|8 March 2001, Volume 20, Number 10, Pages 1203-1211|
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|Caspase-dependent cleavage of the hematopoietic specific adaptor protein Gads alters signalling from the T cell receptor|
|Donna M Berry1, Sally J Benn1, Alec M Cheng2 and C Jane McGlade1|
1The Arthur and Sonia Labatt Brain Tumour Research Centre, The Hospital for Sick Children, Toronto, Ontario, Canada. M5G 1X8
2Division of Rheumatology, Department of Medicine, Washington University School of Medicine, St. Louis MO, USA
Correspondence to: C Jane McGlade, The Hospital for Sick Children, 555 University Avenue, Toronto, Ontario, Canada M5G 1X8
Gads is a SH2 and SH3 domain-containing, hematopoietic-specific adaptor protein that functions in signalling from the T cell receptor. Gads acts by linking SLP-76, bound by the carboxy-terminal Gads SH3 domain, to tyrosine phosphorylated LAT which contains binding sites for the Gads SH2 domain. Gads is distinguished from Grb2 and the closely related Grap protein by the presence of a 120 amino acid unique region between the SH2 domain and the carboxy terminal SH3 domain. Here we demonstrate that the unique region of Gads contains a capase cleavage site. Induction of apoptosis in lymphocytes results in detectable Gads cleavage by 60 min. Gads cleavage is blocked in vivo by treating cells with a caspase 3 inhibitor. A putative caspase 3 cleavage site was identified within the unique region and mutation of this site prevented Gads cleavage in vitro, and in vivo. The Gads cleavage products retained the predicted binding specificity for SLP-76 and LAT. Expression of the Gads cleavage products in Jurkat T cells inhibited NFAT activation following TCR cross linking. These findings indicate that cleavage of Gads in vivo could function to alter signalling downstream of the T cell receptor by disrupting cross talk between SLP-76 and LAT. Oncogene (2001) 20, 1203-1211.
Gads; caspase; T cell receptor; SH2 domain; SH3 domain
Apoptosis is important in the normal development and homeostasis of multicellular organisms. The process of apoptosis is tightly regulated by a number of biochemical signalling pathways. The Caenorhabditis elegans cell death gene ced-3 and its mammalian homologue interleukin-1B-converting enzyme (ICE/Caspase-1) are prototypical members of a family of cysteine-dependent aspartate directed proteases (caspases) (Nicholson et al., 1995; Yuan et al., 1993). This family of proteases has 13 known members which have been shown to play an important role in mediating apoptotic cell death (Earnshaw et al., 1999; Fadeel et al., 2000; Villa et al., 1997). In non-apoptotic cells, caspases exist as zymogens, which become active through either self-proteolysis or cleavage by other caspase family members. Current data suggest that cell death signals result in the activation of caspase-8, 9 or 2 which then mediate cleavage and activation of downstream effector caspases (3, 6 and 7) (Earnshaw et al., 1999). Effector caspases are responsible for the cleavage of specific target proteins, thereby mediating the events associated with cell death. To date, a number of effector caspase targets have been identified including structural components of the cytoskeleton and nucleus, DNA repair enzymes and signalling proteins involved in the regulation of cell cycle and survival (Earnshaw et al., 1999).
T cell receptor (TCR)-mediated apoptosis, or activation-induced cell death (AICD) plays an important role in the negative selection of immature T cells within the thymus and maintaining the function of mature peripheral T cells (Brunner et al., 1995 Ju et al., 1995). Activation of the T cell receptor leads to increased transcription of both the Fas receptor (CD95) and its extracellular ligand FasL (Russell, 1995). The interaction of Fas and FasL initiates the cell death signalling cascade which mediates AICD by promoting receptor aggregation and the recruitment of the adaptor protein FADD and its associated caspase-8 (Martin et al., 1998; Muzio et al., 1998; Yang et al., 1998). Increased concentration of caspase-8 in proximity to the receptor leads to auto-activation of caspase-8 and subsequent activation of downstream effector caspases and cleavage of cellular proteins responsible for controlled cellular destruction (Martin et al., 1998; Muzio et al., 1998; Yan et al., 1998).
