The T cell receptor (TCR) is a versatile receptor able to generate different signals that result in distinct T cell responses. The pattern of early signals is determined by the TCR binding kinetics that control the ability of the ligand to coengage TCR and coreceptor. Coengagement of TCR and CD4 results in an agonist signaling pattern with complete tyrosine phosphorylation of TCR subunits, and recruitment and activation of ZAP-70. In contrast, TCR engagement without CD4 coengagement causes a partial agonist type of signaling, characterized by distinct phosphorylation of TCR subunits and recruitment but no activation of ZAP-70. The pathways triggered by partial agonist signaling are unknown. Here, we show that agonists cause association of active lck and active ZAP-70 with p120-GTPase–activating protein (p120-GAP). These associations follow engagement of CD4 or CD3, respectively. In contrast, partial agonists do not activate lck or ZAP-70, but induce association of p120-GAP with inactive ZAP-70. Despite these differences, both agonist and partial agonist signals activate the mitogen-activated protein kinase (MAPK) pathway. However, MAPK activation by partial agonists is transient, supporting a kinetic, CD4-dependent model for the mechanism of action of variant TCR ligands. Transient MAPK activation may explain some of the responses to TCR partial agonists and antagonists.

The current paradigm of T cell activation proposes that signal transduction from the TCR is initiated after ligand binding by src-kinase–dependent phosphorylation of immune receptor, tyrosine- based activation motifs (ITAMs)1 in the cytoplasmic domains of the CD3/TCR ζ chains (for review see reference 1). Phosphorylation of the ITAMs leads to recruitment and activation of ζ-associated protein (ZAP)-70 through its SH2 domain, and subsequent recruitment and activation of other proteins such as p36 (linker for activation of T cells) (LAT), vav, and SLP-76, through SH2–SH3 domain interactions. This leads to the formation of multimolecular complexes that activate several signaling cascades such as the phospholipase C-γ1 (PLC-γ1)–dependent cascade (2), and the ones emanating from activation of p21ras such as the mitogen-activated protein kinase (MAPK) pathway and the phosphatidyl-inositol 3-kinase (PI-3K) pathway (36). These signaling cascades ultimately converge on the nucleus resulting in the changes of gene expression that characterize T cell activation.

Implicit in this paradigm is the assumption that similar levels of TCR occupancy by different ligands will lead to identical signal transduction events. However, recent evidence indicates that the TCR is not an off/on signal transduction complex, but rather is able to transduce different patterns of signals that can cause dramatic effects on T cell function (7, 8). These distinct patterns of early TCR-mediated signaling are induced by variant TCR ligands that have conservative substitutions in the TCR contact points of the peptide or the MHC molecule (9, 10). As a result, they act as partial agonist or antagonist ligands of the TCR depending on whether they cause partial activation of the T cell or they inhibit agonist-induced activation (for review see references 11, 12). The differential T cell activation induced by these variant TCR ligands correlates with a distinct pattern of early TCR-mediated signaling that is characterized by predominant appearance of the p21 form of phospho-ζ with little phosphorylation of CD3-ε, and with recruitment of ZAP-70 to the TCR-activating complex but no phosphorylation nor activation of this protein tyrosine kinase (13, 14). This distinct pattern of TCR-mediated signal results in an array of intracellular changes that include increase in cell size (10), induction of acidification of the extracellular environment (15, 16), transient or partial calcium signals (17, 18), modulation of TCR expression (19), and upregulation of some cell surface molecules such as IL-2 receptor and leukocyte function–associated antigen 1 (7, 8, 12, 20). These cellular changes translate functionally into inhibition of agonist-induced cell proliferation and/or cytokine production, split cytokine production profiles, and/or induction of T cell anergy. Together, this evidence strongly suggests that the distinct pattern of TCR-mediated signaling induced by partial agonist ligands can activate, at least, some signaling pathways emanating from the TCR.

It is not known how the partial agonist type of early TCR-mediated signaling is transmitted to downstream pathways. Two general possibilities can be considered. One is that this differential signaling may transduce distinct or diminished signals along each one of the pathways emanating from the TCR, resulting in less signal being delivered to the nucleus. Alternatively, the early TCR-mediated signaling in partial agonist mode could translate into dissociated delivery of signals, such as only some of the pathways emanating from the TCR are activated whereas others are not. Here, we explore this issue in relation to one of the pathways emanating from the TCR. Some of the functional responses induced by TCR partial agonist ligands, such as TCR downmodulation, can be attributed to serine phosphorylation (2123). Therefore, we examined the effects of TCR partial agonists on the Ras-MAPK pathway of signal transduction, because it primarily involves serine/ threonine kinases and regulates cell proliferation and differentiation (24). Here, we show that signaling from the TCR in a partial agonist mode causes recruitment of p120-GTPase–activating protein (p120-GAP), a potential Ras effector molecule, to the receptor, and is capable of activating the extracellular regulated kinase (ERK)-1 and ERK-2 MAPKs. However, it does so in a more transient fashion than full agonist ligands of the TCR do. Our results provide the first experimental evidence for activation of the MAPK after partial agonist signaling from the TCR. Activation of the Ras-MAPK pathway may explain some of the effects seen after TCR engagement with these variant ligands, such as downmodulation of TCR or split cytokine production without proliferation.

Materials And Methods

Cells.

3C6 is a Th1, CD4+ murine T cell clone specific for pigeon cytochrome c fragment 81–104 [PCC(81-104)] bound to the I-Ek class II MHC molecule (25). This clone was maintained by cycles of antigen stimulation with irradiated spleen cells, IL-2– induced cell expansion, and rest, as previously described (14). 3C6 T cells were used at least 10–14 d after last seeing antigen. L cells (P13.9), which had been transfected with cDNA constructs encoding Eα and Eβ chains of I-Ek, as well as the costimulatory molecules CD54 (ICAM-1) and CD80 (B7-1), were used as APCs (14, 26, 27).

Reagents.

The following peptide was used for these experiments: PCC(81-104) (IFAGIKKKAERADLIAYLKQATAK). This peptide was commercially provided by Procyon Biopharma Inc. (London, Ontario, Canada) and purified to >85% by HPLC before use. Pervanadate was prepared by dissolving 100 μl of 0.1 M sodium orthovanadate (Sigma Chemical Co., St. Louis, MO) into 900 μl of double distilled water and 3.3 μl of 30% hydrogen peroxide, and kept at room temperature for 15 min before use.

Monoclonal Antibodies.

