cell line expressing the CD4 molecule HUT78 CD4+. This association is present in nonactivated cells and increases after anti-CD3 activation. It is dependent on CD4 expression and, in part, on the association of CD4 with p56lck, as shown by the strongly decreased association of Sam68 with p120GAP in the CD4− mutants, HUT78 CD4−, and by the reduced association of Sam68 with both p120GAP and p56lck in the HUT78 T cell line expressing a CD4 mutant unable to interact with p56lck, HUT78 C420/22. We propose that recruitment of Sam68, via CD4/p56lck, to the inner face of the plasma membrane may permit, via its docking properties, the correct association of key signaling molecules including PLCγ1 and p120GAP. This formation of transduction modules will enable the activation of different signaling cascades including the p21ras pathway and an array of downstream events, ultimately leading to T cell activation.

Ras proteins are small guanine nucleotide-binding proteins that associate with the plasma membrane and cycle between an inactive GDP-bound form and a biologically active GTP-bound form (1, 2). Ras activation is of particular importance in coupling receptor tyrosine kinases to downstream signaling pathways involved in the control of cell proliferation and differentiation (3, 4, 5). Tyrosine kinases induce the exchange of GDP for GTP on Ras (reviewed in 6 . GTP-bound Ras then undergoes conformational changes (7) enabling it to associate with downstream targets, notably the Raf protein serine/threonine kinase, which activates the mitogen-activated protein (MAP)3 kinase kinase cascade (8, 9, 10).

Ras-GTP also interacts specifically with GTPase-activating proteins (GAPs), which act to enhance the weak intrinsic Ras-GTPase activity accelerating the hydrolysis rate of bound GTP to GDP (2). P120GAP is a GAP that regulates p21ras activity in mammalian cells (11, 12, 13). This protein is of particular interest in tyrosine kinase signaling because the N-terminal region of p120GAP possesses two SH2 domains, which flank a single SH3 domain (14). Thus, p120GAP binds in a phosphotyrosine/SH2-dependent fashion to activated growth factor receptors (14). It also binds to intracellular phosphotyrosine containing proteins of 62 and 190 kDa in cells stimulated with a variety of growth factors or transformed by oncogenic tyrosine kinase variants (15, 16). The GAP-associated p62 protein becomes tyrosine phosphorylated in response to platelet-derived growth factor (PDGF (17)), epidermal growth factor (EGF (18)), and insulin (19) and associates with GAP through the GAP SH2 domain (16). Several tyrosine-phosphorylated proteins of ∼62 kDa have been shown to interact with p120GAP. A 60-kDa tyrosine-phosphorylated protein substrate of the insulin receptor kinase has been shown to associate with p120GAP (20). More recently, another protein of 62 kDa, p62Dok, was cloned and found to bind to p120GAP (21, 22). A cDNA coding for a 62-kDa protein, hump62, was cloned in 1992 (23). It was shown to be tyrosine phosphorylated and to associate with p120GAP in cells transformed by tyrosine kinase oncogenes (15, 16). In 1994, two other groups identified a 68-kDa phosphoprotein in mitotic NIH-3T3 murine fibroblasts overexpressing Src. This protein, named Sam68, has RNA-binding properties (24, 25, 26) and associates with Src through its SH2 and SH3 domains. The cDNA encoding for Sam68 was found to be homologous to hump62 (23). However, both groups found that Sam68 did not associate with p120GAP and was not tyrosine phosphorylated in interphase cells (24, 25). This controversy was apparently resolved when it was found that hump62 encodes Sam68 protein, but not the p120GAP-associated p62, and that the two proteins are not related (27).

In T lymphocytes, the TCR is coupled via its cytoplasmic domain to a protein tyrosine kinases cascade (28) that couples the TCR to the guanine nucleotide-binding protein, p21ras (13). This, in turn, leads to the activation of Raf-1 and the MAP kinase ERK-2 (29, 30), finally leading to IL-2 production and cell proliferation (31, 32). In T cells, p120GAP activity has been shown to be inhibited by T cell triggering, leading to p21ras activation (13). Sam68 was shown to coprecipitate in T cells with various cellular proteins such as SHP-1, PLCγ1, the phosphatidylinositol-3 (PI 3)-kinase p85 subunit, and Grb2 (33, 34). We have previously described, in CD4+ T cells, an association of Sam68 with PLCγ1 (35). Moreover, we showed that there is an association in these cells of PLCγ1 with p120GAP, and that both the Sam68/PLCγ1 and the PLCγ1/p120GAP associations are inhibited by CD4 ligands (35). This inhibition correlated with an inhibition of the PLCγ1 activity (35) as well as with an inhibition of the Raf/Erk-2 signaling cascade (36). We therefore investigated whether Sam68 could coprecipitate with p120GAP in T cells and looked for the molecular partners necessary for this association. We demonstrate herein that Sam68 associates with p120GAP in human peripheral CD4+ T lymphocytes and in a T cell line expressing the wild-type CD4 molecule. In addition, we show that this association is dependent on the CD4 molecule expression and that the interaction of p56lck with the intracytoplasmic domain of CD4 plays a role in this association. We suggest that these associations may play a role in the formation of multifunctional activation modules ultimately leading to T cell activation.