Initiation of signalling events from the TCR involves phosphorylation of tyrosine residues in the intracellular domains of the receptor CD3 complex by Src-family kinases including Lck and Fyn (Chan and Shaw, 1996; Wange and Samelson, 1996). The phosphorylation of these residues creates a SH2 binding site for the protein tyrosine kinase ZAP-70, which is subsequently phosphorylated and activated by Lck (Chan et al., 1995; Wange et al., 1993). The activation of ZAP-70 initiates multiple signalling pathways through phosphorylation of multiple downstream substrates, including enzymes such as Phospholipase Cl (Weiss et al., 1991), and Vav (Bustelo et al., 1992), hematopoietic specific adaptor and docking proteins including the linker of activated T cells (LAT) (Buday et al., 1994; Nel et al., 1995; Weber et al., 1998; Zhang et al., 1998b) and SH2 domain-containing leukocyte protein of 76 kDa (SLP-76) (Jackman et al., 1995). The SLP-76 and LAT adaptor proteins are fundamental in coordinating the activation of both Ras-Erk and Ca2+-mobilization pathways and both are essential for T cell development and activation (Finco et al., 1998; Jackman et al., 1995; Yablonski et al., 1998; Zhang et al., 1998b, 1999).
Gads is a hematopoietic adaptor protein with amino and carboxy terminal SH3 domains flanking a central SH2 domain (Asada et al., 1999; Law et al., 1999; Liu and McGlade, 1998). Gads is most closely related to the adaptors Grb2 and Grap but contains a unique region rich in glutamine and proline residues for which a function has not been determined. Gads has been shown to play an important role in signalling from the T cell receptor (TCR) by linking the adaptor proteins SLP-76 and LAT upon TCR engagement. SLP-76 is constitutively associated with Gads through the carboxy-terminal SH3 domain of Gads, while LAT is inducibly associated via the SH2 domain (Liu et al., 1999). Overexpression of Gads can synergize with SLP-76 to augment NFAT and IL-2 activation, while a SH2-inactivated form of Gads inhibits this activation (Asada et al., 1999; Law et al., 1999; Liu et al., 1999).
Here we show that in T lymphocytes, cell death triggered by activation of the Fas receptor results in the cleavage of Gads. We show that this cleavage is mediated by caspase 3 and that the conserved cleavage site lies within the unique linker region of Gads which distinguishes it from Grb2 and Grap. In addition, we have found SLP-76 and LAT are also cleaved with similar kinetics. Due to the important role of the Gads-SLP-76-LAT complex in TCR signalling, we also investigated the effects of the Gads caspase cleavage products on distal signalling events. The expression of either the C-terminal or N-terminal cleavage products inhibited NFAT activity, suggesting that caspase-mediated cleavage of Gads alters signalling from the activated TCR.
FasL stimulation of T cells induces proteolytic cleavage of Gads
During the course of our analysis of Gads protein expression in primary lymphocytes, a faster migrating protein which was immunoreactive with anti-Gads antisera was observed (Figure 1a). We considered that this Gads reactive band might be generated by proteolytic cleavage as a result of caspase activation, a consequence of programmed cell death which is triggered when primary cells are placed in culture. Therefore, we tested whether similar proteolytic cleavage of Gads occurred in Jurkat T cells stimulated with Fas ligand (FasL), which activates the cell death receptor Fas and initiates a caspase cascade. Following treatment with FasL for up to 150 min, Jurkat cells were harvested and protein lysates analysed by SDS-PAGE (Figure 1b). We found the appearance of anti-Gads reactive cleavage product by 90 min, at the same time that cleavage of PARP, a known substrate of caspase 3 is detected. In addition, when the Gads blot was stripped and reprobed for the known Gads-associated protein SLP-76, it was discovered that it too undergoes proteolytic cleavage after Fas receptor activation. Microscopic examination of the cells confirmed that FasL stimulation resulted in approximately 60% of cells undergoing apoptotic cell death after 90 min. Other agents known to trigger apoptotic cell death, such as etoposide and staurosporine also resulted in cleavage of Gads in Jurkat T cells (Figure 1c). Treatment of the K562 leukaemia cell line with etoposide did not result in any observable cell death and also did not cause cleavage of Gads. These results suggest that initiation of programmed cell death in T cells triggers proteolytic cleavage of Gads.
In T cells Gads resides within specific protein complexes, some constitutive and some induced following TCR stimulation. Therefore, we tested whether FasL stimulation also resulted in cleavage of other components of these complexes. Following FasL stimulation for the indicated times whole cell lysates were prepared and immunoblotted with the indicated anti-sera (Figure 2). Cleavage products were detected when protein lysates were blotted for the known Gads associated protein LAT, and to a lesser extent the SLP-76 associated protein SLAP-130 (Figure 2). Other LAT associated molecules including Grb2, SOS and PLC did not appear to be cleaved following FasL stimulation, suggesting that Gads complexes are specifically cleaved.
