Abstract
Engagement of the TCR determines the fate of T cells to activate their functional programs, proliferate, or undergo apoptosis. The intracellular signal transduction pathways that dictate the specific outcome of receptor engagement have only been partially elucidated. The adapter protein, Shc, is involved in cytokine production, mitogenesis, transformation, and apoptosis in different cell systems. We found that Shc becomes phosphorylated on tyrosine residues upon stimulation of the TCR in DO11.10 hybridoma T cells; therefore, we investigated the role of Shc in activation-induced cell death in these cells by creating a series of stably transfected cell lines. Expression of Shc-SH2 (the SH2 domain of Shc) or Shc-Y239/240F (full-length Shc in which tyrosines 239 and 240 have been mutated to phenylalanine) resulted in the inhibition of activation-induced cell death and Fas ligand up-regulation after TCR cross-linking. Expression of wild-type Shc or Shc-Y317F had no significant effect. In addition, we found that Shc-SH2 and Shc-Y239/240F, but not Shc-Y317F, inhibited phosphorylation of extracellular signal-regulated protein kinase and production of IL-2 after TCR cross-linking. These results indicate an important role for Shc in the early signaling events that lead to activation-induced cell death and IL-2 production after TCR activation.
Stimulation through the TCR/CD3 complex initiates an intracellular signaling cascade that involves the activation and inactivation of a number of proteins of diverse function. This includes activation of tyrosine kinases (Lck, Fyn, and ZAP-70), tyrosine phosphatases (SHP-1 and SHP-2), serine/threonine kinases (protein kinase C isoforms, Raf, mitogen-activated protein kinases (MAPKs)4), serine/threonine phosphatases (calcineurin and protein phosphatase 2A), and phosphoinositide kinases and phosphatases (phosphoinositide 3-kinase and SH2-containing 5′-inositol phosphatase, respectively). In addition, a class of adapter molecules that mediate protein-protein interactions, but have no apparent catalytic function, have also been shown to play critical roles in T cell signaling. Integration of signals initiated or regulated by these various molecules determines the functional responses of T cells, such as proliferation, IL-2 production, or activation-induced cell death (AICD).
To understand how intracellular signaling events initiated by TCR engagement mediate these various outcomes, we have examined the role of the adapter protein, Shc, in the regulation of AICD and the synthesis of IL-2. Shc exists as 46- and 52-kDa isoforms in T cells and is composed of an amino-terminal phosphotyrosine binding domain, a central collagen homology domain, which contains TCR-stimulated tyrosine phosphorylation sites, and a carboxyl-terminal SH2 domain, but it has no apparent catalytic domain (1). Interaction of the phosphotyrosine binding or SH2 domain of Shc with activated, tyrosine-phosphorylated receptors or other molecules and/or binding of other proteins to phosphorylated Shc have been implicated in the assembly of signaling complexes at or near the activated receptor that lead to downstream signaling events. Recent studies have described an anti-apoptotic role for Shc in IL-3 withdrawal-induced apoptosis in pro-B cells (2, 3).
Several studies have suggested a role for Shc in T cell activation. Shc is rapidly phosphorylated on tyrosine residues in response to TCR engagement, and phosphorylated Shc subsequently binds to Grb2 and mSos, two proteins involved in Ras activation, in both T cell hybridomas and normal human PBL (4). A physical interaction between the SH2 domain of Shc and the TCR ζ-chain has been demonstrated, suggesting that a Shc:Grb2:mSOS complex localized to the activated TCR would be one mechanism of Ras activation in T cells (5, 6). A constitutive, physical association between Shc and TCR-ζ has been observed in CTLA-4-deficient mice, which is correlated with lymphoproliferation in these mice (7). Shc associates with the SH2-containing 5′-inositol phosphatase, SHIP, which plays a negative regulatory role in other receptor systems (4).
In this report we describe a functional role for Shc in two events that occur during T cell activation, AICD and IL-2 production, in a T cell hybridoma line that has been previously shown to undergo apoptosis and produce IL-2 upon cross-linking of the TCR/CD3 complex. Through expression of mutant Shc proteins, we demonstrate here that the SH2 domain of Shc and specific tyrosines within the collagen homology domain of Shc that become phosphorylated upon TCR activation are critical for Shc function in the regulation of apoptosis and IL-2 production.
