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Molecular and Cellular Biology, February 2004, p. 1747-1757, Vol. 24, No. 4
0270-7306/04/$08.00+0     DOI: 10.1128/MCB.24.4.1747-1757.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

p66SHC Promotes Apoptosis and Antagonizes Mitogenic Signaling in T Cells{dagger}

Sonia Pacini,1,{ddagger} Michela Pellegrini,1,{ddagger} Enrica Migliaccio,2 Laura Patrussi,1 Cristina Ulivieri,1 Andrea Ventura,2 Fabio Carraro,3 Antonella Naldini,3 Luisa Lanfrancone,2 Piergiuseppe Pelicci,2 and Cosima T. Baldari1*

Department of Evolutionary Biology,1 Department of General Physiology, University of Siena, 53100 Siena,3 European Institute of Oncology, 20141 Milan, Italy2

Received 1 October 2003/ Returned for modification 11 November 2003/ Accepted 19 November 2003


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ABSTRACT
 
Of the three Shc isoforms, p66Shc is responsible for fine-tuning p52/p46Shc signaling to Ras and has been implicated in apoptotic responses to oxidative stress. Here we show that human peripheral blood lymphocytes and mouse thymocytes and splenic T cells acquire the capacity to express p66Shc in response to apoptogenic stimulation. Using a panel of T-cell transfectants and p66Shc-/- T cells, we show that p66Shc expression results in increased susceptibility to apoptogenic stimuli, which depends on Ser36 phosphorylation and correlates with an altered balance in apoptosis-regulating gene expression. Furthermore, p66Shc blunts mitogenic responses to T-cell receptor engagement, at least in part by transdominant inhibition of p52Shc signaling to Ras/mitogen-activated protein kinases, in an S36-dependent manner. The data highlight a novel interplay between p66Shc and p52Shc in the control of T-cell fate.


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INTRODUCTION
 
Initially identified as components of receptor tyrosine kinase pathways leading to Ras activation, Shc proteins have been implicated in a variety of processes regulating cell commitment to proliferation, survival, or apoptosis (2, 16, 30). Shc is expressed as three isoforms encoded by the same genetic locus (17). All isoforms share a common domain structure, which includes an SH2 domain, a proline-rich CH1 domain, and a PTB domain, which not only permits interactions with tyrosine phosphoproteins but also binds membrane phospholipids (37). The CH1 domain has three phosphorylatable tyrosine residues, YY239/240 and Y317 in human p52Shc, which mediate Grb2 recruitment (8, 31, 34) and have been proposed to be differentially coupled to the fos mitogenic pathway and to the myc survival pathway (8). p66Shc has an additional N-terminal proline-rich CH2 domain containing a phosphorylatable serine residue at position 36, which is required for coupling Shc to stress responses leading to apoptosis (18).

While the molecular basis of Shc as a mediator of mitogenic responses has been well established (31), the role of p66Shc in this process is still awaiting full characterization. With the exception of hematopoietic and neuronal cells, Shc is expressed in a wide variety of transformed and normal cells, although the ratio of p66Shc to p52/p46Shc is variable among cell types. Interestingly, in fibroblasts p66Shc appears to antagonize mitogenic signals elicited by p52/p46Shc, thereby providing a negative control loop (17, 22). More recently, analysis of a gene-targeted mouse lacking p66Shc has highlighted a role for p66Shc in p53-dependent stress responses leading to apoptosis (18, 32).

As in other hematopoietic cells, Shc is expressed in T cells as p52/p46Shc. Following T-cell receptor (TCR) engagement, Shc is recruited to the TCR through both a PTB domain-mediated interaction with ZAP-70 and an SH2 domain-mediated interaction with the tyrosine-phosphorylated CD3{zeta} immunoreceptor tyrosine-based activation motifs (19, 23, 28). Shc is phosphorylated on tyrosine residues in response to TCR triggering and as such not only recruits Grb2/Sos complexes but also enhances Grb2 association with Sos (29). An inducible association of Shc with lipid rafts has been described previously (26, 33), supporting the notion that Shc participates in the supramolecular signaling complex assembled in this location following TCR engagement (13). Although a plethora of pathways activated by the TCR result in juxtaposition of Grb2/Sos complexes to Ras at the plasma membrane, Shc appears to play a nonredundant role in TCR signaling to Ras (1, 9, 19, 26, 27). Furthermore, the recent observation that thymocytes are blocked at the double-negative stage in mice carrying a conditional mutation of the Shc locus suggests that Shc is also required for pre-TCR signaling (36).

Expression of p52/p46Shc and p66Shc is controlled by different promoters (17, 35). We have recently shown that, when tested outside of the chromatin context, the p66Shc promoter is capable of driving high levels of constitutive transcription in T cells. Furthermore, p66Shc expression can be correlated to the state of promoter methylation and the activity of histone deacetylases (35). Here we show that, although undetectable in normal resting T cells, p66Shc can be inducibly expressed in these cells in response to apoptogenic stimuli. Using a panel of T-cell transfectants expressing p66Shc or mutants thereof, as well as p66Shc-/- T cells, we provide evidence for a role of p66Shc in the negative regulation of T-cell activation and survival.


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MATERIALS AND METHODS
 
Mice, cells, plasmids, and antibodies. The p66Shc-/- mouse was described previously (18). Age-matched 129 mice (Charles River Italia) were used as controls. The age of the mice ranged from 2 to 6 months.

Mouse splenic T cells were purified by immunomagnetic sorting using anti-panB antibody-conjugated beads (Dynal Biotech, Oslo, Norway) after depletion of monocytes/macrophages by adherence and subsequently checked by flow cytometry with anti-CD3 and anti-CD22 monoclonal antibodies (MAb). Human peripheral blood mononuclear cells were purified from whole blood by density gradient centrifugation on Ficoll-Paque (Amersham Pharmacia Italia srl, Milan, Italy) and subsequently depleted of monocytes by adherence. The T-lymphoma Jurkat line as well as JSL1, a Jurkat variant lacking Shc expression (9), were used for the generation of stable transfectants.

