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*
Division of Pediatric Oncology, Dana-Farber Cancer Institute, and
Department of Pediatrics, Harvard Medical School, Boston, MA 02115;
Division of Hematology and Oncology, Beth Israel Deaconess Medical Center, Boston, MA 02215; and
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Beirne Carter Center for Immunology, University of Virginia, Charlottesville, VA 22908
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 
-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-
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.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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-PLC
1 (06-152; Upstate Biotechnology, Lake Placid, NY);
anti-mouse CD3
(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 x 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 x 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 x [(experimental release - spontaneous release)/(total release - spontaneous release)].
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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).
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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. 2A).
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. 2B. 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.
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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.
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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. 4A, 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. 4B). 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.
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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. 5A, 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.
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1 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 PLC1 (Fig. 5B)
and LAT (Fig. 5C). 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).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(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 PLC (Fig. 5B) or LAT (Fig. 5C), 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-
(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. 1A), 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 PLC
(Fig. 5B) or LAT (Fig. 5C); 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-
B, and Oct-1 (37).
Promoter elements identified to date that regulate the FasL
promoter are NF-AT, Egr-3, NF-B, 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.
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Acknowledgments |
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Footnotes |
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2 J.C.P. and M.R.V. contributed equally to this work.
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:
4 Abbreviations used in this paper: MAPK, mitogen-activated protein kinase; AICD, activation-induced cell death; PI, propidium iodide; PLC1, phospholipase C1; FasL, Fas ligand; wt, wild type; LAT, linker for activation of T cells.
Received for publication December 11, 1998. Accepted for publication June 25, 1999.
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References |
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chain of the T cell receptor upon T cell activation. Science 262:902. chain tyrosine-based activation motif. J. Biol. Chem. 270:13981.B sites in mouse CD95 ligand (Fas ligand) promoter: functional analysis in T cell hybridoma. J. Immunol. 161:3469.This article has been cited by other articles:
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  A. J. Giles, T. P. Bender, and K. S. Ravichandran The Adaptor Protein Shc Plays a Key Role during Early B Cell Development J. Immunol., November 1, 2009; 183(9): 5468 - 5476. [Abstract] [Full Text] [PDF]
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 T. O. Yarovinsky, M. M. Monick, and G. W. Hunninghake Integrin Receptors Are Crucial for the Restimulation of Activated T Lymphocytes Am. J. Respir. Cell Mol. Biol., May 1, 2003; 28(5): 607 - 615. [Abstract] [Full Text] [PDF]
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 M. Iwashima, M. Takamatsu, H. Yamagishi, Y. Hatanaka, Y.-Y. Huang, C. McGinty, S. Yamasaki, and T. Koike Genetic evidence for Shc requirement in TCR-induced c-Rel nuclear translocation and IL-2 expression PNAS, April 2, 2002; 99(7): 4544 - 4549. [Abstract] [Full Text] [PDF]
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 R. X.-D. Song, R. A. McPherson, L. Adam, Y. Bao, M. Shupnik, R. Kumar, and R. J. Santen Linkage of Rapid Estrogen Action to MAPK Activation by ER{alpha}-Shc Association and Shc Pathway Activation Mol. Endocrinol., January 1, 2002; 16(1): 116 - 127. [Abstract] [Full Text] [PDF]
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M. Iwashima, M. Takamatsu, H. Yamagishi, Y. Hatanaka, Y.-Y. Huang, C. McGinty, S. Yamasaki, and T. Koike Genetic evidence for Shc requirement in TCR-induced c-Rel nuclear translocation and IL-2 expression PNAS, April 2, 2002; 99(7): 4544 - 4549. [Abstract] [Full Text] [PDF]
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