The linker region of Gads contains a caspase 3 cleavage site
Inspection of the Gads amino acid sequence revealed that the unique linker region between the SH2 domain and the carboxy terminal SH3 domain contains a potential caspase cleavage site (Figure 3a). Although the amino acid sequence throughout the linker region of human and murine Gads is not absolutely conserved, the sequence of the putative cleavage site at amino acids 232-235 is identical in the mouse and human sequences (unpublished observation). Cleavage of the Gads protein at this site would yield an anti-Gads immunoreactive protein with a predicted molecular weight of approximately 24 kDa, in agreement with the size of the cleavage product observed in Figure 1. Since the sequence in Gads most closely matched the consensus cleavage site for the caspase 3 family of proteases, we tested whether specific caspase inhibitors would block Gads cleavage following FasL stimulation of Jurkat T cells. Figure 3b shows that treatment of Jurkat cells with both a pan-caspase inhibitor as well as a caspase 3 specific inhibitor prevented Gads cleavage. An inhibitor of Caspase 1 (ICE) family members did not protect Gads from cleavage, suggesting that Gads is likely a caspase 3 target.
In order to confirm that Gads was a caspase 3 substrate and to identify the cleavage site, an in vitro cleavage assay was performed using recombinant caspases and in vitro translated 35S-methionine labeled Gads protein (Figure 4a). A mutant form of Gads in which aspartic acid 235, within the putative cleavage site, was changed to alanine, was also used in the in vitro assay. Caspase 3 efficiently cleaved wild type Gads but not Gads D235A confirming that this site is a caspase 3 cleavage site. Caspase 7 appeared to cleave Gads very inefficiently, suggesting that Gads is a specific target of caspase 3 in vitro. When expressed in Jurkat T cells, only the wild-type form of Gads but not the D235A mutant was cleaved following FasL treatment, confirming that the DIND235 sequence within the Gads linker region is also required for caspase cleavage of Gads in vivo (Figure 4b).
Binding specificity of the Gads cleavage products
Gads functions as an adaptor protein that plays a critical role in assembling a complex containing LAT and SLP-76, via its SH2 and carboxy-terminal SH3 domain respectively, that is important for signalling downstream of the T cell receptor (Liu et al., 1999). Cleavage of Gads at D235 would result in the release of the carboxy terminal SH3 domain of Gads from the remainder of the protein consisting of the amino terminal SH3 domain and the central SH2 domain. Therefore, we examined how such a cleavage event could alter the binding properties of the Gads SH2 and SH3 domains and their interaction with targets following T cell receptor activation. Epitope tagged Gads proteins representative of the two cleavage products were expressed in Jurkat T cells. Following TCR activation the Gads construct representing the amino terminal cleavage product (CL-N) was found to associate with LAT while the carboxy terminal product (CL-C) did not (Figure 5). Similarly, the carboxy terminal cleavage product efficiently associated with SLP-76 independent of TCR activation. Furthermore, examination of the tyrosine phosphorylated proteins bound to each cleavage product following TCR activation did not reveal the presence of any new co-immunoprecipitating proteins, suggesting the specificity of the Gads SH2 and SH3 domains is retained even in the cleaved form of the molecule. These results suggest that cleavage of Gads could uncouple SLP-76 and LAT downstream of the TCR.
Gads cleavage products alter TCR signalling
To examine the effects of Gads cleavage on downstream signalling from the T cell receptor, we measured IL-2 promoter activity in Jurkat T cells following TCR cross linking in the presence of transiently expressed Gads cleavage products. The transient over expression of SLP-76 has previously been shown to enhance transcription of a luciferase reporter gene driven by the NFAT-binding region of the IL-2 promoter in response to TCR ligation (Motto et al., 1996). Co-expression of either of the Gads caspase cleavage products with SLP-76 inhibited this enhanced anti-CD3 dependent activation of NFAT (Figure 6a). No difference in the maximum luciferase units induced by ionomycin and PMA was observed between transfectants, suggesting that the effect of the Gads cleavage products is proximal to the TCR (data not shown). Previously we have shown that over expression of Gads alone has no observable effect on a NFAT driven reporter, while co-expression of Gads with SLP-76 results in synergistic activation of NFAT (Liu et al., 1999). Expression of the Gads cleavage products was also able to inhibit the synergistic activation of NFAT observed in the presence of transient SLP-76 and wild type Gads (Figure 6a). Furthermore, when the cleavage products are transiently over expressed together, a dosage dependent inhibition of SLP-76 enhanced NFAT activation was observed (Figure 6b). These results indicate that the Gads cleavage products function as dominant inhibitors of SLP-76 dependent signalling and suggests that, in vivo, cleavage of Gads molecules could suppress signalling from the TCR.