The cDNA encoding p66Shc was cloned into pcDNA3 (Invitrogen, Leek, The Netherlands). The initiation codons for both p52Shc and p46Shc were both mutagenized from ATG to TTG in order to achieve expression of only p66Shc (17). This mutant as well as the p663F and the p66SA mutants were generated by site-specific mutagenesis of p66Shc cDNA using standard procedures. The isolated CH2 domain was obtained by PCR amplification of a fragment of the p66Shc cDNA spanning amino acids 1 to 110 and cloned into pcDNA3 after engineering of a TAA termination codon at the 3' end. A pcDNA3 construct encoding hemagglutinin-tagged p52Shc (18) and the FasL luciferase reporter (14) were previously described.

p66Shc and mutants thereof, as well as the isolated CH2, were immunoprecipitated using a rabbit polyclonal antiserum raised against a CH2-glutathione S-transferase (GST) fusion protein (17). Alternatively, rabbit polyclonal antisera specific for either the PTB (19) or the SH2 (Upstate Biotechnology Inc., Boston, Mass.) domain were used. Shc was probed by immunoblotting using either monoclonal (Transduction Laboratories, Mamhead, United Kingdom) or polyclonal (Upstate Biotechnology Inc.) anti-SH2 antibodies. A polyclonal antiserum recognizing phosphorylated S36 was purchased from Alexis Biochemicals (San Diego, Calif.). MAb against phosphorylated Y317 or YY239/240 were purchased from Nanotools (Teningen, Germany). Phospho-specific polyclonal antibodies against the active forms of Erk and p38 mitogen-activated protein kinase (MAPK) were purchased from Cell Signaling Technologies (Beverly, Mass.). Anti-phosphotyrosine, anti-Ras, and anti-ZAP-70 MAbs, as well as an agonistic anti-Fas MAb, were purchased from Upstate Biotechnology Inc.; polyclonal anti-ZAP-70, anti-Erk, anti-p38, anti-Grb2, and monoclonal anti-CD3{zeta} antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, Calif.). Immunoglobulin G from OKT3 and 145-2c11 (anti-human and anti-mouse CD3, respectively; American Type Culture Collection) and B66.6 (anti-CD4) (3) hybridoma supernatants were affinity purified on Mabtrap (Amersham Pharmacia Italia srl) and titrated by flow cytometry. Fluorochrome-labeled anti-human CD69, anti-mouse CD69, CD3, and CD22, and annexin V were purchased from Becton Dickinson Biosciences. Secondary unlabeled antibodies were purchased from Cappel (Durham, N.C.), and secondary peroxidase-labeled antibodies were obtained from Amersham Pharmacia Biotech. Agarose-conjugated GST-Raf was purchased from Upstate Biotechnology Inc.

Transfections, luciferase assays, RNase protection assays, and flow cytometry. Transfections were carried out as described previously, using a modification of the DEAE-dextran procedure (19). All samples were set up in duplicate. A plasmid encoding ß-galactosidase under the control of the cytomegalovirus enhancer was cotransfected as a control of transfection efficiency. Cells were allowed to recover for 22 h before activation. Luciferase activity was quantitated 8 h after activation and normalized to the levels of ß-galactosidase activity. pcDNA3 or the pcDNA3-p66Shc expression constructs were introduced into Jurkat cells by electroporation, and stably transfected cells were selected in medium containing 1 mg of G418 (Gibco BRL, Life Technologies Italia srl, Milan, Italy)/ml.

Total RNA was isolated by RNAwiz (Ambion, Austin, Tex.). DNA templates were purchased from Pharmingen (San Diego, Calif.) as a multiprobe template set (hStress-1; Riboquant RNase protection assay system). RNase protection assays were carried out as described previously (20), using antisense L32 transcripts to normalize the amounts of loaded RNA. Cell lysates and RNA probes were hybridized, and the products of the RNase protection assay were separated on denaturing polyacrylamide gels. Gels were transferred to positively charged nylon membranes, and nonisotopic detection was performed by using a BrightStar BioDetect kit (Ambion). Gel electrophoretic autoradiographs were quantitated by Sigma Gel analysis software (Jandel Scientific, Chicago, Ill.).

CD3, Fas, and CD69 surface expression on Jurkat transfectants was quantitated by flow cytometry using fluorescein isothiocyanate (FITC)-labeled MAb. Mouse T cells were doubly labeled with fluorochrome-conjugated anti-CD3 and anti-CD69 MAb. Samples were processed using a FACScan flow cytometer (Becton Dickinson, San Jose, Calif.).

Activations, immunoprecipitations, in vitro binding assays, and immunoblotting. Activations by cross-linking of anti-CD3 were carried out as described previously (19). Cells (1 x 106 to 5 x 106/sample for direct immunoblot analysis and 1 x 107 to 5 x 107/sample for immunoprecipitations or in vitro binding assays) were lysed in 1% (vol/vol) Triton X-100 in 20 mM Tris-HCl (pH 8), 150 mM NaCl (in the presence of 0.2 mg of Na orthovanadate/ml, 1 µg of pepstatin, leupeptin, and aprotinin/ml, and 10 mM phenylmethylsulfonyl fluoride), and postnuclear supernatants were probed as such or immunoprecipitated using the appropriate polyclonal or monoclonal antibodies and either protein A-Sepharose or agarose conjugated with anti-mouse antibodies (Sigma Italia). In vitro binding assays were carried out as described elsewhere (19). p66Shc immunodepletion was carried out by four rounds of immunoprecipitation with anti-CH2 antibodies. The effectiveness of the immunodepletion was checked by immunoblotting. For the in vitro analysis of p66Shc-p52Shc competition, Shc was immunoprecipitated from lysates of untreated Jurkat cells transfected with empty vector. The immunoprecipitates were subsequently used for in vitro binding assays in the presence of graded amounts of a lysate from JSL1 cells stably expressing p66Shc and activated by CD3 ligation, added with a complementary amount of a lysate from CD3-activated parental JSL1 cells to normalize total protein levels. Cell lysis was carried out in 3% Triton X-100 to disrupt preexisting protein complexes. The Triton X-100 concentration was subsequently adjusted to 1% for the in vitro binding experiment. Immunoblotting was carried out using a chemiluminescence detection system (Pierce, Rockford, Ill.). Prestained molecular weight markers were purchased from Life Technologies Italia srl.