Materials and Methods

Plasmids and DNA-mediated gene transfer

The different HUT 78 T cell lines were obtained as previously described (37). The HUT78 CD4− T cell line was transfected with wild-type CD4 cDNA (HUT78 CD4+). The HUT78 C420/22 T cell line was transfected with a mutant CD4 molecule in which C420 and C422 were replaced with two adenosines (A). We have previously demonstrated (37) that 1) all of the transfected HUT78 T cell lines expressed comparable levels of CD4 by flow cytometric analysis, 2) these cell lines expressed equivalent amounts of active p56lck in Western blot analysis, and 3) the association between the wild-type CD4 and p56lck was confirmed by coprecipitation experiments and Western blotting, with anti-CD4 followed by anti-p56lck Abs. The same experiments were used to verify the absence of association between the mutated form of CD4 and p56lck.

Cell preparation

Human T cells were obtained from peripheral blood of healthy volunteers as previously described (35). Briefly, PBMC were isolated by Ficoll-Hypaque (Pharmacia, Uppsala, Sweden) density sedimentation. T cells were then enriched by rosetting with neuraminidase-treated sheep erythrocytes.

The three HUT78 T cell lines were cultured in RPMI 1640 medium (Life Technologies/Biocult, Paisley, U.K.) supplemented with 10% FCS.

Reagents

MAb UCHT1 (IgG1), specific for the CD3 ε-chain (ascitic fluid) was used at a final concentration of 1:300. Anti-PLCγ1 and antiphosphotyrosine 4G10 mAbs were from Upstate Biotechnology (Lake Placid, NY). Anti-GAP (B4F8) and anti-p56lck mAbs as well as polyclonal anti-Sam68 Abs were from Santa Cruz Biotechnology (Santa Cruz, CA), and anti-Sam68 P and C were kindly provided by S. Courtneidge (Sugen, Redwood City, CA).

Cell activation and immunoprecipitation experiments

T cells were washed in RPMI 1640 (Life Technologies) and stimulated for 3 min with the UCHT1 mAb or left unactivated. Activation was stopped by centrifugation for 1 min at 10,000 rpm at 4°C, and cells were immediately lysed for 40 min at 4°C in lysis buffer (20 mM Tris-HCl, pH 7.5, 140 mM NaCl, 1% digitonin, 50 U/ml aprotinin, 1 mM sodium orthovanadate, and 1 mM PMSF) or for 20 min at 4°C in Nonidet P-40 1% lysis buffer (20 mM Tris-HCl, pH 7.4, 140 mM NaCl, 1% Nonidet P-40, 2 mM EDTA, 1 mM PMSF, 1% aprotinin, 1 mM sodium orthovanadate, 50 mM NaF, and 2 μg/ml of antipain, pepstatin, and leupeptin). Nuclei and cellular debris were removed by centrifugation for 20 min at 10,000 rpm. The protein concentration was determined in postnuclear supernatants by using the Bio-Rad kit with BSA as standard (Bio-Rad, Richmond, CA). The same amount of each extract was precleared at 4°C by rocking for 1 h with 50 μl of packed protein A-Sepharose beads (Pharmacia). Lysates were incubated overnight with 2 μg of anti-GAP Ab, 10 mg of anti-Sam68 Ab, or 5 ml of anti-Sam68 (C). Immunoprecipitates were recovered by incubation with 50 μl of protein A-Sepharose beads for 1 h at 4°C, then washed three times in lysis buffer. Proteins were then eluted and dissolved by boiling for 5 min in Laemmli sample buffer before resolution on standard 8% SDS-PAGE gel.

Western blot analysis

Protein from total lysates or immunoprecipitates were electrophoretically transferred for 2 h at 120 V to a polyvinylidene difluoride membrane (Immobilon P; Millipore, Bedford, MA). After blocking with 5% BSA, the blots were incubated with 0.1 μg/ml of antiphosphotyrosine mAb 4G10, anti-GAP 0.5 μg/ml, anti-Sam68 (Santa Cruz Biotechnology), or anti-Sam68 P or C used at a final concentration of 1/3000, followed by horseradish peroxidase-conjugated secondary Abs (Amersham, Little Chalfont, U.K.). Blots were revealed by enhanced chemiluminescence according to the manufacturer’s instructions (Amersham).