Materials and Methods
Abs and reagents
The Abs used in this study include anti-mouse IL-2, mouse anti-hamster cross-linking Ab, and PE-conjugated anti-CD95 (clone MFL3; PharMingen, San Diego, CA); anti-LAT and anti-PLCγ1 (06-152; Upstate Biotechnology, Lake Placid, NY); anti-mouse CD3ε (145-2C11) and anti-phospho-MAPK (New England Biolabs, Cambridge, MA); anti-Grb2 (Santa Cruz Biotechnology, Santa Cruz, CA); and anti-Shc (Transduction Laboratories, Lexington, KY). Anti-phosphotyrosine Ab (4G10) was a gift from Dr. Tom Roberts (Dana-Farber Cancer Institute, Boston, MA). 51C was purchased from New England Nuclear (Boston, MA). Propidium iodide (PI) and PMA were obtained from Sigma (St. Louis, MO). Ionomycin was purchased from Calbiochem (San Diego, CA). Murine FasIg-Fc fusion protein was provided by Dr. Shyr-Te Ju (Boston University Hospital, Boston, MA).
Plasmids
The GST-Shc constructs used in these studies were generated by PCR and subcloned into the pEBG vector, as described previously (8, 9).
Cells and transfections
The murine T cell hybridoma, DO11.10 (10), was provided by Dr. Barbara Osborne (University of Massachusetts, Amherst, MA). The B cell hybridoma line, LK 35.2 (11), was obtained from Dr. Christoph Klein (Children’s Hospital, Boston, MA). Cells were grown in RPMI 1640 containing 10% heat-inactivated FCS, 100 U/ml penicillin, 100 μg/ml streptomycin, and 2 mM l-glutamine. Cells (107) were transfected at 800 μF/250 V using a BRL (Gaithersburg, MD) electroporator. Twenty micrograms of each of the GST-Shc constructs were transfected. Geneticin-resistant transfectants were tested for expression of the Shc construct by Western blot analysis.
T cell activation
For phosphorylation studies, cells were incubated with anti-CD3 Ab (1 μg/ml) for 10 min on ice followed by a 10-min incubation on ice with anti-hamster cross-linking Ab. Samples were subsequently incubated for 2 min at 37°C. For IL-2 production and apoptosis studies, cells were stimulated by plate-bound anti-CD3 Ab for the indicated times.
Immunoprecipitations and immunoblotting
Unstimulated or stimulated cells were lysed in buffer containing 0.5% Triton X-100, 50 mM Tris (pH 7.6), 150 mM NaCl, 1 mM Na3V4O7, 10 mM NaF, 10 mM sodium pyrophosphate, and protease inhibitors. Lysates were subjected to immunoprecipitation with anti-Shc Abs and protein A-Sepharose or glutathione-Sepharose beads. For phospho-MAPK studies, total cell lysates from 1 × 106 cells were analyzed. Proteins were subjected to SDS-PAGE separation, transferred to a polyvinylidene difluoride membrane, and immunoblotted with the appropriate Abs.
IL-2 production assays
IL-2 production was measured by a standard ELISA according to the manufacturer’s protocol (Genzyme, Cambridge, MA). Cells were stimulated for 12 h, as indicated in the figure legends, and plates were read at 450 nm on a Bio-Rad (Richmond, CA) plate reader.
Apoptosis assays
Cells were left unstimulated or were stimulated with plate-bound anti-CD3 for the indicated periods of time. Apoptotic cells were analyzed by flow cytometry after staining with hypotonic PI solution as described previously (12).
FasL-mediated lysis assays
The functional activity of FasL was determined by the ability of FasL-expressing cells to induce apoptosis in Fas+ LK35.2 target cells as described previously (13). Briefly, 5 × 106 LK 35.2 cells were labeled for 1 h at 37°C with 20 μCi 51Cr. Stably transfected DO11.10 cells were incubated (105 cells/well) for 3 h at 37°C in anti-CD3-coated 96-well plates before 104 51Cr-labeled LK 35.2 cells were added. In FasL blocking studies, activated DO11.10 cells were incubated with 10 μg/ml murine FasIg-Fc fusion protein before incubation with the LK35.2 target cells. After an additional 6-h incubation, 100 μl of supernatant was removed from each well and counted in a gamma counter to determine experimental release. The percent specific lysis was calculated with the following formula: % lysis = 100 × [(experimental release − spontaneous release)/(total release − spontaneous release)].