Cell viability, apoptosis, and proliferation assays. Jurkat transfectants, human peripheral blood lymphocytes (PBL), or mouse thymocytes or purified splenic T cells were treated with proapoptotic stimuli for 8 to 72 h. Pharmacological stimuli included H2O2, A23187 (Roche Diagnostics SpA, Milan, Italy), and dexamethasone (Sigma Italia srl). Fas ligation was achieved using an agonistic anti-Fas MAb and plate-bound secondary antibodies as described previously (19). Activation-induced cell death by sequential ligation of CD4 and CD3 was achieved by cross-linking CD4 for 15 min at 37°C in solution using saturating amounts of the anti-CD4 MAb B66.6 and 25 µg of anti-mouse antibodies/ml, followed by incubation of the cells with OKT3 and plating on plastic-bound secondary antibodies for 8 to 24 h. Alternatively, death was induced by prolonged cross-linking of CD3 using OKT3 and plate-bound secondary antibodies in the absence of costimulation.

Cell viability was assessed by trypan blue exclusion. Apoptosis was analyzed by flow cytometry by labeling cells with FITC-labeled annexin V and 50 µg of propidium iodide (PI)/ml as described elsewhere (11) and gating on annexin V+ PI- cells. Alternatively, apoptotic cells were visualized by fluorescence microscopy on a Leica Microsystems confocal microscope (Heidelberg, Germany) after labeling with TdT and FITC-labeled dUTP (Roche Diagnostics SpA) as described previously (7).

Mouse splenic T-cell proliferation was measured by flow cytometric analysis of carboxyfluorescein succinimidyl ester (CFSE)-labeled cells. Cells were resuspended at 20 x 106/ml in phosphate-buffered saline and stained with 10 µM CFSE (Molecular Probes Europe BV, Leiden, The Netherlands) for 8 min at room temperature. Cells were subsequently washed twice in RPMI-7.5% fetal calf serum, resuspended at 5 x 106/ml, and plated on plastic-bound anti-CD3 MAb. Cells were analyzed 24 to 96 h after stimulation.


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RESULTS
 
Expression of p66Shc in human PBL and mouse T cells in response to apoptogenic stimulation. The reactivation of the p66Shc-encoding locus in cells lacking p66Shc expression in the presence of demethylating agents or histone deacetylase inhibitors (35) suggests that under certain conditions these cells might acquire the capacity to express p66Shc. Since p66Shc has been implicated in apoptotic responses to stress in fibroblasts, we analyzed p66Shc expression in human PBL treated with a variety of apoptogenic stimuli. These included H2O2, the calcium ionophore A23187, Fas ligation, and sequential engagement of CD4 and CD3. As shown in Fig. 1A, p66Shc expression was upregulated in T cells treated with all proapoptotic stimuli. A low but detectable basal expression of p66Shc, which was upregulated by treatment with dexamethasone (Fig. 1B) or A23187 (data not shown), was also observed in both mouse thymocytes and purified splenic T cells. The possibility of cross-reactivity to the antibody of an unrelated protein was ruled out by immunoblot analysis of p66Shc-/- cells (Fig. 1B). Hence, although under normal conditions T lymphocytes lack p66Shc expression, they acquire the capacity to express this protein in response to apoptogenic stimulation, suggesting a link between p66Shc and apoptosis.



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  		FIG. 1. Apoptogenic stimuli induce p66Shc expression in PBL. (A) Immunoblot analysis with anti-Shc antibodies of postnuclear supernatants from human PBL treated for the indicated times with different apoptogenic stimuli. CD3, anti-CD3 MAb; CD4, anti-CD4 MAb; CD3 + 4, sequential ligation using specific MAbs for CD4 and CD3. The calcium ionophore A23187 was used at 500 ng/ml. (B) On the left is an immunoblot analysis with anti-Shc antibodies of postnuclear supernatants from untreated control (+/+) or p66Shc-/- (-/-) mouse thymocytes and splenocytes. The right panel shows an immunoblot analysis with anti-Shc antibodies of postnuclear supernatants from thymocytes and purified splenic T cells from control mice treated with either carrier or 0.25 µM dexamethasone (dex) for 20 h. Control blots of the same filters with anti-Vav Ab are shown below. Representative experiments are shown (n >= 3).

Expression of p66Shc correlates with increased T-cell susceptibility to apoptogenic stimuli. At variance with PBL, Jurkat T cells do not express p66Shc even after apoptogenic stimulation (data not shown), in agreement with full methylation of the p66Shc gene promoter in these cells, compared to its partial methylation in PBL (35). Therefore, we used Jurkat T cells for the biochemical and functional characterization of p66Shc in T-cell apoptosis. A panel of Jurkat lines stably transfected with expression constructs encoding either p66Shc or mutants thereof was generated (Fig. 2A). Mutated versions of p66Shc included a mutant lacking S36 (p66SA), a mutant lacking YY239/240 and Y317 (p663F), and the isolated CH2 domain (CH2). All transfectants expressed similar levels of surface CD3 (data not shown).