Results

Sam68 is associated with p120GAP and PLCγ1 in human T cells

In T cells and in fibroblasts, p120GAP is associated with tyrosine-phosphorylated proteins migrating at a molecular mass of 60 to 68 kDa in a phosphotyrosine- and SH2 domain-dependent fashion (20, 21, 22, 23). We have previously demonstrated in human peripheral CD4+ T cells that Sam68 is associated with PLCγ1 (35), using a commercial Ab directed against the central portion of the predicted hump62 protein (23, 27). We have also demonstrated that p120GAP and PLCγ1 were associated in a multifunctional complex (35). To further characterize the p120GAP-associated 62- to 68-kDa protein that we described previously, immunoprecipitation experiments using anti-p120GAP, anti-Sam68, and anti-PLCγ1 Abs were performed on lysates from HUT 78 CD4+ T cells (Fig. 1⇓). Cells were activated for 3 min with the anti-CD3 UCHT1 mAb or were left unactivated, then lysed in 1% digitonin lysis buffer. Whole-cell lysates (Fig. 1⇓, lane 1 and 2), anti-GAP (lane 3 and 4), anti Sam68 (Santa Cruz Biotechnology; lane 5 and 6), and anti-PLCγ1 (lane 7 and 8; rabbit polyclonal Ab in panel A, mouse monoclonal Ab in panel B) immunoprecipitates were analyzed for the presence of Sam68. As shown in Figure 1⇓, A and B, a band migrating at a molecular mass of 66 to 68 kDa was immunoreactive with two anti-Sam68 Abs (anti-P in panel A, anti-C in panel B) in all lanes. Conversely, the presence of p120GAP in anti-Sam68 and anti-PLCγ1 immunoprecipitates (Fig. 1⇓A) was detected, showing the association of Sam68 with both p120GAP and PLCγ1. Coprecipitation of p120GAP with Sam68 was also observed in control peripheral T cells lysed in 1% digitonin lysis buffer (Fig. 2⇓). In these cells, the association of Sam68 with p120GAP was present in nonactivated T cells, increased at 3 min after anti-CD3 activation, and returned to basal level after 10 min of activation.

FIGURE 1.
FIGURE 1.

Sam68 coprecipitates with p120GAP and PLCγ1 in HUT78 CD4+ T cells. T cells (5 × 106 HUT78 CD4+) were stimulated for 3 min with UCHT1 (1:300) or left unstimulated. Cells were subsequently lysed in a 1% digitonin lysis buffer, and lysates were precleared with mouse IgG (1 μg/500 μg of lysates), then immunoprecipitated with 2 μg of anti-p120GAP (B4F8), 2 μl of anti-Sam68 (C), or 2 μg of anti-PLCγ1 (rabbit polyclonal Ab (A) and mouse monoclonal Ab in (B)). Immunoprecipitates and whole lysates were resolved on an 8% polyacrylamide gel. After transfer, the membrane was cut into two parts. The upper part of panel A shows an anti-p120GAP Western blot and the lower part an anti-Sam68 (P) blot of the same membrane. B shows an anti-Sam68 (C) blot performed on a different membrane. One representative experiment of four is shown.

FIGURE 2.
FIGURE 2.

Sam68 coprecipitates with p120GAP in human peripheral T cells. T cells (30 × 106) were activated for 3 or 10 min with UCHT1 (1:300) or left unstimulated. Cells were lysed in a 1% digitonin buffer, and lysates were precleared with 1 μg of mouse IgG, then immunoprecipitated with 2 μg of anti-p120GAP (B4F8) or 2 μl of anti-Sam68 (C). After transfer, the membrane was cut into two parts. The upper panel shows an anti-p120GAP blot and the lower panel an anti-Sam68 (C) blot of the same membrane. One representative experiment of three is shown.

The use in our experiments of a mild lysis buffer (1% digitonin) that preserves unstable or weak protein associations, instead of the stronger lysis buffer used by other groups (24, 25), may account for the association of Sam68 with p120GAP that we observed. We thus performed the same experiments on lysates of anti-CD3-activated HUT 78 CD4+ T cells obtained with a 1% Nonidet P-40 lysis buffer. Coprecipitation of Sam68 with p120GAP and PLCγ1 was also observed in these conditions. Moreover, the association of Sam68 with p120GAP and PLCγ1 was more easily detected, probably due to a more efficient lysis of cells with the Nonidet P-40 lysis buffer (data not shown).

Sam68 association with p120GAP in human T cells is dependent on the presence of the CD4 molecule