Results
Shc is rapidly phosphorylated upon TCR/CD3 cross-linking of DO11.10 hybridoma cells
To study the role of Shc in AICD, we chose the murine T cell hybridoma line, DO11.10, which undergoes AICD and produces IL-2 upon TCR engagement (10, 14). Previous studies have indicated that Shc becomes rapidly phosphorylated on tyrosine residues following stimulation of T cells with several stimuli, including cross-linking with anti-CD3 and anti-TCR Abs (5, 15). We initially confirmed that Shc becomes phosphorylated on tyrosine residues in DO11.10 cells following cross-linking of the TCR/CD3 complex. Cells were left unstimulated or were stimulated by anti-CD3 cross-linking. Tyrosine phosphorylation of Shc was analyzed by anti-phosphotyrosine immunoblotting. As shown in Fig. 1⇓A (lane 2), Shc is rapidly phosphorylated upon TCR/CD3 cross-linking. To confirm this finding in primary cells, CD4+ murine splenocytes that were induced to undergo AICD (16) were examined for tyrosine phosphorylation of Shc. Our results indicated that Shc is phosphorylated on tyrosine residues in primary cells under conditions that induce apoptosis (data not shown).
Shc is phosphorylated on tyrosine residues following TCR stimulation of DO11.10 hybridoma cells. A, DO11.10 cells were left unstimulated (−) or were stimulated (+) for 2 min by anti-CD3 cross-linking as described in Materials and Methods. Shc proteins were immunoprecipitated with anti-Shc Ab and analyzed by anti-phosphotyrosine immunoblotting. B, Expression of GST-Shc proteins in stably transfected DO11.10 cell lines was determined by immunoprecipitation and Western blot analysis with anti-Shc Abs. Specific bands corresponding to transfected Shc proteins are indicated by arrows.
Generation of stable T cell lines that express wild-type and mutant forms of Shc
To dissect the specific role of Shc in DO11.10 cells, we produced stable transfectants of DO11.10 cells that express GST-tagged wild-type Shc (wt-Shc) and Shc mutants that include the SH2 domain alone of Shc (Shc-SH2), full-length Shc with a point mutation at tyrosine 317 (Shc-Y317F), and full-length Shc with point mutations at tyrosines 239 and 240 (Shc-Y239/240F). The mutant Shc proteins were chosen for specific reasons. In epidermal growth factor receptor signal transduction studies, the SH2 domain of Shc (Shc-SH2) has been shown to function as a dominant interfering protein that blocks endogenous Shc function (15, 17, 18). The tyrosine mutants were designed because Y239, Y240, and Y317 constitute the three major phosphorylation sites on Shc following TCR stimulation (19). Since tyrosine phosphorylation of Shc is important for its interaction with Grb2 and for mediating its downstream signaling effects (18, 20), the Shc-Y317F and Shc-Y239/240F mutants were expected to elucidate which, if any, of these tyrosines are responsible for Shc function during T cell activation. Several clones that stably express the wild-type and mutant Shc proteins were generated and analyzed. Expression levels of both the TCR (data not shown) and mutant Shc proteins (Fig. 1⇑B) of representative clones were comparable.
Expression of dominant interfering Shc mutants inhibits apoptosis in DO11.10 hybridoma cells
DO11.10 cells undergo apoptosis in response to TCR-mediated stimulation. If Shc is essential for mediating early TCR-initiated signaling events, the effect of the dominant interfering mutants could be manifested by their influence on AICD. To test this possibility, the stable cell lines expressing mutant Shc proteins were examined for their ability to undergo AICD upon TCR cross-linking. Representative clones with high stable expression of each of the transfected genes were analyzed by PI staining after 8 or 12 h of stimulation with anti-CD3 Ab (Fig. 2⇓A). Flow cytometric analysis of the subdiploid peak, which represents cells undergoing apoptosis, revealed that cells that expressed Shc-Y317F underwent apoptosis to an extent comparable to cells that express wt-Shc in response to anti-CD3 cross-linking. In contrast, expression of dominant-interfering Shc-SH2 or Shc-Y239/240F markedly inhibited apoptosis. In all experiments, <5% of the unstimulated cells underwent apoptosis. To establish that these differences weren’t due to clonal variation, at least five clonal cell lines expressing each Shc construct were tested for their ability to undergo AICD after 12 h of anti-CD3 cross-linking. The pooled results are shown in Fig. 2⇓B. These data strongly suggest that Shc plays a critical role in early signaling events that involve the Shc-SH2 domain and interactions mediated by Y239/240 of Shc.