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  		FIG. 2. p66Shc expression promotes apoptosis in Jurkat T cells. (A) Top: scheme of the mutant or chimeric Shc cDNA constructs used in this study. Bottom: immunoblot analysis using anti-Shc antibodies of postnuclear supernatants of Jurkat cells stably transfected with the cDNA constructs shown above. J, Jurkat; pc, cells stably transfected with pcDNA3; p66, wild-type p66Shc; p66SA, p66Shc carrying a serine-to-alanine mutation at position 36; p663F, p66Shc carrying tyrosine-to-phenylalanine substitutions at positions 239/240 and 317; CH2, isolated CH2 domain of p66Shc. (B and C) Quantitation of cell death by trypan blue exclusion in Jurkat cells stably transfected with either empty vector or the same vector encoding p66Shc. Cells were treated for the indicated times with 200 µM H2O2 or 200 to 500 ng of A23187/ml or, alternatively, plated on plastic-immobilized anti-Fas (Fas) or anti-CD3 (CD3) MAb (n >= 3). (D) Left: flow cytometric analysis of parental Jurkat cells or cells expressing p66Shc treated for 4 h with 500 ng of A23187/ml and stained with FITC-labeled annexin V and PI. Apoptotic cells are annexin V+ PI-. Right: analysis by fluorescence microscopy of p66Shc-expressing untreated cells labeled by TUNEL. Percentages of TUNEL-positive untreated cells were as follows: control cells, 3.5% ± 0.5%; p66Shc-expressing cells, 8.9% ± 0.4%; p66SA-expressing cells, 2.5% ± 0.5% (n = 2; percentages were calculated on 100 cells/sample). (E) Quantitation of cell death by trypan blue exclusion in thymocytes (n = 7) and splenic T cells (n = 4) from control and p66Shc-/- mice treated with carrier (-dex) or 1 µM dexamethasone (+dex) for 5 h. In each experiment a pool of cells from two to three mice was used. Similar results were obtained with splenic T cells after 20 h of treatment (20.2% ± 1.7% death in control untreated cells versus 14.0% ± 0.1% in p66Shc-/- cells; 62.6% ± 4.6% death in dexamethasone-treated control cells versus 45.5% ± 1.5% in p66Shc-/- cells; n = 4). The differences in the basal levels of apoptosis were confirmed by TUNEL staining of freshly isolated thymocytes and splenocytes (percent TUNEL-positive cells in a representative experiment: 11% control versus 8.5% p66Shc-/- splenocytes; 36% control versus 17% p66Shc-/- thymocytes [n = 2; percentages were calculated based on 300 cells/sample]). (F) Quantitation of cell death by trypan blue exclusion in Jurkat cells stably transfected with either empty vector or the same vector encoding different p66Shc mutants. Cells were treated for 16 h with 200 µM H2O2 or 200 to 500 ng of A23187/ml (n >= 3).

Cell viability was measured by trypan blue exclusion following treatment with a variety of proapoptotic agents, including H2O2, the calcium ionophore A23187, ligation of Fas using an agonistic MAb, and prolonged CD3 cross-linking in the absence of costimulation. Slight, yet significantly higher levels of spontaneous mortality were consistently found in p66Shc-expressing cells compared to control cells (Fig. 2B and C). Terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) staining showed that death occurred by apoptosis (Fig. 2D). Expression of p66Shc correlated with increased levels of cell death in response to all stimuli used, as evaluated both by trypan blue exclusion (Fig. 2B and C) and staining with annexin V and PI (Fig. 2D and data not shown). The effect was unique to this Shc isoform, as p52Shc overexpression did not affect either basal or agonist-induced apoptosis (see Fig. S1 in the supplemental material). Of note, the differences in the apoptotic responses of control and p66Shc-expressing cells showed a trend to become less pronounced with longer times of treatment (Fig. 2C [anti-Fas MAb] and data not shown [for other stimuli]), suggesting that cells expressing p66Shc are in a state of priming to apoptosis, which results in accelerated death in response to apoptogenic stimuli. Hence, p66Shc expression results in increased cell susceptibility to apoptosis.

To investigate the physiological contribution of p66Shc to T-cell apoptosis, we analyzed thymocytes and splenic T cells from wild-type and p66Shc-/- mice (18). Cells were treated with dexamethasone (5 h), and death was quantitated both by trypan blue exclusion and by TUNEL. Significantly lower levels of spontaneous death, as well as decreased responses to apoptogenic treatment, were observed in p66Shc-/- splenic T cells (Fig. 2E and data not shown). The differences between p66Shc-/- cells and control cells were detectable even at a longer duration of dexamethasone treatment (20 h), when cell mortality was maximal in wild-type cells (Fig. 2E legend). A reduction in the levels of A23187-induced apoptosis was also observed in p66Shc-/- T cells (data not shown). A similar trend was observed with p66Shc-/- thymocytes (Fig. 2E). Together, the data strongly support a role for p66Shc in T-cell apoptosis.

To address the role of S36 in p66Shc-dependent T-cell priming to apoptosis, S36 phosphorylation was analyzed by immunoblotting with a phospho-specific antibody. S36 phosphorylation was detectable in the absence of stimulation, and it increased further in response to treatment with H2O2 or A23187, as well as Fas ligation or prolonged CD3 engagement (Fig. 3A and B). S36 was also phosphorylated in cells expressing either p66Shc3F (Fig. 3B) or the isolated CH2 domain (Fig. 3C), indicating that S36 phosphorylation was independent of tyrosine phosphorylation and only required the CH2 domain.



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  		FIG.3. Apoptogenic stimuli promote S36 phosphorylation on p66Shc. The immunoblot analysis was performed using an S36 phospho-specific antibody of p66Shc-specific immunoprecipitates from Jurkat T-cell transfectants expressing either wild-type or mutant p66Shc treated with different apoptogenic stimuli. The lower part of each panel shows the same filter reprobed after stripping either with an anti-SH2 Shc MAb (A and B) or with an anti-CH2 Shc antiserum (C). 0, no treatment; CD3, CD3 cross-linking using anti-CD3 MAb for 10 min; Fas, Fas cross-linking using agonistic anti-Fas MAb for 5 min; A, A23187 at 500 ng/ml; H2O2, H2O2 at 220 µM. Representative experiments are shown (n >= 3).

The enhanced susceptibility to apoptogenic stimuli was completely annulled by substitution of S36 (Fig. 2F), thereby mapping a critical determinant of the proapoptotic function of p66Shc to this residue. Notwithstanding the presence of phosphorylated S36, cells expressing either p66Shc3F or the isolated CH2 domain harbored similar levels of death compared to control cells (Fig. 2F), indicating that both full-length p66Shc and integrity of the phosphorylatable tyrosine residues are required to prime T cells to apoptosis.