The association of Sam68 with p120GAP is still a matter of controversy. Two different groups have shown in transformed fibroblasts that Sam68 does not associate with p120GAP (25, 26, 27). Protein interactions and signaling pathways may differ in different cell types. One hypothesis explaining the association of Sam68 and p120GAP in human T cells and not in fibroblasts could be the requirement for a T cell-specific coreceptor such as CD4, allowing the presence of Sam68 in a cellular compartment where it can interact with p120GAP. To address this issue, we performed anti-p120GAP immunoprecipitations in HUT 78 CD4− cells, which do not express CD4, and in CD4-transfected HUT78 CD4+ cells. To assess protein associations in resting cells and in anti-CD3-activated cells, both T cell lines were starved in RPMI 1640 medium supplemented with 3% FCS for 18 h and then in RPMI alone for 1 h at 37°C before stimulation (Fig. 3). Activation by the anti-CD3 mAb UCHT1 for 3 min (Fig. 3⇓, lanes 2 and 4) induced the association of several tyrosine-phosphorylated proteins, including a doublet migrating at 62 to 64 kDa in both HUT78 CD4− and HUT78 CD4+ T cells, as revealed by the 4G10 immunoblot (Fig. 3⇓, middle panel). However, a highly tyrosine-phosphorylated band with a higher molecular mass of 66 to 68 kDa was present in p120GAP immunoprecipitates from nonactivated HUT78 CD4+ T cells (lane 3). Its tyrosine phosphorylation was enhanced after anti-CD3 activation (lane 4). Anti-Sam68 (Santa Cruz Biotechnology) immunobloting of the lower part of the same membrane (Fig. 3⇓, lower panel) revealed that this band was Sam68. The same amount of p120GAP was present in each lane, as demonstrated by the anti-p120GAP immunoblot performed on the same membrane (upper panel). The proteins precipitated in association with p120GAP were specific as demonstrated by their absence in nonspecific immunoprecipitates performed with mouse immunoglobulins (Fig. 3⇓, lanes 5 to 8). These data indicate that the presence of the CD4 molecule at the surface of HUT78 T cells enables the association of Sam68 with p120GAP.

FIGURE 3.
FIGURE 3.

Sam68 is associated with p120GAP in HUT78 CD4+ T cells and not in HUT78 CD4− T cells. Association of both proteins is increased by the anti-CD3-induced tyrosine phosphorylation of Sam68. 5 × 106 HUT78 CD4+ T cells or HUT78 CD4− T cells were activated for 3 min with UCHT1 (1:300) or left unstimulated. Cells were lysed in a 1% Nonidet P-40 lysis buffer and lysates were treated as described in Figure 1⇑. The middle panel shows a 4G10 immunoblot of the membrane. The arrow indicates Sam68. The upper panel shows an anti-p120GAP blot and the lower panel an anti-Sam68 (C) blot performed on the same membrane after stripping. One representative experiment of three is shown.

p56lck association with the CD4 molecule is involved in Sam68 interaction with p120GAP

The CD4 molecule is noncovalently associated with the p56lck tyrosine kinase (38). We assessed the role of the association of CD4 with p56lck in the formation of complexes between p120GAP and Sam68. P120GAP immunoprecipitations were performed on lysates from UCHT1-activated HUT78 CD4+ (Fig. 4⇓, lane 2), HUT78 C420/22 (a T cell line transfected with a cDNA coding for a mutated CD4 molecule abolishing the association between p56lck and CD4 (lane 5)), and HUT78 CD4− T cells (lane 8). Whole cell lysates (lanes 1, 4, and 7) were run in parallel as a positive control of the amount of Sam68 in the lysate of each cell type (lanes 3, 6, and 9) (Fig. 4). In HUT78 CD4+ cells, Sam68 was present in the anti-p120GAP immunoprecipitate (lane 2). There was a small amount of Sam68 present in p120GAP immunoprecipitates performed on HUT78 C420/22 T cells (lane 5), and Sam68 was almost absent in HUT78 CD4− cells (lane 8). The same amount of protein was present in each lane (lanes 2, 5, and 8, upper panel).

FIGURE 4.
FIGURE 4.

Sam68 association with p120GAP is partly dependant on p56lck association with the CD4 molecule. 5 × 106 HUT78 CD4+ T cells, HUT78 C420 T cells, or HUT78 CD4− T cells were activated for 3 min with UCHT1 (1:300). Cells were lysed in a 1% Nonidet P-40 lysis buffer, and lysates were treated as described in the legend to Figure 1⇑. After transfer, the membrane was cut into two parts. The upper panel shows an anti-p120GAP blot and the lower panel an anti-Sam68 (C) blot performed on the same membrane. One representative experiment of three is shown.

Sam68 is phosphorylated in HUT 78 C420/22 cells but has a decreased association with p56lck

The decreased association of p120GAP with Sam68 that we observed in the HUT78 C420/22 cell line could be due to a decrease in tyrosine phosphorylation of Sam68 because of the lack of CD4/p56lck association in this cell line. To investigate this hypothesis, we first assessed the tyrosine phosphorylation status of Sam68 in HUT78 CD4+ and HUT78 C420/22 T cells (Fig. 5). In both cell lines, UCHT1 activation for 3 or 10 min induced the tyrosine phosphorylation of several proteins including a band migrating at 66 to 68 kDa. In whole cell lysates (Fig. 5⇓, lanes 1 to 6) of both cell lines, a highly tyrosine-phosphorylated band migrating at 66 kDa was detected among other tyrosine phosphorylated proteins after CD3 activation. The pattern of tyrosine phosphorylation was very similar in both cell lines. In anti-Sam68 immunoprecipitates (Fig. 5⇓, lanes 7 to 12), a tyrosine-phosphorylated triplet migrating between 66 and 68 kDa was weakly detected in nonactivated cells in both cell lines (lanes 7 and 10). Anti-CD3 activation of both cell lines increased the tyrosine phosphorylation of this triplet by the same level. This triplet was shown to be Sam68 (data not shown). The tyrosine-phosphorylated band migrating at 100 to 105 kDa in the anti-Sam68 immunoprecipitates (Fig. 5⇓, lanes 7 to 12) was also present in the nonspecific immunoprecipitates (data not shown). The same results were obtained in HUT78 CD4− T cells (data not shown).