4G10 (provided by Dr. B. Druker, Oregon Health Sciences University, Portland, OR) is a mouse IgG2b monoclonal antibody against phosphotyrosine, B4F8 is a mouse IgG2a monoclonal antibody against p120-GTPase activating protein (p120-GAP; Santa Cruz Biotechnology, Santa Cruz, CA), rabbit immune sera against lck and ZAP-70 were provided by L.E. Samelson (CBMB, NICHD, National Institutes of Health, Bethesda, MD), and H57-597 is a hamster IgG against a common epitope of the TCR β chain. Anti–ACTIVE MAPK (Promega, Madison, WI) is a rabbit antiserum against dual phosphorylated MAPK-derived peptide and recognizes MAPK2 enzyme bands at 42/44 kD after cell activation.

The anti-CD3-fos, anti-CD4-jun, and the bivalent anti-CD3-CD4 fos–jun antibody reagents were prepared by J. Tso (Protein Design Labs, Palo Alto, CA) and have been previously described (2833). These chimeric antibodies combine a Fab fragment against CD3 or against CD4 linked to fos or jun, respectively. fos and jun, separately, will form homodimers and will lead to bivalent engagement of CD3 in the case of stimulation with anti-CD3-fos, or to bivalent engagement of CD4 for T cells incubated with anti-CD4-jun. However, when anti-CD3-fos and anti-CD4-jun chimeric antibodies are mixed, the equilibrium reaction is skewed towards fos–jun heterodimerization, and this will result in CD3-CD4 coengagement (34).

T Cell Stimulation.

Ag stimulation of T cells was done as previously described (14, 33). In brief, P13.9 cells (106 cells/group) were plated in 24-well plates with or without PCC(81-104), and incubated at 37°C, overnight. The next day, T cells (107 cells/ group) were added. Plates were then spun down (1,000 rpm for 30 s) and incubated at 37°C for 10 min. T cell stimulation with heterofunctional antibodies was performed in a final volume of 100 μl (107 cells/group) in Eppendorf tubes with the indicated heterofunctional antibodies (10 μg/ml) or different amounts of pervanadate for 10 min at 37°C. After stimulation, T cells were harvested in cold PBS containing sodium orthovanadate (400 μM) and EDTA (400 μM), and lysed in 1× lysis buffer (1% Triton X-100, 150 mM NaCl, 10 mM Tris, pH 7.6, 5 mM EDTA, 1 mM sodium orthovanadate, 10 μg/ml leupeptin, 10 μg/ml aprotinin, and 25 μM p-nitrophenyl-p′-guanidino-benzoate).

Tyrosine Phosphorylation Analysis.

Detection of tyrosine phosphorylation of proteins in ZAP-70 or p120-GAP immunoprecipitates was performed as described (14). 4G10 monoclonal antibody was used for immunoblotting. Rabbit antiserum to ZAP-70 was used to immunoprecipitate ZAP-70 and associated proteins, and mouse monoclonal antibody against p120-GAP was used for immunoprecipitation of p120-GAP. Immunoprecipitations were performed from 107 cells/group (14). Signal detection was performed by chemiluminescence (Boehringer Mannheim, Laval, Quebec, Canada) and intensity was quantitated using an imaging densitometer (model GS 700; BioRad Labs., Hercules, CA) and the Molecular Analyst® software (version 1.0, BioRad Labs.).

Detection of activated ERK-1 and ERK-2 was performed by Western blotting of cell lysates with the anti–ACTIVE MAPK (Promega) rabbit antiserum. In brief, 107 cells/sample was stimulated with the appropriate antibodies at different concentrations or for different times at 37°C. Next, cells were washed once with PBS containing sodium orthovanadate, and subsequently lysed in 1× lysis buffer (500 μl) on ice for 30 min. Lysates cleared of nuclear debris by centrifugation at 14,000 rpm at 4°C for 10 min, and 300 μl of supernatant was mixed with 4× sample buffer. 30 μl of this solution (4.5 × 105cell equivalents/group) were run in 12% SDS-PAGE, transferred to polyvinylidene difluoride membranes, and blotted with anti–ACTIVE MAPK antiserum (Promega) using chemiluminescence (Boehringer Mannheim).

In Vitro Immune Complex Kinase Assays.

T cells (107/sample) were stimulated with either the anti-CD3-CD4 fos–jun antibodies, the anti-CD3 fos antibodies, or the anti-CD4 jun antibodies for 10–12 min at 37°C, mixing gently on a thermomixer. Cells were lysed with 1× lysis buffer for 30 min on ice. In vitro immune complex kinase assays of lck, ZAP-70, and p120-GAP immunoprecipitates were performed as previously described (14). MAPK activity assay was performed according to manufacturer specifications (New England Biolabs, Boston, MA). In brief, collected cell lysates were immunoprecipitated with a phospho-specific p42/p44 MAPK monoclonal antibody on protein A–Sepharose beads with gentle rocking, overnight at 4°C. The next day, the bead pellets were washed twice with 1× lysis buffer and twice with 1× kinase buffer (25 mM Tris, pH 7.5, 5 mM β-glycerolphosphate, 2 mM DTT, 0.1 mM sodium orthovanadate, and 10 mM magnesium chloride) and then incubated with the kinase reaction mix (1× kinase buffer containing 200 μM ATP) containing 2 μg of Elk-1 fusion protein for 30 min at 30°C. The reaction was terminated with 4× sample buffer and the samples were resolved on a 12% SDS-PAGE gel. The separated proteins were transferred onto PVDF membrane and the membrane was blocked for 1 h at room temperature. After blocking, the membrane was incubated in primary antibody solution (a monoclonal antibody against serine phosphorylated Elk-1) at 4°C overnight. The next morning, the membrane was washed three times in TBST and then incubated with a horseradish peroxidase–conjugated secondary antibody for 1 h at room temperature. The membrane was again washed three times with TBST and then incubated for 1 min with chemiluminescence reagent before film exposure for 5–10 s.

Flow Cytometric Analysis of TCR Downmodulation.

3C6 T cells were incubated with different concentrations of heterofunctional antibodies for 1–3 h at 37°C. Subsequently, T cells were harvested and stained with FITC-labeled monoclonal antibody against mouse TCR-α/β (H57) or isotype-matched control antibody. Percentage of downmodulated receptors can be calculated as the ratio of mean fluorescence value of experimental samples over the mean fluorescence value of untreated control for the same time period (35).

Results

Agonist and Partial Agonist Signaling Patterns from the TCR Induced by CD3–CD4 Coengagement or by Bivalent CD3 Engagement, Respectively.

As we have previously reported (33, 34), coengagement of CD3 and CD4 induces an agonist pattern of early signaling from the TCR. This pattern is characterized by the presence of two forms of phosphorylated TCR-ζ (p21 and p23), phosphorylation of CD3-ε, and recruitment and phosphorylation of ZAP-70 in phosphotyrosine blots of ZAP-70 immunoprecipitates (Fig. 1). In contrast, phosphotyrosine blots of ZAP-70 immunoprecipitates from T cells stimulated by bivalent engagement of CD3 show a partial agonist pattern of tyrosine phosphorylation. This is identified by predominant appearance of the p21 form of phospho-ζ, with little or no phospho-ε, and recruitment but no phosphorylation of ZAP-70 tyrosine kinase. In previous studies (14, 33), we have shown that the two patterns of early TCR-mediated signaling are not the result of quantitative differences in TCR engagement because they do not overlap after titration of antigenic peptide or stimulating antibody. Bivalent engagement of CD4 alone did not induce significant tyrosine phosphorylation of TCR subunits in ZAP-70 immunoprecipitates.