Enhanced expression of apoptosis-regulating genes in cells expressing p66Shc. p66Shc regulates the levels of reactive oxygen species (ROS) (21, 32). In agreement with this notion, measurement of intracellular ROS showed that p66Shc-expressing cells have higher levels of ROS compared to control cells (V. Petronilli, P. Bernardi and C. T. Baldari, unpublished results). Since oxidants can modulate gene expression (10), we quantitated transcription of proapoptotic genes potentially implicated in p66Shc-mediated apoptosis. FasL gene transcription was evaluated in transient-transfection assays using a luciferase reporter driven by the FasL enhancer. p66Shc expression resulted in increased luciferase levels, both in the absence of stimulation and in response to treatment with A23187, alone or in combination with phorbol myristate acetate (PMA), suggesting that Fas-FasL interactions underlie at least in part p66Shc-dependent apoptosis. This effect was completely annulled by substitution of S36 (Fig. 4A). The levels of Fas were not affected by p66Shc expression (data not shown).



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  		FIG. 4. Enhanced FasL enhancer activity and increased transcription of proapoptotic genes in cells expressing p66Shc. (A) Relative luciferase activity in parental Jurkat cells or cells expressing p66Shc (top) or p66SA (bottom) and transiently cotransfected with a FasL/luciferase reporter and cytomegalovirus ß-galactosidase control vector. Cells were treated for 8 h with 500 ng of A23187/ml, alone or in combination with 100 ng of PMA/ml (PMA/A). Luciferase activity was normalized to the ß-galactosidase activity in the same samples to correct for variations in transfection efficiencies. A representative experiment, carried out in duplicate, is shown (n = 3 for p66Shc; n = 2 for p66SA). (B) The relative levels of Bcl-xL, GADD45, and Bax mRNA in unstimulated cells were quantitated in RNase protection assays and normalized to an internal housekeeping control (L32). For each gene, the data are expressed as the percentage of the levels of transcription in p66Shc-expressing cells compared to that in parental cells, taken as 100% (n = 6). Similar results were obtained when glyceraldehyde-3-phosphate dehydrogenase was used as an alternative internal housekeeping control.

Expression of other genes involved in the control of apoptosis was analyzed by RNase protection. Lower levels of expression of the antiapoptotic gene Bcl-xL and increased levels of expression of the proapoptotic genes Bax and GADD45 were observed in p66Shc-expressing cells compared to control cells (Fig. 4B). Collectively, the data suggest that the state of priming to apoptosis induced by p66Shc expression results from an alteration in the balance of antiapoptotic and proapoptotic gene expression.

p66Shc antagonizes p52Shc signaling to Ras/MAPK in T cells. To assess the impact of p66Shc on p52/p46Shc-dependent mitogenic signaling, we analyzed Erk activation. p66Shc expression correlated with a decrease in both basal and TCR-dependent Erk phosphorylation (Fig. 5A), but not of the related MAPK p38 (data not shown). This effect was observable also in an independent transfectant expressing about half the level of p66Shc (data not shown). No differences in phosphorylation of LAT, one of the principal mediators of signaling to Ras/MAPK, were detected (Fig. 5A). In agreement with the reduction of Erk activation, Ras activation was inhibited in p66Shc-expressing cells (Fig. 5B), as evaluated by in vitro binding assays using a GST-Raf fusion protein. The negative impact of p66Shc on MAPK activation was paralleled by an almost complete inhibition of TCR-dependent expression of CD69, an early activation marker which requires Erk activity (Fig. 5C). TCR-dependent MAPK activation and CD69 expression were not reduced by p52Shc overexpression, indicating that this effect was unique to the p66 isoform (see Fig. S1 in the supplemental material). Inhibition of both Erk activation and CD69 expression was strictly dependent on the integrity of S36; however, no inhibition was observed in the presence of the isolated CH2 domain (Fig. 5A and C). Hence, p66Shc antagonizes TCR-dependent Ras/MAPK activation. This activity requires, in addition to S36, also the portion of the protein common to all Shc isoforms, suggesting a mechanism of competitive inhibition. Of note, no significant differences in TCR-dependent Erk activation (data not shown) or CD69 expression (Fig. 5D) were observed between mouse p66Shc-/- and control splenic T cells. However, enhanced proliferative responses to CD3 ligation were consistently observed in p66Shc-/- splenic T cells (Fig. 5E), both at saturating and at limiting concentrations of anti-CD3 MAb, supporting a blunting activity of p66Shc on mitogenic signaling in vivo.



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  		FIG. 5. p66Shc blunts Ras/Erk activation, CD69 expression, and T-cell proliferation. (A) Left and middle panels: immunoblot analysis of postnuclear supernatants of control Jurkat cells (parental [Jurkat] or transfected with empty vector [pc]) or cells expressing wild-type or mutant p66Shc or the isolated CH2 domain using phospho-specific antibodies against the active forms of Erk. Right panel: antiphosphotyrosine immunoblot of LAT-specific immunoprecipitates from control or p66Shc-expressing cells. For each filter the control blot is shown below. 0, nonactivated; CD3, activation by TCR-CD3 cross-linking for 1 min using anti-CD3 MAb. (B) Immunoblot analysis using an anti-Ras MAb of in vitro binding assays of postnuclear supernatants of control or p66Shc-expressing cells to agarose-bound GST-Raf. Total cell lysates separated on the same gel are shown. 0, nonactivated; CD3, activation by TCR-CD3 cross-linking for 1 min using anti-CD3 MAb. (C) Flow cytometric analysis of Jurkat cells stably transfected with empty vector or p66Shc expression constructs using FITC-labeled anti-CD69 MAb. 0, nonactivated (thin line); CD3, activated for 16 h by CD3 cross-linking on plate-bound antibody (thick line); PMA, 100 ng of PMA/ml (dotted line). The shaded area shows cells labeled with FITC-labeled isotype control. (D) Flow cytometric analysis of control (top) or p66Shc-/- (bottom) mouse splenocytes using FITC-labeled anti-CD3 MAb and phycoerythrin-labeled anti-CD69 MAb. CD69 expression was analyzed on gated CD3+ cells. 0, nonactivated (thin line); CD3, activated for 16 h by CD3 cross-linking on plate-bound antibody (thick line); PMA, 100 ng of PMA/ml (shaded area). (E) Flow cytometric analysis of CFSE-labeled splenocytes from control (top) or p66Shc-/- (bottom) mice stimulated for 96 h with different concentrations of plate-bound anti-CD3 MAb corresponding, respectively, to ~70, 55, and 40% saturation of surface CD3. CFSE fluorescence was analyzed on gated CD3+ cells (shaded histogram). CFSE staining in nonstimulated CD3+ cells is shown as an empty histogram. Representative experiments are shown (n >= 3).