Dominant interfering forms of Shc have an anti-apoptotic effect. A, The effect of expression of Shc mutants on apoptosis was determined by flow cytometric analysis of cells treated with hypotonic PI 8 or 12 h after anti-CD3 cross-linking as described in Materials and Methods. B, Data from several experiments, using at least five individual clonal cell lines for each Shc mutant after stimulation for 12 h, are expressed as the percent apoptosis. Error bars indicate SEs. Statistical analyses were performed using the nonparametric Mann-Whitney U test. Differences between DO11.10 and Shc-SH2 (p < 0.0015) and DO11.10 v. Shc-Y239/240F (p < 0.0001) are statistically significant.
Expression of Shc-SH2 and Shc-Y239/240F inhibits expression of FasL
AICD in DO11.10 cells (and many other T cells) occurs through the up-regulation of FasL on the activated cells and the subsequent interaction of FasL with Fas expressed on the same or neighboring T cells (reviewed in Ref. 21). The Fas:FasL interaction leads to suicide or fratricide through the ensuing Fas-mediated death pathway. To determine whether the inhibition of apoptosis mediated by mutant Shc proteins is due to the inhibition of FasL up-regulation, we used a sensitive bioassay to determine the effect of Shc on FasL that relies upon the ability of FasL+ cells to lyse 51Cr-labeled Fas-sensitive LK35.2 cells (13). Fig. 3⇓ shows that Fas-mediated cytolysis of LK35.2 cells was induced during AICD of DO11.10 cells, and this cytolysis was not affected by the expression of wt-Shc. This result indicates that DO11.10 cells up-regulate their FasL expression during AICD. The cytolytic effect of DO11.10 cells on LK35.2 was completely inhibited by preincubation of the DO11.10 cells with murine FasIg-Fc fusion protein, indicating that the killing was mediated by the Fas-FasL interaction (data not shown). FasL expression was inhibited by >50% with expression of Shc-SH2 or Shc-Y239/240F, but not Shc-Y317F. Stimulation of the cells with PMA plus ionomycin, which bypasses TCR stimulation and Shc activation, resulted in comparable induction of FasL cell surface expression (42–54%) in all stable cell lines analyzed (data not shown). The inhibition of FasL expression by mutant Shc proteins correlates with the effects of these proteins on apoptosis of DO11.10 cells. These results suggest that Shc plays an important role in TCR-induced up-regulation of FasL and the subsequent induction of apoptosis.
Shc-SH2 and Shc-Y239/240F inhibit expression of FasL. Cells were stimulated for 3 h by anti-CD3 cross-linking and were subsequently incubated for 6 h with Fas+ 51Cr-labeled LK35.2 target cells. Assays were performed in triplicate. 51Cr release, which indicates Fas-mediated lysis of target cells, was measured in a gamma counter. One representative experiment of three performed is shown.
TCR-dependent IL-2 production is regulated by Shc
Concomitant with the induction of apoptosis, TCR engagement of DO11.10 cells leads to the secretion of IL-2. To determine whether Shc plays a role in the events that lead to IL-2 production, cells were stimulated for 12 h by anti-CD3 cross-linking, and IL-2 production was quantitated by ELISA. As shown in Fig. 4⇓A, anti-CD3-stimulated IL-2 production in DO11.10 cells and that in Shc-Y317F-expressing cells were comparable. However, in cells that express Shc-SH2 or Shc-Y239/240F, IL-2 production was inhibited by approximately 50%. These cell lines showed comparable levels of IL-2 production in response to PMA and ionomycin, suggesting that the Shc-SH2- and Shc-Y239/240F-expressing cells are capable of IL-2 production if the Shc-dependent component of the pathway is bypassed (Fig. 4⇓B). These data demonstrate that Shc plays a role in TCR-mediated synthesis of IL-2, and this is dependent upon Shc-SH2 and the phosphorylation of Shc on Y239/240 residues.