FIGURE 5.
FIGURE 5.

After anti-CD3 activation, Sam68 is tyrosine phopshorylated in HUT78 C420 T cells to the same level as in HUT78 CD4+ T cells. Its association with p56lck is decreased in HUT78 C420 T cells. 5 × 106 HUT78 CD4+ or HUT78 C420 T cells were activated for 3 or 10 min with UCHT1 (1:300) or left unstimulated. Cells were lysed in a 1% Nonidet P-40 lysis buffer, and lysates were treated as described in the legend to Figure 1⇑. The upper left panel shows a 4G10 blot performed on whole lysates and the upper right panel a 4G10 blot performed on anti-Sam68 (C) immunoprecipitates. The lower panel represents anti-p56lck immunoprecipitates revealed with an anti-Sam68 (C) blot. The arrow indicates Sam68. One representative experiment of three is shown.

We then assessed the level of Sam68 associated with p56lck in HUT78 CD4+ and HUT78 C420/22 cell lines. Anti-p56lck immunoprecipitations were performed on lysates from nonactivated and UCHT1-activated cells, and precipitated proteins were revealed with anti-Sam68 C Ab (Fig. 5⇑, lower panel). A protein migrating at 66 to 68 kDa immunoreactive with an anti-Sam68 (C) Ab, was coprecipitated with p56lck in both cell lines. The association was present in nonactivated cells (Fig. 5⇑, lanes 1 and 4) and was slightly increased after UCHT1 activation (lanes 2, 3, 5, and 6). However, the amount of Sam68 associated with p56lck was markedly decreased in HUT78 C420/22 (Fig. 5⇑, lanes 4 to 6) as compared with the amount associated with p56lck in HUT78 CD4+ T cells (lanes 1 to 3). The same amount of Sam68 was present in both cell lines as demonstrated by the anti-Sam68 immunoprecipitates run in parallel.

Discussion

We have previously reported in CD4+ human peripheral T cells the existence of multifunctional complexes containing several transducing proteins that play a role in T cell activation. We showed that activation of CD4+ T cells through the TCR induced the association of p120GAP with PLCγ1 and other unidentified tyrosine-phosphorylated proteins, and we described an association of PLCγ1 with Sam68. CD4 ligand binding induced defective formation of these mutifunctional complexes, which correlated with the inhibition of signaling through the TCR (36). The aim of the present study was to investigate whether Sam68 was associated with p120GAP in CD4+ T cells and to characterize the molecule(s) responsible for this association.

Several groups have reported the association of tyrosine-phosphorylated proteins of 62 to 68 kDa with p120GAP in both T cells and other cell types (20, 21, 22, 23, 33, 39). A 62-kDa protein associated with p120GAP was first cloned and named hump62 (23). Two other groups described a highly tyrosine-phosphorylated 68-kDa protein, Sam68, in transformed murine and human fibroblasts: they reported this protein as an effector of the tyrosine kinase p60c-Src in mitosis and also showed that it interacts with the SH2 and SH3 domains of p60c-Src during the S phase and translocates thereafter to the nucleus (24, 25). They also showed that it does not associate with p120GAP in NIH-3T3 cells, based on the lack of coimmunoprecipitation between both proteins in interphase cells (27). Finally, they demonstrated that hump62 cDNA encodes Sam68 and not the p120GAP-associated p62 protein (27). However, the interaction of Sam68 with the SH2 domain of p120GAP in a human CD4+ T cell line has been reported (33).

We demonstrate herein that p120GAP is associated with Sam68 in human CD4+ peripheral T cells and in a T cell line (HUT78) expressing the wild-type CD4 molecule. In contrast, no association of Sam68 with p120GAP was observed in the same T cell line lacking CD4 expression. Moreover, we obtained the same results in a HUT78 T cell line transfected with a cDNA coding for a CD4 molecule without a cytoplasmic domain (data not shown). In addition, in the HUT78 T cell line transfected with a cDNA coding for a CD4 molecule mutated in the domain that interacts with p56lck, HUT78 C420/422 (37), the association of Sam68 with p120GAP was strongly reduced, although still detectable. Altogether, these results suggest that the CD4 cytoplasmic domain is necessary for Sam68/p120GAP association and that the CD4-associated pool of p56lck may play a role in this association. However, our data do not allow us to state that there is a direct interaction between p120GAP and Sam68, as they may be coprecipitated through other protein interaction.