Further qualitative differences between the agonist pattern and the partial agonist pattern of early TCR-mediated signaling were seen when the kinase activity associated with the TCR complex under conditions of bivalent CD3 engagement or after CD3 and CD4 coengagement were examined. As genetic and biochemical approaches have previously demonstrated (36, 37), TCR-mediated signaling involves recruitment and activation of lck and ZAP-70 tyrosine kinases. Consistent with this, we found increased kinase activity associated with lck in T cells activated in an agonist pattern after CD3 and CD4 coengagement (Fig. 2). In these conditions, immune complex kinase assays after lck immunoprecipitation showed increased autophosphorylation of lck and phosphorylation of a 70-kD band as well as two bands of 23 and 32 kD, bands compatible with ZAP-70, TCR-ζ, and CD3-ε, respectively (Fig. 2). ZAP-70 immunoprecipitates showed significant autophosphorylation of ZAP-70 (Fig. 2). In contrast, no significant increase of lck phosphorylation or kinase activity was detected in lck immunoprecipitates or ZAP-70 immunoprecipitates from cell lysates of T cells activated in a partial agonist mode after bivalent CD3 engagement alone (Fig. 2). From these experiments, we concluded that bivalent engagement of CD3 and coengagement of CD3–CD4 fully reproduce the distinct patterns of early TCR signaling seen after engagement of TCR with ligands with partial agonist or agonist properties, respectively (13, 14).

Despite these differences in early TCR-mediated signaling, we found that both agonist ligand and partial agonist ligand–induced similar loss of surface TCR expression as measured by flow cytometric analysis using a monoclonal antibody against mouse TCR (H57-597; Fig. 3). This finding implies that some signals from the TCR are equally induced by both types of ligands since TCR downmodulation is a reflection of T cell activation (19, 38) and protein kinase C–dependent serine phosphorylation of CD3 chains (23, 3943).

Differential Effects of CD3 or CD3–CD4 Engagement on Recruitment and Phosphorylation of p120-GAP.

To explore the downstream effects of these two qualitatively distinct patterns of early signaling, we initially looked at recruitment and activation of different molecules involved in the Ras-MAPK pathway of signal transduction. Among the many Ras effector molecules, we concentrated on p120-GAP for three reasons. First, Ag-induced T cell activation induces tyrosine phosphorylation of p120-GAP (44). Second, p120-GAP regulates Ras function by promoting GTPase activity (4547). And third, p120-GAP may be itself a Ras effector molecule (48). We performed p120-GAP immunoprecipitates and examined phosphorylation and association of p120-GAP after agonist and partial agonist conditions of stimulation. T cell activation in an agonist mode (either by peptide–MHC molecule complexes on the surface of an APC or by heterofunctional antibodies against CD3 and CD4) induced a rapid, dose-dependent increase in phosphotyrosine content of p120-GAP (Fig. 4). In contrast, partial agonist signaling from the TCR did not induce tyrosine phosphorylation of p120-GAP. In addition, bivalent CD4 engagement alone did not induce tyrosine phosphorylation of p120-GAP either. Despite the differences in p120-GAP tyrosine phosphorylation, p120-GAP immunoprecipitation showed a tyrosine-phosphorylated 70-kD band that was immunoreactive with an antiserum against ZAP-70 in T cells stimulated in full agonist mode (Fig. 5, a and b). Surprisingly, this 70-kD protein was also associated to p120-GAP in T cells stimulated in partial agonist mode (Fig. 5,b), although it was not phosphorylated (Fig. 5 a), indicating that either pattern of early TCR-mediated signaling can induce p120-GAP recruitment to ZAP-70, but only agonists can induce its phosphorylation.

To further support that this 70-kD band was ZAP-70, the protein was depleted from the lysate by sequential rounds of immunoprecipitation with an antiserum against ZAP-70 (Fig. 6). Interestingly, the data suggest that the proportion of ZAP-70 that is associated with p120-GAP is relatively low compared with the total amount of ZAP-70 in the cells. Reblotting of the same membrane with an antiphosphotyrosine antibody showed a similar result for phospho–ZAP-70. In addition, in vitro immune complex kinase assays on p120-GAP immunoprecipitates showed phosphorylated lck and ZAP-70 bands as well as phosphorylation of the cdb3 exogenous substrate for ZAP-70 after CD3–CD4 coengagement (Fig. 7). In contrast, and consistent with our previous findings on kinase assays of ZAP and lck immunoprecipitates from partial agonist-stimulated T cells, no kinase activity was detected in p120-GAP immunoprecipitates from these cells. The association between ZAP-70 and p120-GAP is unlikely due to coassociation of these molecules through TCR subunits because these proteins were not detected in p120-GAP immunoprecipitates. In these kinase assays, p120-GAP was tyrosine phosphorylated only in the immunoprecipitates from T cells stimulated by agonist ligands.

Previous reports have demonstrated that p120-GAP can associate with lck and is a substrate of this src-kinase (49, 50). Given that lck is noncovalently associated with CD4 (51), the lack of coengagement of CD3 and CD4 might translate into lack of association between p120-GAP and lck and could explain the lack of tyrosine phosphorylation of p120-GAP. To test this hypothesis, we examined whether the two patterns of early TCR-mediated signaling differed in the ability of p120-GAP to associate with lck. As shown in Fig. 5,c, there is no association between p120-GAP and lck under basal conditions. However, after TCR-mediated signaling by the agonist, there was significant association of these two molecules. Both the p56 and the activation-dependent p59 form of lck were present in p120-GAP immunoprecipitates of T cells stimulated by coengagement of CD3 and CD4 confirming that T cell activation had occurred. Partial agonist type of TCR-mediated signaling resulted in no association of these two molecules. Of note was the observation that bivalent engagement of CD4 alone did induce the association of p120-GAP with lck as well as tyrosine phosphorylation of p120-GAP in immune complex kinase assays (Fig. 7); however, under these conditions only the resting p56 form of lck was detected.

Thus, our results indicate that p120-GAP is recruited and associated to ZAP-70 after engagement of the TCR in vivo. Early TCR signaling in agonist mode induces kinase activity of lck and ZAP and that correlates with tyrosine phosphorylation of the recruited p120-GAP. In contrast, early TCR-mediated signaling in partial agonist mode fails to induce kinase activity of lck and ZAP-70, and therefore it is not able to induce phosphorylation of the recruited p120-GAP.