Expression of dominant interfering forms of Shc inhibits anti-TCR-induced IL-2 production in DO11.10 cells. A, Stable cell lines were left unstimulated or were stimulated for 12 h by anti-CD3 cross-linking. A standard ELISA was performed, and plates were read at 450 nm to detect IL-2 production. Pooled results from four separate experiments are shown. B, IL-2 production of cells stimulated with PMA plus ionomycin. Error bars indicate SEs. Differences between DO11.10 and Shc-SH2 (p < 0.05) and between DO11.10 and Shc-Y239/240F (p < 0.0047) are statistically significant.
Mutation of Y239/240F of Shc inhibits interaction with Grb2
Many of the functional effects of Shc are mediated by interaction of Shc with Grb2 and activation of the Ras signaling pathway. Shc contains two potential binding sites for Grb2 within its CH domain: Y239/240 and Y317. Stable Shc-transfected DO11.10 cell lines were tested for the association of endogenous Grb2 with GST-Shc by anti-Grb2 immunoblotting of proteins that precipitated with glutathione-Sepharose beads. As shown in Fig. 5⇓A, anti-TCR cross-linking led to the association of Grb2 with wt-Shc. A comparable amount of Grb2 was associated with Shc-Y317F. In contrast, very little Grb2 associated with Shc-Y239/240F. This finding suggests that Y239/240 of Shc is the major Grb2 binding site in TCR-stimulated DO11.10 cells. The inability of Grb2 to bind to Shc-Y239/240F may play a role in the inhibitory effects observed in cells expressing this protein.
Mutation of Y239/240 of Shc inhibits interaction with Grb2. Stable cell lines were left unstimulated or were stimulated with anti-CD3 Ab for 2 min at 37°C. A, Proteins that associate with the mutant and wild-type Shc proteins were precipitated by glutathione-Sepharose beads. Grb2 that associated with Shc was visualized by immunoblotting with anti-Grb2 Ab. PLCγ1 (B) and LAT (C) were specifically immunoprecipitated, and their phosphorylation status was determined by immunoblot analysis with anti-phosphotyrosine Ab.
Cross-linking the TCR on DO11.10 cells also induces phosphorylation on tyrosine residues of several other signaling proteins, including PLCγ1 and LAT. To demonstrate the specificity of the effect of Shc mutants on signaling pathways, we analyzed the phosphorylation status of these two proteins in the different stable cell lines in response to TCR cross-linking. Expression of dominant-interfering Shc proteins had no effect on the phosphorylation state of PLCγ1 (Fig. 5⇑B) and LAT (Fig. 5⇑C).
Mutation of Y239/240F of Shc inhibits phosphorylation of ERK
Many of the functional effects of Shc observed in cell systems are mediated by activation of the Ras-Raf-MEK1/2-ERK1/2 signaling pathway. ERK activation is correlated with phosphorylation on tyrosine residues, which can be detected by an Ab that specifically recognizes tyrosine-phosphorylated ERK1 and ERK2 (tyrosine 204). To determine whether activation of ERK correlated with the functional effects observed by expression of mutant Shc proteins in T cells, phosphorylation of ERK in the cell lines that express Shc proteins was assessed. As shown in Fig. 6⇓, anti-TCR cross-linking led to robust phosphorylation of ERK in DO11.10 cells and cell lines with stable expression of Shc-wt and Shc-Y317F. In contrast, expression of Shc-SH2 and Shc-Y239/240F inhibited phosphorylation of ERK in response to anti-CD3 stimulation. This finding suggests that the inhibitory effect of Shc-SH2 and Shc-Y239/240F on IL-2 production and AICD in TCR-stimulated DO11.10 cells is mediated through inhibition of the ERK pathway. Confirmation of this finding was evidenced by analysis of the effect of Shc mutants on activation of MEK, an upstream activator of ERK. Expression of Shc-SH2 and Shc-Y239/240F inhibited phosphorylation of MEK in response to anti-CD3 cross-linking. In contrast, expression of Shc-wt and Shc-Y317F had no effect (data not shown).