Gads is a member of a family of SH2 and SH3 domain-containing adaptors which function in tyrosine kinase mediated signalling cascades. Gads is distinguished from the other two members of this family, Grb2 and GRAP, by the presence of a unique region between the central SH2 domain and the carboxy terminal SH3 domain. We have shown that one consequence of the presence of this unique region is to confer sensitivity to caspase cleavage following the initiation of programmed cell death in T cells. Cleavage of Gads at this site results in the separation of the carboxy terminal SH3 domain from the rest of the Gads molecule, effectively uncoupling Gads SH3 binding proteins such as SLP-76 from the tyrosine phosphorylated proteins which bind the Gads SH2 domain. In addition, we have found that the presence of the Gads cleavage products inhibits signalling from the T cell receptor.
In the periphery, reactive T cells are eliminated by activation induced cell death (AICD), a process mediated by Fas and FasL (CD95/CD95L) during the down regulation of an immune response (Brunner et al., 1995; Ju et al., 1995; Russell, 1995). Gads plays a central role in T cell activation by linking the membrane protein LAT, with SLP-76 (Liu et al., 1999). The assembly of a SLP-76-Gads-LAT complex is required for efficient transduction of downstream signals such as calcium mobilization, JNK kinase activation and NFAT activation of the IL-2 promoter (Ishiai et al., 2000; Zhang et al., 2000). Cleavage of Gads, would result in both the loss of its adaptor function as well as the production of cleavage products which can act in a dominant inhibitory manner. Therefore, Gads cleavage would effectively result in the uncoupling of signalling pathways downstream of the T cell receptor during apoptosis and cessation of proliferation, survival and activation signals.
In addition to Gads we have found that the Gads associated-molecules SLP-76 and LAT as well as SLP-76 associated SLAP-130/Fyb are cleaved following FasL stimulation of Jurkat cells. SLP-76, LAT and SLAP-130/Fyb all contain potential caspase 3 sites which if cleaved would yield proteolytic products with molecular weights consistent with the electrophoretic mobilities of those observed in this study (unpublished observations). For example, SLP-76 contains four putative caspase 3 cleavage sites including the sequence DEDD (amino acids 320-323). Cleavage at this site would yield a 30 kDa product and a 46 kDa product containing the anti-SLP-76 antibody immunoreactive region in agreement with the putative proteolytic product detected in Figure 1b. Whether the physical association of these molecules in this complex influences their susceptibility to cleavage remains to be determined.
Other components of the T cell receptor signalling cascade such as Vav and HPK1 have also been identified as caspase 3 substrates in lymphocytes (Chen et al., 1999; Hofmann et al., 2000). Cleavage of these signalling enzymes leads to alterations in their catalytic activity. In the case of HPK1, separation of the C-terminal regulatory domain from the N-terminal kinase domain leads to enhanced kinase activity, and the cleaved peptides have a reduced capacity to associate to HPK1 binding partners Grb2 and Crk (Chen et al., 1999). The caspase-dependent cleavage of Vav1 does not affect its ability to activate JNK, a kinase which has been implicated as a positive regulator in apoptosis, but the cleaved form of Vav1 completely fails to induce IL-2 transcription, suggesting that the cleavage of Vav1 may inhibit signalling from the TCR (Hofmann et al., 2000). Gads is a unique example of an adaptor protein that is a substrate for caspase cleavage. The functional consequence of caspase cleavage of Gads is likely the uncoupling of specific signalling pathways proximal to the TCR, rather than deregulation of a signalling enzyme or disassembly of a structural complex.
In addition to inducing apoptosis in T cells, there is some evidence that Fas mediated signalling may also be involved in proliferation. Inhibition of the FADD adaptor protein through either gene targeting or the expression of a dominant negative form of FADD has revealed some surprising effects on T cell proliferation. Though FADD knock-out mice were embryonic lethal (Yeh et al., 1998; Zhang et al., 1998a), thymic reconstitution of RAG-1 deficient chimeric mice revealed the expected defects in Fas-induced T cell apoptosis, but surprisingly cells were also resistant to activation induced proliferation (Zhang et al., 1998a). Additional evidence for FADDs proliferative role is provided by studies examining mice which express a dominant negative form of FADD; T cells from these mice also have defects in proliferation and calcium mobilization (Hueber et al., 2000; Zornig et al., 1998). The paradoxical effect of cell death pathways on T cell proliferation may be mediated via the cleavage of signalling molecules downstream of the T cell receptor which could modulate the spectrum of pathways activated in response to a given signal.