Activation of ERK-1 and ERK-2 upon Early TCR-mediated Signaling in Agonist and Partial Agonist Modes.

Activation of p21ras leads to recruitment and activation of Raf-1, which then activates MEK-1 and MEK-2, which subsequently activate ERK-1 and ERK-2 (24). Thus, we assessed the activation of these stages of the Ras-MAPK pathway by looking at the levels of double phosphorylated ERK-1 and ERK-2, as these are the active forms of ERK (24), and at the kinase activity of ERK-1 and ERK-2 measured as serine phosphorylation of exogenous Elk-1 as substrate. Immunoblotting with a monoclonal antibody against dual phosphorylated ERK-1 (p44) and ERK-2 (p42) showed a significant increase in these forms upon short stimulation with either anti-CD3-fos or with anti-CD3-fos–anti-CD4-jun (Fig. 8 a). ERK-1 and ERK-2 activation remained high for at least 10 min in T cells stimulated by the agonist ligand as well as in those stimulated by the partial agonist ligand. However, upon CD3 and CD4 coengagement, ERK-1 and ERK-2 activation did not start to significantly decrease until after 30 min and was still detectable after 60 min of stimulation. In contrast, ERKs activation decreased more rapidly after partial agonist signaling, with significantly decreased levels by 10 min after activation with bivalent CD3 engagement and disappearance after 30 min. The possibility of differential activation of p42 or p44 at the initial time points is unlikely given that the signal ratio for p44/p42 is similar for both groups. As ERK activation decreases, the loss of p44 phosphorylation occurs faster than the loss of p42 phosphorylation in both experimental groups.

These results were confirmed using in vitro kinase assays for the ERKs (Fig. 8,b). Coengagement of CD3 and CD4 for 2 min induced a significant increase in kinase activity of ERKs as indicated by increasing serine phosphorylation of exogenous Elk-1 (Fig. 8,b). Under these conditions, ERK kinase activity remained high for up to 60 min. Bivalent engagement of CD3 alone also induced a substantial increase in phosphorylation at 2 min, which was equal to that observed after full agonist TCR-mediated signaling. Therefore, the initiation of ERK activation is similar after either agonist or partial agonist engagement of the TCR. However, the kinetics of phosphorylation and phosphoprotein decay of this pathway are distinctly different, with significant loss of kinase activity after 10 min and almost complete loss of activity after 30 min under conditions of partial agonist signaling from the TCR. The increase in ERK kinase activity seen after TCR-mediated signaling in agonist mode or partial agonist mode were both dose dependent (Fig. 8 c).

Discussion

Variant TCR ligands with partial agonist properties induce a pattern of early TCR-mediated signaling that is distinct from the signals induced by agonist ligands (for reviews see references 12, 20). Although agonist ligands induce appearance of two forms of phospho–TCR-ζ (p21 and p23), phospho–CD3-ε, and subsequent recruitment and activation of ZAP-70, partial agonists of the TCR only induce the p21 form of phospho–TCR-ζ and fail to activate recruited ZAP-70. This distinct signaling pattern correlates with restricted T cell responses such as limited cytokine production or induction of T cell anergy (7, 8). Characterization of the steps linking the initial events after receptor engagement with the downstream effector responses is important to understand T cell differentiation after TCR ligation. The results presented in this paper demonstrate that stimulation of T cells in a partial agonist mode after bivalent engagement of CD3 causes recruitment and association of p120-GAP with ZAP-70, and activation of the Ras-MAPK pathway. However, in contrast to full agonist pattern of TCR-mediated signaling, partial agonist TCR-mediated signaling activates ERK-1 and ERK-2 only transiently, either because of lack of sustained stimulation and normal decay or because of faster inactivation of these serine/threonine kinases.

We have shown that p120-GAP can associate with ZAP-70 in vivo after early TCR-mediated signaling in agonist mode and in partial agonist mode. However, only full agonist type of signaling induced phosphorylation of p120-GAP. This is expected since only agonist ligands induce activation of lck and of ZAP-70 (13, 14), and lck can act as a kinase on ZAP-70 (36) and on p120-GAP (50). On the other hand, the partial agonist pattern of TCR-mediated signaling failed to activate the kinase activity of lck and did not induce association of p120-GAP to this src-kinase. This could explain the lack of tyrosine phosphorylation of ZAP-70 and of p120-GAP (34). The dissociated recruitment and activation of signaling molecules after partial agonist signaling from the TCR may have profound functional consequences. This is well established for ZAP-70, for which recruitment and activation are two separate, discrete events, and for which tyrosine phosphorylation correlates with kinase activity (14, 36, 52, 53). The biological implications of tyrosine phosphorylation of p120-GAP are still unknown.

What is the molecular basis for the association between p120-GAP and these kinases? In the agonist type of signaling, both lck and ZAP-70 are tyrosine phosphorylated and active. Given that p120-GAP has two SH2 domains (48), the association of p120-GAP to these molecules can be explained on the basis of SH2-mediated interactions with phospho-tyrosine-based motifs, as previously reported (54). However, the molecular mechanism by which the ZAP-70–p120-GAP association occurs after early TCR signaling in partial agonist mode is not clear because, in these conditions, ZAP-70 is not phosphorylated, excluding SH2-mediated interactions between these two molecules. Two possible mechanisms can be proposed. One is that p120-GAP associates with ZAP-70 through an interaction involving the SH3 domain of p120-GAP and a proline-rich region in ZAP-70. Close examination of the ZAP-70 sequence shows that it contains a PTLPAHP stretch between residues 267 and 274, downstream of the SH2(C) domain (52). This sequence conforms to the consensus sequence for SH3 domain binding (55). This claim is further supported by the similarity between this sequence and the proline-rich sequence in G3BP (PQRPQR) that likely mediates the high affinity binding between this protein and the SH3 domain of p120-GAP (56). The PTLPAHP sequence only contains relatively minor substitutions between the proline residues. This mechanism would be similar to that reported for MAPK recruitment to the TCR via SH3 interactions with lck (57). Alternatively, the association between ZAP-70 and p120-GAP may occur through an intermediate molecule to which both bind through their SH2 domains. One potential candidate could be a TCR subunit such as TCR-ζ or a CD3 chain because these are phosphorylated in tyrosines after partial agonist type of signaling from the TCR, and we know that this is sufficient for ZAP-70 recruitment (14). However, we have failed to detect the presence of TCR subunits by direct blotting of p120-GAP immunoprecipitates as well as by phosphotyrosine blotting of these samples. Another candidate could be p62-Sam68 (58, 59), although we do not have any indication of a tyrosine phosphorylated protein of this range in p120-GAP immunoprecipitates.