Mutation of Y239/240 of Shc inhibits phosphorylation of MAPK. DO11.10 cell lines were either left unstimulated or were stimulated by cross-linking with anti-CD3 for 10 min at 37°C. Cells were lysed, and proteins were separated by SDS-PAGE, transferred to a polyvinylidene difluoride membrane, and immunoblotted with anti-phospho-MAPK Ab.
Discussion
Shc was initially identified as an oncogene that induced a transformed phenotype in cultured fibroblasts and tumor formation in nude mice (1). Since then, the role of Shc in epidermal growth factor signaling and mitogenesis as well as its role in the regulation of cell cycle progression and integrin signaling have been well characterized (22, 23). An anti-apoptotic role for Shc in IL-3 withdrawal-induced apoptosis of the murine pro B cell line BaF3 has been described (3). In contrast, our studies demonstrate a positive role for Shc in the early signaling events that lead to AICD. Although the pathways involved in apoptosis in these two systems have not been completely elucidated, it has become increasingly clear that distinct mechanisms regulate apoptosis in different cell types. Cytokine withdrawal-mediated apoptosis does not activate the Fas pathway, whereas AICD involves Fas-mediated apoptosis. Several studies have shown that the up-regulation of FasL is pivotal for AICD and is dependent on Lck, ZAP-70, CD45, calcineurin, and Ras activities and an intact CD3-ζ (24, 25, 26, 27, 28). Subsequent to its up-regulation, FasL interacts with Fas and activates the Fas death pathway.
Our data suggest that Shc is required for the up-regulation of FasL during AICD. The contradictory roles of Shc in cytokine withdrawal-mediated apoptosis and AICD further emphasize the differences in these two mechanisms of apoptosis. A similar contradictory role for c-Myc in apoptosis has been described. c-Myc serves as a survival factor in cytokine withdrawal-induced apoptosis, whereas FasL expression during AICD requires c-Myc expression (29). We found that expression of dominant interfering Shc-SH2 or Shc-Y239/240F diminished FasL up-regulation as determined by a Fas-mediated cytolysis assay, indicating that Shc affects signaling events upstream of FasL expression. We did not find an inhibitory effect by mutation of Y317 of Shc, which suggests that the signaling pathway elicited through this tyrosine is not involved in these functional events. This was surprising, as many of the previous studies on the function of Shc had attributed its effects to its interaction with Grb2 through Y317. However, a role for Grb2 in the Shc functions described here cannot be ruled out, as it has also recently been shown that Grb2 can bind to Shc through tyrosine 239 and lead to the formation of a Shc:Grb2:mSos complex (19, 30). In our studies we have found that mutation of Y317 did not diminish the amount of Grb2 that associated with Shc, while mutation of Y239/240 severely blocked this association. This finding correlates with the inhibition of apoptosis, IL-2 production, and up-regulation of FasL activity observed in cells that express Shc-Y239/240F, but not Shc-Y317F. The Shc-SH2 construct is also unable to bind to Grb2, but can still potentially interact with other critical signaling molecules. Therefore, the inability to form a productive signaling complex that involves an Shc-Grb2 association results in the dominant negative effects observed by expression of the Shc mutants. This Shc-Grb2 complex is necessary for phosphorylation of MEK and its substrate, Erk, which, in turn, are required for downstream signaling events. As expected, we did not detect an effect of mutant forms of Shc on phosphorylation of PLCγ (Fig. 5⇑B) or LAT (Fig. 5⇑C), indicating that the effect of the Shc mutants on T cell signaling is specific for the Grb2-Mek-MAPK pathway.
There has been much controversy regarding the functional role of Shc in T cells. Although reports have demonstrated phosphorylation of Shc in response to TCR activation and the association of Shc with TCR-ζ (4, 5, 6), studies in Jurkat T cells have failed to confirm these results. Our data in DO11.10 cells clearly indicate that Shc plays a role in early TCR-mediated signaling events that ultimately result in AICD and IL-2 production. This is in apparent contrast to reports that Shc is not involved in TCR-stimulated NF-AT activation in Jurkat T cells (15, 31). We have also been unable to detect involvement of Shc in IL-2 production in two distinct Jurkat cell lines (data not shown). We believe that this may be due to poor tyrosine phosphorylation of Shc in Jurkat cells following TCR/CD3 stimulation, in contrast to the robust phosphorylation of Shc that can be detected in normal peripheral blood T cells and several other murine and human T cell lines. In addition to the detection of tyrosine-phosphorylated Shc in anti-TCR stimulated DO11.10 cells (Fig. 1⇑A), we have seen robust phosphorylation of Shc in CD4+ splenocytes under conditions that induce AICD (16) (data not shown).