p52Shc is recruited to the TCR signaling complex through its association with ZAP-70 and CD3{zeta} (19, 28). To understand whether p66Shc can compete with p52Shc for binding to these molecules, p66Shc-specific immunoprecipitates were probed for ZAP-70. p66Shc was found associated with ZAP-70 even in the absence of stimulation, and this association increased following CD3 ligation (Fig. 6A). TCR-dependent association of p66Shc with phospho-CD3{zeta} was also detected (data not shown). Similar levels of ZAP-70 (Fig. 6B) or phospho-CD3{zeta} (data not shown) were found to inducibly associate with p66Shc and p52Shc, indicating the possibility of an effective competition between the two isoforms. To further address this point, Shc was immunoprecipitated from control or p66Shc-expressing cells previously immunodepleted of p66Shc using a CH2 domain-specific antibody. Dramatically lower levels of ZAP-70 were found associated with p52Shc in cells coexpressing p66Shc (Fig. 6C). To measure directly the effect of p66Shc on the capacity of p52Shc to bind ZAP-70, Shc proteins were recovered from cells transfected with empty vector. The Shc-specific immunoprecipitates were then incubated with graded amounts of lysate from a Jurkat Shc-negative variant (JSL1 [9]), stably transfected with the p66Shc-expression construct, as a source of p66Shc (Fig. 6D, middle panel). The total amount of proteins was normalized using increasing amounts of lysate from JSL1 parental cells (Fig. 6D, lower panel). The amounts of added lysate from parental and JSL1-p66Shc cells were adjusted to obtain a range of p66Shc/p52Shc ratios from ~1:1 to ~1:10. Both parental and p66Shc-expressing JSL1 cells were activated by TCR cross-linking to induce ZAP-70 phosphorylation and lysed in 3% Triton X-100 to disrupt preexisting protein interactions. The resulting complexes were analyzed by immunoblotting using anti-ZAP-70 antibodies. As shown in Fig. 6D (upper panel), p66Shc prevented p52Shc binding to ZAP-70 even at the lowest concentration, supporting an effective competition between p66Shc and p52Shc for a limiting number of ZAP-70 molecules available for interaction with Shc.



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  		FIG. 6. Interaction of p66Shc with ZAP-70 and Grb2. (A) Immunoblot analysis using anti-ZAP-70 MAb of p66Shc-specific immunoprecipitates of Jurkat cells expressing wild-type or mutant p66Shc. (B) Immunoblot analysis with anti-ZAP-70 MAb of Shc-specific immunoprecipitates. Immunoprecipitation from p66Shc-expressing cells was carried out using an anti-CH2 antibody to selectively recover p66Shc, while p52/p46Shc was immunoprecipitated from cells transfected with empty vector using an anti-PTB antibody. The immunoprecipitates were resolved on the same gel. (C) Immunoblot analysis with anti-ZAP-70 MAb of Shc-specific immunoprecipitates from control (pc) or p66Shc-expressing cells. p66Shc-expressing cells were depleted from p66Shc by immunoprecipitation with an anti-CH2 antibody. The levels of ZAP-70 in the CH2-specific immunoprecipitate from activated p66Shc-expressing cells are shown in the last lane on the right. (D) Immunoblot with anti-ZAP-70 MAb of an in vitro binding assay of Shc-specific immunoprecipitates from lysates of unstimulated control cells (pc), in the presence of graded amounts of p66Shc (JSL1-p66). The two lowest panels shows anti-Shc and anti-ZAP-70 immunoblots of the input cell lysate used for each sample (graded amounts of JSL1-p66Shc lysate were added with a complementary amount of parental JSL1 lysate to achieve identical amounts of total proteins, including ZAP-70). Both parental JSL1 and JSL1-p66 were activated by TCR cross-linking (1 min). All cells were lysed in 3% Triton X-100 to disrupt preexisting protein complexes. (E) Left: immunoblot analysis with anti-Grb2 MAb of Shc-specific immunoprecipitates. Immunoprecipitation from p66Shc-expressing cells was carried out using an anti-CH2 antibody to selectively recover p66Shc, while p52/p46Shc was immunoprecipitated from cells transfected with empty vector using an anti-PTB antibody. The immunoprecipitates were resolved on the same gel. Right: immunoblot analysis with anti-Grb2 MAb of p66Shc-specific immunoprecipitates from cells expressing p66Shc or p66SA. (F) Immunoblot analysis with anti-Grb2 MAb of Shc-specific immunoprecipitates from control (pc) or p66Shc-expressing cells. p66Shc-expressing cells were either first immunodepleted from p66Shc using an anti-CH2 antibody or directly submitted to immunoprecipitation with an anti-PTB antibody. A control blot with anti-Shc MAb is shown below. The effectiveness of the immunodepletion is documented in the bottom panel, which shows an anti-Shc immunoblot of the input lysates. Proteins were separated under nonreducing conditions for a better discrimination of Grb2. 0, nonactivated; CD3, activation by TCR-CD3 cross-linking for 1 min using anti-CD3 MAb. Control blots of all experiments are provided. Representative experiments are shown (n >= 3).