How does activation of ERK-1 and ERK-2 occur after TCR-mediated signaling in partial agonist mode? Our results indicate that coengagement of CD4 with CD3 is not necessary for activation of the Ras-MAPK pathway, but it is required for optimal and sustained activation of this pathway (60). Coengagement of CD3 and CD4 may be especially important when the affinity of the TCR for its ligand is low, such as in the case of weak agonist ligands (61) or partial agonists and antagonists of the TCR (62, 63). Under these conditions, CD4 coengagement may increase the stability of TCR–peptide–MHC molecule complex engagement and provide sustained TCR-induced signaling (64). Our finding of transient activation of MAPKs in the absence of CD3–CD4 coengagement is therefore compatible with our previous model in which the efficiency of TCR– CD4 coengagement determines the quality of TCR-mediated signaling and partial agonism (33, 34). The transient kinetics of MAPK activation after partial agonist stimulation supports a CD4-dependent kinetic basis for differential TCR-mediated signaling by partial agonist ligands. TCR antagonism may represent conditions of a much lower range of TCR affinity that correlate with very transient or undetectable ERK activation (65).

It is important to note that bivalent engagement of CD3 may be a critical feature in the induction of partial agonist signaling in our experimental model, in line with recent claims that oligomerization of the TCR may be needed for initiation of signaling (66). The specific CD3 dimerization induced by our chimeric antibodies, in contrast to antibody-induced cross-linking (59), could explain the differences between our data showing lck-independent activation of ERKs and previous reports claiming that ERK activation is primarily lck dependent (67, 68). Some ligands, including the chimeric molecules used here, may induce receptor dimerization with the proper architecture to cause PKC-dependent activation of Raf-1 and ERK (6972) in the absence of lck activation (68, 73). In vivo data would support this model. For example, although lck-deficient mice have a drastic reduction in the total number of double positive thymocytes, they still have a significant proportion of these cells (74). This would support a lck-independent pathway for MAPK activation given that differentiation into CD4+CD8+ thymocytes is critically dependent on activation of the MAPK pathway (7577).

Based on the above mentioned considerations, we propose that partial agonist-induced differential phosphorylation of TCR subunits causes activation of Ras. This could be the result of activation of a kinase other than lck (possibly fyn; reference 78), which would concomitantly recruit ZAP-70 and activate some other signaling molecules like p95vav that shares homology with some guanine nucleotide releasing factors involved in Ras activation (79, 80). Ras activation would then recruit different effector molecules to the cell membrane, including Raf (81) and p120-GAP. Activation of Ras and Raf-1 would then result in activation of ERK-1 and ERK-2. In addition, the presence of p120-GAP in proximity to engaged and differentially phosphorylated TCR would favor the association of p120-GAP with recruited, TCR-associated ZAP-70. Although we have not ruled out an indirect association between these two molecules, we suggest that this would occur through proline-rich–SH3 domain interactions. An effector function of p120-GAP other than acting as GTPase is still unknown (45).

The demonstration of the flexibility of TCR-mediated recognition and consequent T cell responses raises an interesting question related to downstream signaling by variant TCR ligands. That is, does differential early TCR-mediated signaling result in a similar decrease in activation of all downstream pathways? Or conversely, does this differential signaling correlate with preserved activation of some pathways but not others? Our data would support a model of dissociated activation of some signaling pathways, but not others. This could reflect different requirements of sustained signaling for each signaling pathway emanating from the TCR. Such a model is also supported by two recent reports indicating that partial agonists of the TCR can induce low and transient calcium signals that do not correlate with detectable generation of inositol trisphosphates (17, 18). The different thresholds of activation for downstream pathways would then correlate with different patterns of effector T cell responses as recently reported (82). The ability to dissociate signaling pathways may have fundamental biological implications in the design of immunomodulatory strategies that target specific signaling pathways whose activation results in split T cell responses. In this sense, selective activation of the Ras-MAPK pathway may reproduce some of the effects seen after TCR engagement with partial agonist ligands.

Acknowledgments

We thank Dr. Ron L. Wange and the members of the Madrenas lab for helpful suggestions and comments.

This work is supported by grants from the Medical Research Council of Canada, the Kidney Foundation of Canada, the Leukemia Research Fund of Canada, and the London Health Sciences Centre. J. Madrenas is a Scholar from the Kidney Foundation of Canada.

Abbreviations used in this paper

     
  • ERK

    extracellular regulated kinase

  •  
  • MAPK

    mitogen-activated protein kinase

  •  
  • p120-GAP

    p120-GTPase– activating protein

  •  
  • PCC(81-104)