It has been shown that Sam68 is tyrosine phosphorylated after cell activation and that this event enables its interaction with signaling molecules in a phosphotyrosine/SH2-dependent fashion (33). Thus, a reduced tyrosine phosphorylation of Sam68 in the HUT78CD4− and HUT78C420/422 T cell lines could explain its reduced association with p120GAP. However, Sam68 was equally phosphorylated in the three T cell lines in response to TCR activation. These data suggest that Sam68 can be phosphorylated by another tyrosine kinase or by a pool of p56lck not associated with the CD4 molecule. Indeed, it has recently been shown that p59fyn, a TCR/CD3-associated tyrosine kinase of the src family, can phosphorylate Sam68 in T cells (34). Moreover, we observed an association of Sam68 with p56lck in the HUT78C420/422, although to a much lesser extent than in the HUT78 CD4+ T cell line. This result suggests that alternative pathways to the CD4 could facilitate, to a lesser degree, p56lck or p59fyn interaction with Sam68. The CD2 molecule, which is exclusively expressed on T and NK cells, is a potential candidate. It has been shown that cross-linking of the CD2 by anti-CD2 Abs induced the coimmunoprecipitation of a 62- to 66-kDa protein with p120GAP in a Jurkat T cell line (39). These observations could account for the residual association of Sam68 with p120GAP and the tyrosine phosphorylation of Sam68 in the HUT78 CD4− and HUT78 C420/422 T cell lines. It is worth noting that we observed an association of p56lck with Sam68 as well as a tyrosine phosphorylation of this protein in nonactivated T cells. Both the association and the phosphorylation were stronger in T cells activated by the TCR/CD3. The preassociation of Sam68 with the p56lck could be due to a proline-rich, SH3-dependent association of Sam68 with the Src kinases as reported elsewhere (33). p21ras plays a central role in the regulation of cellular signal transduction processes leading to cell growth and differentiation.

The activation of p21ras is positively regulated by guanine nucleotide exchange factors, which allow GTP binding to the protein, and is negatively regulated by GAPs, which inactivate p21ras by accelerating the slow, intrinsic rate of GTP hydrolysis (11, 12, 15). T cell activation has been shown to activate p21ras in a protein tyrosine kinase-dependent way (14). In T lymphocytes, p120GAP seems to be the major regulator of p21ras (14). The catalytic activity of p120GAP is regulated, at least in part, by its interaction with phosphotyrosine proteins including p190 and several proteins migrating at an apparent molecular mass of 62 to 68 kDa (20, 21, 22, 23, 33). We have previously demonstrated that preincubation of CD4+ T lymphocytes with CD4 ligands inhibits the activation of targets of the p21ras pathway; i.e., Erk-2 and the c-jun-N-terminal kinases ultimately leading to inhibition of IL-2 transcription (40). These inhibitions correlated with an inhibition by the CD4 ligands of the formation of multifunctional complexes containing PLCγ1, Sam68, and p120GAP (35). The data presented herein show that CD4 is necessary for the association of Sam68 with p120GAP and that the cytoplasmic domain of CD4 and its associated pool of p56lck plays a role in this association. We propose that recruitment of Sam68 via CD4/p56lck to the inner face of the plasma membrane may permit, via its docking properties, the correct association of key signaling molecules including PLCγ1, p120GAP, and Grb2. This formation of transduction modules will enable activation of different signaling cascades including the p21ras pathway. CD4 ligand binding, by precluding the formation of these signaling modules, may prevent correct T cell activation. The role of p56lck, Sam68, and p120GAP in this inhibition is currently under investigation.

Acknowledgments

We thank Jane E. Peake for checking the English and for critical reading of the manuscript. We also thank Jim Di Santo for helpful discussions and critical reading of the manuscript.

Footnotes

  • ↵1 Address correspondence and reprint requests to Dr. Nada Jabado, Unité d’Immuno-Hématologie, Hôpital Necker-Enfants Malades, 149 rue de Sèvres, 75015 Paris, France. E-mail address: Jabado{at}necker.fr

  • ↵2 S.J. is the recipient of a grant from the Ligue Nationale Contre Le Cancer.

  • ↵3 Abbreviations used in this paper: MAP, mitogen-activated protein; GAP, GTPase-activating protein.

  • Received November 17, 1997.
  • Accepted May 19, 1998.