Probing of p66Shc-specific immunoprecipitates with antiphosphotyrosine antibodies, as well as with phospho-specific antibodies, showed that following TCR engagement p66Shc was detectable as a tyrosine phosphoprotein migrating with a slightly slower mobility compared to ZAP-70 (Fig. 7A and data not shown). Substitution of S36 did not affect p66Shc phosphorylation on tyrosine residues (Fig. 7A). Apoptogenic stimulation, while inducing S36 phosphorylation (Fig. 3), failed to significantly affect either tyrosine phosphorylation of p66Shc (Fig. 7B) or its association with ZAP-70 (data not shown). A time course analysis of tyrosine and S36 phosphorylation of p66Shc following TCR engagement showed that the two events were staggered, with a transient tyrosine phosphorylation peaking at 1 min and a sustained S36 phosphorylation beginning 3 min after stimulation, when tyrosine phosphorylation was declining (Fig. 7C). The different timing of tyrosine and S36 phosphorylation, as well as their complementary patterns in cells expressing either the S36 or the 3F mutant (Fig. 3A and B and 7A) underlines that tyrosine and S36 phosphorylation of p66Shc are independent.



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  		FIG. 7. Sequential phosphorylation of p66Shc on tyrosine and S36. (A and B) Antiphosphotyrosine and control anti-Shc immunoblot of p66Shc-specific immunoprecipitates from Jurkat transfectants expressing wild-type or mutant p66Shc. Cells were either activated by TCR cross-linking (A) or treated with apoptogenic stimuli (B). The migration of phosphorylated ZAP-70 in the antiphosphotyrosine immunoblot was confirmed by probing the same filter with anti-ZAP-70 MAb. 0, nonactivated; CD3, activated by CD3 cross-linking for 1 min; A, 500 ng of A23187/ml; Fas, Fas ligation with agonistic anti-Fas MAb for 5 min. (C) Immunoblot analysis of tyrosine or S36 phosphorylation on p66Shc-specific immunoprecipitates of Jurkat cells expressing p66Shc. Cells were activated by CD3 cross-linking for different times as indicated. A control blot of the same filter with anti-Shc MAb is shown. Representative experiments are shown (n = 2 to 3).

Phosphorylation of p66Shc on both Grb2 binding sites (as revealed by immunoblotting with phospho-specific antibodies [data not shown]) suggests a potential interaction of p66Shc with Grb2. Probing p66Shc-specific immunoprecipitates for Grb2 showed that TCR engagement resulted in Grb2 recruitment (Fig. 6E). p66Shc and p52Shc coprecipitated similar levels of Grb2 in p66Shc-expressing cells and control cells, respectively, suggesting the possibility that, when coexpressed, the two isoforms might compete for Grb2 binding (Fig. 6E, left). In support of a transdominant inhibition of the p52Shc interaction with Grb2 by p66Shc, decreased levels of Grb2 association with p52Shc were found in p66Shc-expressing cells previously immunodepleted with a CH2 domain-specific antibody (Fig. 6F). Hence, while itself unable to sustain Ras/MAPK activation, p66Shc competitively inhibits p52Shc interaction with ZAP-70 and Grb2, resulting in defective downstream signaling. Of note, substitution of S36 did not affect Grb2 binding to p66Shc (Fig. 6E, right); however, this interaction does not terminate signaling to Ras (Fig. 5). Collectively, the data suggest that blunting of TCR-dependent Ras activation by p66Shc might result from Grb2 sequestration in a prohibitive conformation, an activity which requires S36 phosphorylation.


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DISCUSSION
 
The expression of multiple Shc isoforms within the same cell, as well as their capacity to all be phosphorylated on tyrosine residues in response to growth factors and to interact with the signaling machinery leading to Ras activation, raises the question of the specificity and potential redundancy of each isoform. While the role of p46Shc remains as yet elusive, the picture which is slowly emerging for the other two isoforms is that, while p52Shc is hard-wired in the cellular mechanisms controlling the proliferative response to growth factors, p66Shc is responsible for the fine-tuning of this response. The study of a gene-targeted mouse differentially lacking p66Shc has added a further level of complexity to the function of Shc proteins. Deletion of p66Shc protects mouse embryo fibroblasts (MEFs) from apoptosis induced by oxidative stress, while overexpression of p66Shc in wild-type MEFs increases their susceptibility to H2O2 (18). Here we have shown that, while normally undetectable in T cells, p66Shc is inducibly expressed in both human PBL and mouse thymocytes and splenic T cells. Forced p66Shc expression results in increased levels of spontaneous mortality and promotes T-cell apoptosis in response to a variety of apoptogenic stimuli, both pharmacological (H2O2 and A23187) and receptor mediated (CD3 ligation in the absence of costimulation, sequential engagement of CD4 and CD3, and Fas ligation). Conversely, significant resistance to both spontaneous and pharmacologically induced apoptosis can be observed in thymocytes and splenic T cells from p66Shc-/- mice. The enhancement in T-cell apoptosis associated with p66Shc expression, together with the complementary phenotype harbored by p66Shc-/- T cells, strongly supports a physiological role of p66Shc in T-cell apoptosis. Of note, reduced ROS levels have been found in p66Shc-/- MEFs (21, 32), linking p66Shc not only to stress-induced but also to homeostatic ROS production. Reduced homeostatic ROS production in p66Shc-/- mouse T cells might account for their lower levels of spontaneous apoptosis, bearing in mind that their normal counterparts express low but measurable levels of p66Shc.

The apoptogenic activity of p66Shc in our T-cell model was completely annulled by S36 substitution, supporting a requirement for S36 phosphorylation. In agreement with the increased levels of spontaneous apoptosis in p66Shc-expressing cells, p66Shc showed detectable levels of S36 phosphorylation—possibly due to the increased ROS levels in p66Shc-expressing cells, which were further enhanced after treatment with proapoptotic stimuli. Interestingly, coexpression of an S36 mutant results in transdominant inhibition of p66Shc-dependent ROS production (21), suggesting that this activity might to be dependent on intermolecular interactions involving phosphorylated S36. Phosphorylation of S36 on the CH2 domain is, however, not sufficient for p66Shc-dependent apoptosis since, notwithstanding S36 phosphorylation, neither p66Shc3F nor the isolated CH2 domain promoted apoptosis in Jurkat cells. Although no enhancement in tyrosine phosphorylation of p66Shc was observed following apoptogenic stimulation, the basal levels of p66Shc phosphorylation, which are likely to result from its constitutive interaction with ZAP-70, might be sufficient to account for the requirement for YY239/240 and Y317 in p66Shc-dependent apoptosis. It should be underlined that we have been unable to detect any significant enhancement of p66Shc association with Grb2 in cells treated with apoptogenic stimuli (data not shown). Whether tyrosine phosphorylation of p66Shc is essential for recruitment of downstream effectors, other than Grb2, implicated in its proapoptotic activity remains to be determined.