    pigeon cytochrome c fragment 81–104

  •  
  • ZAP

    ζ-associated protein

References

References
1
Wange
RL
,
Samelson
LE
Complex complexes: signaling at the TCR
Immunity
1996
5
197
205
[PubMed]
2
Noh
DY
,
Shin
SH
,
Rhee
SG
Phosphoinositide-specific phospholipase C and mitogenic signaling
Biochim Biophys Acta
1995
1242
99
113
[PubMed]
3
Izquierdo
M
,
Leevers
SJ
,
Marshall
CJ
,
Cantrell
D
p21ras couples the T cell antigen receptor to extracellular signal-regulated kinase 2 in T lymphocytes
J Exp Med
1993
178
1199
1208
[PubMed]
4
Weiss
A
,
Littman
DR
Signal transduction by lymphocyte antigen receptors
Cell
1994
76
263
274
[PubMed]
5
Rudd
CE
,
Janssen
O
,
Cai
Y-C
,
da Silva
AJ
,
Raab
M
,
Prasad
KVS
Two-step TCR/CD3-CD4 and CD28 signaling in T cells: SH2/SH3 domains, protein-tyrosine and lipid kinases
Immunol Today
1994
15
225
234
[PubMed]
6
Franklin
RA
,
Tordai
A
,
Patel
H
,
Gardner
AM
,
Johnson
GL
,
Gelfand
EW
Ligation of the T cell receptor complex results in activation of the Ras/Raf-1/MEK/ MAPK cascade in human T lymphocytes
J Clin Invest
1994
93
2134
2140
[PubMed]
7
Sette
A
,
Alexander
J
,
Ruppert
J
,
Snoke
K
,
Franco
A
,
Ishioka
G
,
Grey
HM
Antigen analogs/MHC complexes as specific T cell receptor antagonists
Annu Rev Immunol
1994
12
413
431
[PubMed]
8
Jameson
SC
,
Bevan
MJ
T cell receptor antagonists and partial agonists
Immunity
1995
2
1
11
[PubMed]
9
De Magistris
M
,
Alexander
J
,
Coggeshall
M
,
Altman
A
,
Gaeta
FC
,
Grey
HM
,
Sette
A
Antigen analog-major histocompatibility complexes act as antagonists of the T cell receptor
Cell
1992
68
625
634
[PubMed]
10
Racioppi
L
,
Ronchese
F
,
Matis
LA
,
Germain
RN
Peptide–major histocompatibility complex class II complexes with mixed agonist/antagonist properties provide evidence for ligand-related differences in T cell receptor–dependent intracellular signaling
J Exp Med
1993
177
1047
1060
[PubMed]
11
Kersh
GJ
,
Allen
PM
Essential flexibility in the T-cell recognition of antigen
Nature
1996
380
495
498
[PubMed]
12
Madrenas
J
,
Germain
RN
Variant TCR ligands: new insights into the molecular basis of antigen-dependent signal transduction and T cell activation
Semin Immunol
1996
8
83
101
[PubMed]
13
Sloan-Lancaster
J
,
Shaw
AS
,
Rothbard
JB
,
Allen
PM
Partial T cell signalling: altered phospho-ζ and lack of Zap70 recruitment in APL-induced T cell anergy
Cell
1994
79
913
922
[PubMed]
14
Madrenas
J
,
Wange
RL
,
Wang
JL
,
Isakov
NA
,
Samelson
LE
,
Germain
RN
ζ phosphorylation without ZAP-70 activation induced by TCR antagonists or partial agonists
Science
1995
267
515
518
[PubMed]
15
Beeson
C
,
Rabinowitz
J
,
Tate
K
,
Gütgemann
I
,
Chien
Y
,
Jones
PP
,
Davis
MM
,
McConnell
HM
Early biochemical signals arise from low affinity TCR-ligand reactions at the cell–cell interface
J Exp Med
1996
184
777
782
[PubMed]
16
Rabinowitz
JD
,
Beeson
C
,
Wülfing
C
,
Tate
K
,
Allen
PM
,
Davis
MM
,
McConnell
HM
Altered T cell receptor ligands trigger a subset of early T cell signals
Immunity
1996
5
125
135
[PubMed]
17
Sloan-Lancaster
J
,
Steinberg
TH
,
Allen
PM
Selective activation of the calcium signaling pathway by altered peptide ligands
J Exp Med
1996
184
1525
1530
[PubMed]
18
Wülfing
C
,
Rabinowitz
JD
,
Beeson
C
,
Sjaastad
MD
,
McConnell
HM
,
Davis
MM
Kinetics and extent of T cell activation as measured with the calcium signal
J Exp Med
1997
185
1815
1825
[PubMed]
19
Valitutti
S
,
Muller
S
,
Cella
M
,
Padovan
E
,
Lanzavecchia
A
Serial triggering of many T-cell receptors by a few peptide–MHC complexes
Nature
1995
375
148
151
[PubMed]
20
Sloan-Lancaster
J
,
Allen
PM
Altered peptide ligand-induced partial T cell activation: molecular mechanisms and role in T cell biology
Annu Rev Immunol
1996
14
1
27
[PubMed]
21
Boyer
C
,
Auphan
N
,
Luton
F
,
Malburet
JM
,
Barad
M
,
Bizozzero
JP
,
Reggio
H
,
Schmitt-Verhulst
AM
T cell receptor/CD3 complex internalization following activation of a cytolytic T cell clone: evidence for a protein kinase C–independent staurosporine-sensitive step
Eur J Immunol
1991
21
1623
1634
[PubMed]
22
Sleckman
BP
,
Shin
J
,
Igras
VE
,
Collins
TL
,
Strominger
JL
,
Burakoff
SJ
Disruption of the CD4–p56lck complex is required for rapid internalization of CD4
Proc Natl Acad Sci USA
1992
89
7566
7570
[PubMed]
23
Luton
F
,
Buferne
M
,
Davoust
J
,
Schmitt-Verhulst
AM
,
Boyer
C
Evidence for protein tyrosine kinase involvement in ligand-induced TCR/CD3 internalization and surface redistribution
J Immunol
1994
153
63
72
[PubMed]
24
Seger
R
,
Krebs
EG
The MAPK signaling cascade
FASEB J
1995
9
726
735
[PubMed]
25
Matis
LA
,
Glimcher
LH
,
Paul
WE
,
Schwartz
RH
Magnitude of response of histocompatibility-restricted T-cell clones is a function of the product of the concentrations of antigen and Ia molecules
Proc Natl Acad Sci USA
1983
80
6019
6023
[PubMed]
26
Ronchese
F
,
Schwartz
RH
,
Germain
RN
Functionally distinct subsites on a class II major histocompatibility complex molecule
Nature
1987
329
254
256
[PubMed]
27
König
R
,
Shen
X
,
Germain
RN
Involvement of both major histocompatibility complex class II α and β chains in CD4 function indicates a role for ordered oligomerization in T cell activation
J Exp Med
1995
182
779
787
[PubMed]
28
Kostelny
SA
,
Cole
MS
,
Tso
JY
Formation of a bispecific antibody by the use of leucine zippers
J Immunol
1992
148
1547
1553
[PubMed]
29
Weiner
GJ
,
Kostelny
SA
,
Hillstrom
JR
,
Cole
MS
,
Link
BK
,
Wang
SL
,
Tso
JY
The role of T cell activation in anti-CD3 x antitumor bispecific antibody therapy
J Immunol
1994
152
2385
2392
[PubMed]