References

  1. ↵
    Bourne, H. R., D. A. Sanders, F. McCormick. 1991. The GTPase superfamily conserved structure and molecular mechanism. Nature 349: 117
    OpenUrlCrossRefPubMed
  2. ↵
    Boguski, M. S., F. McCormick. 1993. Proteins regulating ras and its relative. Nature 366: 643
    OpenUrlCrossRefPubMed
  3. ↵
    Dickson, B., E. Hafen. 1994. Genetics of signal transduction in invertebrates. Curr. Opin. Genet. Dev. 4: 64
    OpenUrlCrossRefPubMed
  4. ↵
    Geer, P. V. D., T. Hunter, R. A. Lindberg. 1994. Receptor protein tyrosine kinases and their signal transduction pathways. Annu. Rev. Cell Biol. 10: 251
    OpenUrlCrossRef
  5. ↵
    Pawson, T.. 1995. Protein modules and signalling networks. Nature 373: 573
    OpenUrlCrossRefPubMed
  6. ↵
    Schlessinger, J., D. Bar-Sagi. 1995. Activation of ras and other signaling pathways by tyrosine receptor kinases. Symposia on Quantitative Biology: Molecular Genetics of Cancer 60 Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.
  7. ↵
    Pai, E. F., W. Kabsch, U. Krengel, K. C. Holmes, J. John, A. Wittinhofer. 1989. Structure of the guanine-nucleotide of the Ha-ras oncogene product p21 in the triphosphate conformation. Nature 341: 209
    OpenUrlCrossRefPubMed
  8. ↵
    Moodie, S. A., B. M. Willumsen, M. J. Weber, A. Wolfman. 1993. Complexes of Ras-GTP with Raf1 and mitogen-activated protein kinase kinase. Science 260: 1658
    OpenUrlAbstract/FREE Full Text
  9. ↵
    Vojtek, A. B., S. M. Hollenberg, J. A. Cooper. 1993. Mammalian Ras interacts directly with the serine:threonine kinase Raf. Cell 74: 205
    OpenUrlCrossRefPubMed
  10. ↵
    Warne, P. H., P. R. Viciana, J. Downward. 1993. Direct interaction of Ras and the amino-terminal region of Raf-1 in vitro. Nature 364: 352
    OpenUrlCrossRefPubMed
  11. ↵
    Trahey, M., F. Mc Cormick. 1987. A cytoplasmic protein stimulates normal Nras p21 GTPase, but does not affect oncogenic mutants. Science 238: 542
    OpenUrlAbstract/FREE Full Text
  12. ↵
    Hall, A.. 1990. ras and GAP—who’s controlling whom?. Cell 61: 921
    OpenUrlCrossRefPubMed
  13. ↵
    Downward, J., J. D. Graves, P. H. Warne, S. Rayter, D. A. Cantrell. 1990. Stimulation of p21 ras upon T-cell activation. Nature 346: 719
    OpenUrlCrossRefPubMed
  14. ↵
    Anderson, D. C., A. Koch, L. Grey, C. Ellis, F. Moran, T. Pawson. 1990. Binding of SH2 domains of phospholipase Cγ1, GAP, and Src to activated growth factor receptors. Science 250: 979
    OpenUrlAbstract/FREE Full Text
  15. ↵
    Ellis, C., M. Moran, F. McCormick, T. Pawson. 1990. Phosphorylation of GAP and GAP associated proteins by transforming and mitogenic tyrosine kinases. Nature 343: 377
    OpenUrlCrossRefPubMed
  16. ↵
    Moran, M. F., P. Polakis, F. McCormick, T. Pawson, C. Ellis. 1991. Protein tyrosine kinases regulate the phosphorylation, protein interactions, subcellular distribution and activity of p21ras GTPase-activating protein. Mol. Cell. Biol. 11: 1804
    OpenUrlAbstract/FREE Full Text
  17. ↵
    Kazlaukas, A., C. Ellis, T. Pawson, J. A. Cooper. 1990. Binding of GAP to activated PDGF receptors. Science 247: 1578
    OpenUrlAbstract/FREE Full Text
  18. ↵
    Margolis, B. N., L. A. Koch, M. Mohamadi, D. R. Hurwitz, A. Zilberstein, A. Ullrich, T. Pawson, J. Schlessinger. 1990. The tyrosine phosphorylated carboxy terminus of the EGF receptor is a binding site for GAP and PLCγ1. EMBO J. 9: 4375
    OpenUrlPubMed
  19. ↵
    Pronk, G. J., R. H. Medema, B. M. Burgering, R. Clark, F. McCormick, J. L. Bos. 1992. Interaction between the p21ras GTPase activating protein and the insulin receptor. J. Biol. Chem. 267: 24058
    OpenUrlAbstract/FREE Full Text
  20. ↵
    Hosomi, Y., K. Shii, W. Ogawa, H. Matsuba, M. Yoshida, Y. Okada, K. Yokono, M. Kasuga, S. Baba, R. A. Roth. 1994. Characterization of a 60 kilodalton substrate of the insulin receptor kinase. J. Biol. Chem. 269: 11498
    OpenUrlAbstract/FREE Full Text
  21. ↵
    Carpino, R., D. Wisniewski, A. Strife, D. Morshak, R. Kobayashi, B. Stiltman, B. Clarkson. 1997. p62dok, a constitutively tyrosine-phosphorylated GAP-associated protein in chronic myelogenous leukemia progenitor. Cell 88: 197
    OpenUrlCrossRefPubMed
  22. ↵