The central role of p66Shc in controlling the levels of intracellular oxidants (21, 32) is supported by our finding of increased ROS levels in Jurkat T cells expressing p66Shc. This property of p66Shc is likely to account for the observed changes in the expression of apoptosis-modulating genes. Gene transcription is indeed known to be modulated by oxidants, as effectively exemplified by NF-{kappa}B (25). More recently, a direct link has been established between p66Shc expression, ROS levels, and activity of the forkhead transcription factor (12, 21). Of note, the genes encoding GADD45 and Bax, which are transcriptionally upregulated in p66Shc-expressing Jurkat cells, are both targets of p53 (5), whose activity is controlled by p66Shc through the production of intracellular ROS (32). How p66Shc regulates the steady-state levels of intracellular oxidants has yet to be understood, although the localization of a fraction of p66Shc within mitochondria (32) might provide a clue to this function.

As opposed to p52/p46Shc, overexpression of p66Shc fails to induce fibroblast transformation (17, 24). Furthermore, p52Shc and p66Shc appear to exert opposing activities on the proliferative signals elicited by epidermal growth factor receptor stimulation (17, 22). Here we have shown that, in T cells, increased p66Shc levels correlate with a dramatic blunting of Erk activation and CD69 expression, which is strictly dependent on Ras/MAPK (4). The inhibitory activity of p66Shc on Ras/MAPK activation can be accounted for, at least in part, by a mechanism involving p66Shc competition with p52Shc. When expressed in T cells, p66Shc is indeed coupled to the TCR and can very effectively outcompete the interaction of p52Shc with ZAP-70 induced by TCR engagement. As opposed to p52Shc, a significant interaction between p66Shc and ZAP-70 is found even in the absence of stimulation, supporting a model of transdominant inhibition of p52Shc by p66Shc based on competitive interaction of p66Shc for a limited availability of upstream molecular partners. In this context, lower levels of ZAP-70 (but not of Grb2) were detected in lysates of p66Shc-expressing cells after p66Shc immunodepletion (data not shown). Although overexpression of p66Shc is likely to have significantly amplified the effects of this protein on mitogenic signaling, the constitutive interaction of p66Shc with limiting ZAP-70 is likely to result in p52Shc inhibition even at low levels of p66Shc expression, such as are found in the physiological context of normal preapoptotic T cells. The possibility of an effective competition by a high-affinity interaction, notwithstanding low expression levels of the competitor, can also be hypothesized based on the small fraction of the total cellular complement of cytosolic signaling mediators, including Shc, which is normally recruited to activated receptors. A further level of competition by p66Shc is achieved at the level of Grb2 binding. Interestingly, the S36 mutant also interacts with Grb2, in agreement with its inducible phosphorylation on tyrosine residues, but does not block Ras/MAPK activation, suggesting that when phosphorylated on S36 p66Shc might sequester Grb2 in a nonproductive state. Whether this is the outcome of a close conformation involving phosphorylated S36 or involves the recruitment of a hypothetical inhibitor remains to be determined.

The role of p66Shc as a negative regulator of mitogenic signaling in T cells is further supported by the finding that CD3-dependent proliferation is enhanced in p66Shc-/- splenic T cells. Although p66Shc deficiency does not appear to affect TCR-dependent Erk activation, the higher threshold of Ras activity required for triggering proliferation compared to earlier events, such as Erk activation or CD69 expression, might account for the selective effects of p66Shc deletion on TCR-dependent proliferation. At this stage it cannot, however, be excluded that inhibition by p66Shc of interleukin-2 receptor signaling, which is crucial for T-cell proliferation and requires Shc (6, 15), might contribute to the enhanced proliferative responses of p66Shc-/- cells to TCR ligation. Alternatively, the inhibitory activity of p66Shc on mitogenic signaling might be fully operational only in preapoptotic cells, when p66Shc levels are significantly upregulated. In this scenario, p66Shc might contribute to cell cycle arrest preliminary to initiation of apoptosis.

In summary, our data show that expression of p66Shc increases T-cell susceptibility to apoptosis and blunts TCR-dependent mitogenic signaling. Hence, through the regulation of p66Shc expression, Shc proteins appear to be strategically placed at the cross-road between T-cell activation, survival, and death.


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ACKNOWLEDGMENTS
 
We thank Gary Koretzky for the generous gift of the FasL reporter construct, Makio Iwashima for his kind gift of JSL1 cells, Mario Milco D'Elios for providing buffy coats and valuable input, Silvia Valensin for advice on the flow cytometric analysis of mouse T cells, Susanna Pennacchini, Alfredo Pezzicoli, and Mariateresa Savino for their generous help, and Emanuele Orsini for his assistance with the mice. We are indebted to Paolo Bernardi, Valeria Petronilli, and John L. Telford for productive discussions and critical reading of the manuscript. The technical assistance of Sonia Grassini and secretarial assistance of Giancarlo Benocci are gratefully acknowledged.

This work was generously supported by the Italian Association for Cancer Research. The support of the Consiglio Nazionale delle Ricerche, Telethon (grant E.1161) and the University of Siena is also gratefully acknowledged. S.P. and C.U. are recipients of a FIRC fellowship.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Evolutionary Biology, University of Siena, Via Aldo Moro 2, 52100 Siena, Italy. Phone: 39-0577-234400. Fax: 39-0577-234476. E-mail: baldari{at}unisi.it. Back

{dagger} Supplemental material for this article may be found at http://mcb.asm.org. Back

{ddagger} S.P. and M.P. contributed equally to this work. Back


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Molecular and Cellular Biology, February 2004, p. 1747-1757, Vol. 24, No. 4
0270-7306/04/$08.00+0     DOI: 10.1128/MCB.24.4.1747-1757.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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