30
Weiner
GJ
,
De Gast
GC
Bispecific monoclonal antibody therapy of B-cell malignancy
Leuk Lymphoma
1995
16
199
207
[PubMed]
31
Moreno
MB
,
Titus
JA
,
Cole
MS
,
Tso
JY
,
Le
N
,
Paik
CH
,
Bakacs
T
,
Zacharchuk
CM
,
Segal
DM
,
Wunderlich
JR
Bispecific antibodies retarget murine T cell cytotoxicity against syngeneic breast cancer in vitro and in vivo
Cancer Immunol Immunother
1995
40
182
190
[PubMed]
32
Alegre
ML
,
Tso
JY
,
Sattar
HA
,
Smith
J
,
Desalle
F
,
Cole
M
,
Bluestone
JA
An anti-murine CD3 monoclonal antibody with a low affinity for Fc gamma receptors suppresses transplantation responses while minimizing acute toxicity and immunogenicity
J Immunol
1995
155
1544
1555
[PubMed]
33
Madrenas
J
,
Chau
LA
,
Smith
J
,
Bluestone
JA
,
Germain
RN
The efficiency of CD4 recruitment to ligand-engaged TCR controls the agonist/partial agonist properties of peptide–MHC molecule ligands
J Exp Med
1997
185
219
230
[PubMed]
34
Smith
JA
,
Tso
JY
,
Clark
MR
,
Cole
MS
,
Bluestone
JA
Nonmitogenic anti-CD3 monoclonal antibodies deliver a partial T cell receptor signal and induce clonal anergy
J Exp Med
1997
185
1413
1422
[PubMed]
35
Lee
JE
,
Cossoy
MB
,
Chau
LA
,
Singh
B
,
Madrenas
J
Lck inactivation and loss of TCR-mediated signalling upon persistent engagement with complexes of peptide: MHC molecules
J Immunol
1997
159
61
69
[PubMed]
36
Iwashima
M
,
Irving
BA
,
van Oers
NSC
,
Chan
AC
,
Weiss
A
Sequential interactions of the TCR with two distinct cytoplasmic tyrosine kinases
Science
1994
263
1136
1139
[PubMed]
37
Diez-Orejas
R
,
Ballester
S
,
Feito
MJ
,
Ojeda
G
,
Criado
G
,
Ronda
M
,
Portoles
P
,
Rojo
JM
Genetic and immunochemical evidence for CD4-dependent association of p56lck with the alpha beta T-cell receptor (TCR): regulation of TCR-induced activation
EMBO (Eur Mol Biol Organ) J
1994
13
90
99
[PubMed]
38
Valitutti
S
,
Lanzavecchia
A
Serial triggering of TCRs: a basis for the sensitivity and specificity of antigen recognition
Immunol Today
1997
18
299
304
[PubMed]
39
Cantrell
DA
,
Davies
AA
,
Crumpton
MJ
Activators of protein kinase C down-regulate and phosphorylate the T3/T-cell antigen receptor complex of human T lymphocytes
Proc Natl Acad Sci USA
1985
82
8158
8162
[PubMed]
40
Dietrich
J
,
Hou
X
,
Wegener
AM
,
Geisler
C
CD3 gamma contains a phosphoserine-dependent di-leucine motif involved in down-regulation of the T cell receptor
EMBO (Eur Mol Biol Organ) J
1994
13
2156
2166
[PubMed]
41
Dietrich
J
,
Hou
X
,
Wegener
AM
,
Pedersen
LO
,
Odum
N
,
Geisler
C
Molecular characterization of the di-leucine–based internalization motif of the T cell receptor
J Biol Chem
1996
271
11441
11448
[PubMed]
42
Dietrich
J
,
Kastrup
J
,
Nielsen
BL
,
Odum
N
,
Geisler
C
Regulation and function of the CD3 gamma DxxxLL motif: a binding site for adaptor protein-1 and adaptor protein-2 in vitro
J Cell Biol
1997
138
271
281
[PubMed]
43
Luton
F
,
Legendre
V
,
Gorvel
JP
,
Schmitt-Verhulst
AM
,
Boyer
C
Tyrosine and serine protein kinase activities associated with ligand-induced internalized TCR/CD3 complexes
J Immunol
1997
158
3140
3147
[PubMed]
44
Ericsson
P-O
,
Orchansky
PL
,
Carlow
DA
,
Teh
H-S
Differential activation of phospholipase C-γ1 and mitogen-activated protein kinase in naive and antigen-primed CD4 T cells by the peptide/MHC ligand
J Immunol
1996
156
2045
2053
[PubMed]
45
Marshall
MS
Ras target proteins in eukaryotic cells
FASEB J
1995
9
1311
1318
[PubMed]
46
Marshall
CJ
Ras effectors
Curr Opin Cell Biol
1996
8
197
204
[PubMed]
47
Katz
ME
,
McCormick
F
Signal transduction from multiple Ras effectors
Curr Opin Genet Dev
1997
7
75
79
[PubMed]
48
Tocque
B
,
Delumeau
I
,
Parker
F
,
Maurier
F
,
Multon
MC
,
Schweighoffer
F
Ras-GTPase activating protein (GAP): a putative effector for Ras
Cell Signalling
1997
9
153
158
[PubMed]
49
Ellis
C
,
Liu
XQ
,
Anderson
D
,
Abraham
N
,
Veillette
A
,
Pawson
T
Tyrosine phosphorylation of GAP and GAP-associated proteins in lymphoid and fibroblast cells expressing lck
Oncogene
1991
6
895
901
[PubMed]
50
Amrein
KE
,
Flint
N
,
Panholzer
B
,
Burn
P
Ras GTPase-activating protein: a substrate and a potential binding protein of the protein-tyrosine kinase p56lck
Proc Natl Acad Sci USA
1992
89
3343
3346
[PubMed]
51
Veillette
A
,
Bookman
MA
,
Horak
EM
,
Bolen
JB
The CD4 and CD8 T cell surface antigens are associated with the internal membrane tyrosine-protein kinase p56lck
Cell
1988
55
301
308
[PubMed]
52
Chan
AC
,
Iwashima
M
,
Turck
CW
,
Weiss
A
ZAP-70: a 70 kd protein-tyrosine kinase that associates with the TCR zeta chain
Cell
1992
71
649
662
[PubMed]
53
Wange
RL
,
Kong
AN
,
Samelson
LE
A tyrosine-phosphorylated 70-kDa protein binds a photoaffinity analogue of ATP and associates with both the zeta chain and CD3 components of the activated T cell antigen receptor
J Biol Chem
1992
267
11685
11688
[PubMed]
54
Neumeister
EN
,
Zgu
Y
,
Richard
S
,
Terhorst
C
,
Chan
AC
,
Shaw
AS
Binding of ZAP-70 to phosphorylated T-cell receptor ζ and η enhances its autophosphorylation and generates specific binding sites for SH2 domain-containing proteins
Mol Cell Biol
1995
15
3171
3178
[PubMed]
55
Rickles
RJ
,
Botfield
MC
,
Zhou
XM
,
Henry
PA
,
Brugge
JS
,
Zoller
MJ
Phage display selection of ligand residues important for Src homology 3 domain binding specificity
Proc Natl Acad Sci USA
1995
92
10909
10913
[PubMed]
56
Parker
F
,
Maurier
F
,
Delumeau
I
,
Duchesne
M
,
Faucher
D
,
Debussche
L
,
Dugue
A
,
Schweighoffer
F
,
Tocque
B
A Ras-GTPase–activating protein SH3-domain–binding protein
Mol Cell Biol
1996
16
2561
2569
[PubMed]