    Yamanashi, Y., D. Baltimore. 1997. Identification of the Abl- and rasGAP-associated 62 kDa protein as a docking protein Dok. Cell 88: 205
    OpenUrlCrossRefPubMed
  23. ↵
    Wong, G., O. Müller, R. Clark, L. Conroy, M. F. Moran, P. Polakis, F. McCormick. 1992. Molecular cloning and nuclear acid binding properties of the GAP-associated tyrosine phosphoprotein p62. Cell 69: 551
    OpenUrlCrossRefPubMed
  24. ↵
    Taylor, S. J., D. Shalloway. 1994. An RNA-binding protein associated with Src through its SH2 and SH3 domains in mitosis. Nature 368: 867
    OpenUrlCrossRefPubMed
  25. ↵
    Fumagalli, S., N. F. Totty, J. J. Hsuan, S. A. Courtneidge. 1994. A target for Src in mitosis. Nature 368: 871
    OpenUrlCrossRefPubMed
  26. ↵
    Courtneidge, S. A., S. Fumagalli. 1994. A mitotic function for Src?. Trends Cell. Biol. 4: 345
    OpenUrlCrossRefPubMed
  27. ↵
    Lock, P., S. Fumagalli, P. Polakis, F. McCormick, S. A. Courtneidge. 1996. The human p62 cDNA encodes Sam68 and not the RasGAP-associated p62 protein. Cell 84: 23
    OpenUrlCrossRefPubMed
  28. ↵
    Weiss, A., D. R. Littman. 1994. Signal transduction by lymphocyte antigen receptors. Cell 76: 263
    OpenUrlCrossRefPubMed
  29. ↵
    Izquierdo, M., S. J. Leevers, C. J. Marshall, D. A. Cantrell. 1993. p21ras couples the T cell antigen receptor to extracellular signal-regulated kinase 2 in T lymphocytes. J. Exp. Med. 178: 1199
    OpenUrlAbstract/FREE Full Text
  30. ↵
    Izquierdo, M., S. Bowden, D. A. Cantrell. 1994. The role of Raf-1 in the regulation of extracellular signal-regulated kinase 2 by the T cell antigen receptor. J. Exp. Med. 180: 401
    OpenUrlAbstract/FREE Full Text
  31. ↵
    Rayter, S. I., M. Woodrow, S. C. Lucas, D. A. Cantrell, J. Downward. 1992. p21ras mediates control of IL2 gene promoter function in T cell activation. EMBO J. 11: 4549
    OpenUrlPubMed
  32. ↵
    Baldari, C. T., A. Heguy, J. L. Telford. 1993. ras protein activity is essential for T-cell antigen receptor signal transduction. J. Biol. Chem. 268: 2693
    OpenUrlAbstract/FREE Full Text
  33. ↵
    Richard, S., D. Yu, K. J. Blummer, D. Hausladen, M. W. Oslowy, P. A. Connelly, A. S. Shaw. 1995. Association of p62, a multifunctional SH2- and SH3-domain binding protein, with src family tyrosine kinases, Grb2, and phospholipase Cγ1. Mol. Cell. Biol. 15: 186
    OpenUrlAbstract/FREE Full Text
  34. ↵
    Fusaki, N., A. Iwamatsu, M. Iwashima, J. I. Fujisawa. 1997. Interaction between Sam68 and src family tyrosine kinases Fyn and Lck, in T cell receptor signalling. J. Biol. Chem. 272: 6214
    OpenUrlAbstract/FREE Full Text
  35. ↵
    Jabado, N., A. Pallier, F. LeDeist, F. Bernard, A. Fischer, C. Hivroz. 1997. CD4 ligands inhibit the formation of multifunctional transduction complexes involved in T cell activation. J. Immunol. 158: 13
    OpenUrlAbstract
  36. ↵
    Jabado, N., A. Pallier, S. Jauliac, A. Fischer, C. Hivroz. 1997. gp160 of HIV or anti-CD4 monoclonal antibody ligation of CD4 induces inhibition of JNK and ERK-2 activities in human peripheral CD4+ T lymphocytes. Eur. J. Immunol. 27: 397
    OpenUrlCrossRefPubMed
  37. ↵
    Mazerolles, F., C. Barbat, C. Hivroz, A. Fischer. 1996. Phosphatidylinositol 3-kinase participates in p56lck/CD4-dependent down-regulation of LFA-1-mediated T cell adhesion. J. Immunol. 157: 3844
    OpenUrl
  38. ↵
    Veillette, A., M. A. Bookman, E. M. Horak, J. B. Bolen. 1988. The CD4 and CD8 T cell surface anigens are associated with the internal membrane tyrosine kinase p56lck. Cell 55: 301
    OpenUrlCrossRefPubMed
  39. ↵
    Hubert, P., P. Debré, L. Boumsell, G. Bismuth. 1993. Tyrosine phosphorylation and association with phospholipase Cγ1 of the GAP-associated 62-kD protein after CD2 stimulation in Jurkat T cells. J. Exp. Med. 178: 1587
    OpenUrlAbstract/FREE Full Text
  40. ↵
    Jabado, N., F. Ledeist, A. Fischer, C. Hivroz. 1994. Interaction of HIV gp120 and anti-CD4 antibodies with the CD4 molecule on human CD4+ T cells inhibits the binding activity of NF-AT, NF-κB and AP-1, three nuclear factors regulating interleukin 2 gene enhancer activity. Eur. J. Immunol. 24: 2646
    OpenUrlCrossRefPubMed
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The Journal of Immunology
Vol. 161, Issue 6
15 Sep 1998
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