Expression of the dominant interfering Shc constructs, Shc-SH2 and Shc-Y239/240F, inhibited FasL expression, apoptosis, and IL-2 production by approximately 50% compared with that in parental DO11.10 cells or cells expressing Shc-wt or Shc-Y317F constructs. One possible explanation why a higher degree of inhibition was not observed in these assays is that expression of the dominant interfering constructs did not completely block the ability of endogenous Shc to form productive signaling complexes. Alternatively, it is possible that an Shc-independent signaling pathway contributes to the regulation of Ras activation and subsequent IL-2 production and AICD. LAT is one candidate molecule that has also been shown to play a role in the recruitment of a Grb2:mSOS complex and activation of NF-AT in T cells (32, 33). We did not find an effect of expression of Shc mutants on anti-CD3-stimulated tyrosine phosphorylation of PLCγ (Fig. 5⇑B) or LAT (Fig. 5⇑C); therefore this signaling pathway remains intact in these cells and may contribute to the IL-2 production, apoptosis, and FasL expression that are not inhibited by the dominant interfering Shc constructs. In addition to the signaling pathways that involve Ras-mediated activation of AP-1 in the formation of active NF-AT transcription factor complexes, Vav and SLP-76 have been shown to lead to NF-AT activation in T cells (34). A complex involving SLP-76, LAT, and a recently cloned adapter molecule, Gads, has also been shown to play a role in NF-AT activation in T cells (35). We investigated the potential interaction of Shc with Gads and did not detect an association between these two molecules in DO11.10 cells or Jurkat cells (data not shown). Another pathway that may lead to Ras activation in parallel to an Shc pathway involves protein kinase C (36). Signaling networks that involve these proteins may partially compensate for ineffective Shc signaling when dominant interfering forms are overexpressed.
The distal signaling events that link Shc activation to up-regulation of FasL and IL-2 production remain to be determined. It is possible that Shc acts upstream of a transcription factor necessary for activation of the promoters for each of these genes. Several elements have been described within the promoter for IL-2, including binding sites for NF-AT, AP-1, NF-κB, and Oct-1 (37). Promoter elements identified to date that regulate the FasL promoter are NF-AT, Egr-3, NF-κB, and RE 3 sites (26, 27, 38, 39, 40, 41). Therefore, one downstream component of Shc-mediated signaling may involve activation of one or more of these transcription factors.
Involvement of Shc in early signaling events that lead to ERK phosphorylation, FasL up-regulation, apoptosis, and IL-2 production in T cells underscores the importance of this adapter protein in lymphocyte activation. Further studies are necessary to delineate the additional components of the signaling pathway(s) that lead to these essential T cell functions.
Acknowledgments
We thank Drs. S. Sawasdikosol and J. H. Chang for helpful discussions.
Footnotes
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↵1 This work was supported by the National Institutes of Health Grant CA70758 (to S.J.B.), a fellowship from The Medical Foundation, Inc. (Boston, MA; to J.C.P.), and a Howard Hughes Physician Postdoctoral Fellowship (to M.R.V.).
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↵2 J.C.P. and M.R.V. contributed equally to this work.
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↵3 Address correspondence and reprint requests to Dr. Steven J. Burakoff, Dana 1840, Division of Pediatric Oncology, Dana-Farber Cancer Institute, 44 Binney Street, Boston, MA 02115. E-mail address: steven_burakoff{at}dfci.harvard.edu
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↵4 Abbreviations used in this paper: MAPK, mitogen-activated protein kinase; AICD, activation-induced cell death; PI, propidium iodide; PLCγ1, phospholipase Cγ1; FasL, Fas ligand; wt, wild type; LAT, linker for activation of T cells.
- Received December 11, 1998.
- Accepted June 25, 1999.
- Copyright © 1999 by The American Association of Immunologists
References
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