57
August
A
,
Dupont
B
Association between mitogen-activated protein kinase and the ζ chain of the T cell receptor (TcR) with the SH2,3 domain of p56lck. Differential regulation by TcR cross-linking
J Biol Chem
1996
271
10054
10059
[PubMed]
58
Hubert
P
,
Debre
P
,
Boumsell
L
,
Bismuth
G
Tyrosine phosphorylation and association with phospholipase C gamma-1 of the GAP-associated 62-kD protein after CD2 stimulation of Jurkat T cell
J Exp Med
1993
178
1587
1596
[PubMed]
59
Jabado
N
,
Pallier
A
,
Le Deist
F
,
Bernard
F
,
Fischer
A
,
Hivroz
C
CD4 ligands inhibit the formation of multifunctional transduction complexes involved in T cell activation
J Immunol
1997
158
94
103
[PubMed]
60
Nel
AE
,
Pollack
S
,
Landreth
G
,
Ledbetter
JA
,
Hultin
L
,
Williams
K
,
Katz
R
,
Akerley
B
CD-3–mediated activation of the MAP-2 kinase can be modified by ligation of the CD4 receptor. Evidence for tyrosine phosphorylation during activation of this kinase
J Immunol
1990
145
971
979
[PubMed]
61
Hampl
J
,
Chien
Y
,
Davis
MM
CD4 augments the response of a T cell to agonist but not to antagonist ligands
Immunity
1997
7
379
385
[PubMed]
62
Alam
SM
,
Travers
PJ
,
Wung
JL
,
Nasholds
W
,
Redpath
S
,
Jameson
SC
,
Gascoigne
NRJ
T-cell-receptor affinity and thymocyte positive selection
Nature
1996
381
616
620
[PubMed]
63
Lyons
DS
,
Lieberman
SA
,
Hampl
J
,
Boniface
JJ
,
Chien
Y
,
Berg
LJ
,
Davis
MM
A TCR binds to antagonist ligands with lower affinities and faster dissociation rates than to agonists
Immunity
1996
5
51
59
64
Janeway
CJ
,
Bottomly
K
Signals and signs for lymphocyte responses
Cell
1994
76
275
285
[PubMed]
65
Racioppi
L
,
Matarese
G
,
D'Oro
U
,
De Pascale
M
,
Masci
AM
,
Fontana
S
,
Zappacosta
S
The role of CD4-lck in T-cell receptor antagonism: evidence for negative signaling
Proc Natl Acad Sci USA
1996
93
10360
10365
[PubMed]
66
Reich
Z
,
Boniface
JJ
,
Lyons
DS
,
Borochov
N
,
Wachtel
EJ
,
Davis
MM
Ligand-specific oligomerization of T-cell receptor molecules
Nature
1997
387
617
620
[PubMed]
67
Ettehadieh
E
,
Sanghera
JS
,
Pelech
SL
,
Hess-Bienz
D
,
Watts
J
,
Shastri
N
,
Aebersold
R
Tyrosyl phosphorylation and activation of MAP kinases by p56lck
Science
1992
255
853
855
[PubMed]
68
Gupta
S
,
Weiss
A
,
Kumar
G
,
Wang
S
,
Nel
A
The T-cell antigen receptor utilizes Lck, Raf-1, and MEK-1 for activating mitogen-activated protein kinase. Evidence for the existence of a second protein kinase C–dependent pathway in an Lck-negative Jurkat cell mutant
J Biol Chem
1994
269
17349
17357
[PubMed]
69
Sozeri
O
,
Vollmer
K
,
Liyanage
M
,
Frith
D
,
Kour
G
,
Mark
GED
,
Stabel
S
Activation of the c-Raf protein kinase by protein kinase C phosphorylation
Oncogene
1992
7
2259
2262
[PubMed]
70
Kolch
W
,
Heidecker
G
,
Kochs
G
,
Hummel
R
,
Vahidi
H
,
Mischak
H
,
Finkenzeller
G
,
Marme
D
,
Rapp
UR
Protein kinase C alpha activates RAF-1 by direct phosphorylation
Nature
1993
364
249
252
[PubMed]
71
Siegel
JN
,
June
CH
,
Yamada
H
,
Rapp
UR
,
Samelson
LE
Rapid activation of C-Raf-1 after stimulation of the T-cell receptor or the muscarinic receptor type 1 in resting T cells
J Immunol
1993
151
4116
4127
[PubMed]
72
Ueda
Y
,
Hirai
S
,
Osada
S
,
Suzuki
A
,
Mizuno
K
,
Ohno
S
Protein kinase C activates the MEK-ERK pathway in a manner independent of Ras and dependent on Raf
J Biol Chem
1996
271
23512
23519
[PubMed]
73
August
A
,
Dupont
B
Activation of extracellular signal-regulated protein kinase (ERK/MAP kinase) following CD28 cross-linking: activation in cells lacking p56lck
Tissue Antigens
1995
46
155
162
[PubMed]
74
Molina
TJ
,
Kishihara
K
,
Siderovski
DP
,
Ewijk
W
van, A. Narendran, E. Timms, A. Wakeham, C.J. Paige, K.U. Hartmann, A. Veillette, et al
Profound block in thymocyte development in mice lacking p56lck
Nature
1992
357
161
164
[PubMed]
75
Alberola-Ila
J
,
Forbush
KA
,
Seger
R
,
Krebs
EG
,
Perlmutter
RM
Selective requirement for MAP kinase activation in thymocyte differentiation
Nature
1995
373
620
623
[PubMed]
76
Alberola-Ila
J
,
Hogquist
KA
,
Swan
KA
,
Bevan
MJ
,
Perlmutter
RM
Positive and negative selection invoke distinct signaling pathways
J Exp Med
1996
184
9
18
[PubMed]
77
Crompton
T
,
Gilmour
KC
,
Owen
MJ
The MAP kinase pathway controls differentiation from double-negative to double-positive thymocyte
Cell
1996
86
243
251
[PubMed]
78
van Oers
NSC
,
Killeen
N
,
Weiss
A
Lck regulates the tyrosine phosphorylation of the T cell receptor subunits and ZAP-70 in murine thymocytes
J Exp Med
1996
183
1053
1062
[PubMed]
79
Ramos-Morales
F
,
Druker
BJ
,
Fischer
S
Vav binds to several SH2/SH3 containing proteins in activated lymphocytes
Oncogene
1994
9
1917
1923
[PubMed]
80
Gulbins
E
,
Coggeshall
KM
,
Langlet
C
,
Baier
G
,
Bonnefoy-Berard
N
,
Burn
P
,
Wittinghofer
A
,
Katzav
S
,
Altman
A
Activation of Ras in vitro and in intact fibroblasts by the Vav guanine nucleotide exchange protein
Mol Cell Biol
1994
14
906
913
[PubMed]
81
Stokoe
D
,
Macdonald
SG
,
Cadwallader
K
,
Symons
M
,
Hancock
JF
Activation of Raf as a result of recruitment to the plasma membrane
Science
1994
264
1463
1467
[PubMed]
82
Itoh
Y
,
Germain
RN
Single cell analysis reveals regulated hierarchical T cell antigen receptor signaling thresholds and intraclonal heterogeneity for individual cytokine responses of CD4+T cells
J Exp Med
1997
186
757
766
[PubMed]

Author notes

Address correspondence to J. Madrenas, John P. Robarts Research Institute, Rm. 2-05, PO box 5015, 100 Perth Dr., London, ON, Canada N6A 5K8. Phone: 519-663-5777, Ext. 4242; Fax: 519-663-3789; E-mail: madrenas@rri.on.ca