Materials and methods
Antisera and antibodies
Production of affinity-purified polyclonal Gads antibody has been described previously (Liu et al., 1998). The monoclonal anti-phosphotyrosine antibody 4G10 (Upstate Biotechnology) was used at a dilution of 1 : 1000. Anti-FLAG M2 antibodies (Sigma) were used at a dilution of 1 : 1000 for Western blotting, and 5 g were used in immunoprecipitations. Polyclonal anti-SOS1 and anti-SOS2 antibodies were purchased from Santa Cruz Biotechnology and both were used at a dilution of 1 : 500 for Western blotting. Affinity-purified polyclonal anti-LAT antibody (Upstate Biotechnology), anti-phospholipase C (Upstate Biotechnology) and anti-Grb2 antibody (Transduction Laboratories) were used at a dilutions of 1 : 250, 1 : 1000 and 1 : 500, respectively for Western blotting. Anti-SLP-76 and anti-SLAP-130 polyclonal antisera (a generous gift from G Koretzky) were used at a dilution of 1 : 500 for Western blotting with a donkey anti-sheep-HRP secondary (1 : 25 000). All rabbit polyclonal antibodies were followed with a goat anti-rabbit-HRP from Amersham (1 : 5000) and all monoclonal antibodies were detected with sheep anti-mouse HRP (Amersham).
Plasmids and mutagenesis
Wild type full-length Gads was subcloned, in frame with an amino terminal-FLAG epitope tag, into a modified pEFBOS vector containing a EF-1 promoter, as previously described (Liu et al., 1999). Gads (D235A) was made by substituting aspartic acid residue 235 with an alanine using PCR-based site-directed mutagenesis. Gads (D235A) was subcloned into pcDNA3.1 with an amino-terminal HA-epitope tag and pEF vector with an amino-terminal FLAG epitope tag. The constructs corresponding to the putative Gads caspase cleavage products (Gads-N and Gads-C) were amplified from Gads full-length cDNA and cloned into pEF vector with an amino-terminal FLAG tag.
Jurkat cells were cultured at 37°C with 5% CO2 in RPMI 1640 medium supplemented with 10% (v/v) foetal bovine serum (Wisent), 5 U/ml penicillin C and 5 mg/ml streptomycin sulphate. Primary lymphocytes were isolated and cultured as previously described (Liu et al., 1999).
Apoptosis was induced by culturing cells in media with 15 ng/ml of Fas ligand and 1.5 g ml of a cross-linking monoclonal antibody (Upstate Biotechnology). For the caspase inhibitor experiments, cells were preincubated for 30 min with 50 M AcYVAD-CHO, z-VAD-FMK, or z-DEVD-FMK (dissolved in DMSO), before stimulation with Fas ligand. In a separate experiment, apoptosis was induced by culturing Jurkat cells in the presence of 2 M staurosporine for 3 h or 40 M etoposide for 24 h.
Transient transfection and luciferase assays
For transient transfections 2´107 Jurkat cells were electroporated with 40 g of the indicated plasmids in electroporation buffer (120 mM KCl, 0.5 mM CaCl2, 10 mM K2HPO4, pH 7.4, 25 mM HEPES, pH 7.5, 2 mM EGTA, pH 7.5, 5 mM MgC12) with a Gene Pulser (BioRad) set at 250 V and 960 F. Cells were cultured for 24 h, harvested and processed as described below. For NFAT-luciferase reporter assays, 2´107 Jurkat cells were electroporated with 20 g NFAT luciferase reporter construct, together with 40 g of empty pEF vector or 40 g Gads wt, SLP-76, Gads-N or Gads-C in combinations described for each individual experiment. To control for differences in transfectional efficiency, empty pEF vector was used to be equalize the amount of DNA used in each transfection and equivalent expression was confirmed by Western blot analysis. Twenty-four hours after transfection, 5´105 cells were stimulated (in duplicate) as indicated for 16 h at 37°C and lysed. Luciferase activity was quantified with a luminometer as previously described (Fang et al., 1996) and expressed in arbitrary units relative to cells treated with ionomycin and PMA (maximum stimulation).
Immunoprecipitation and Western blotting
For CD3-stimulation experiments, 20 million Jurkat T cells were resuspended in 500 l of RPMI 1640 medium, prewarmed at 37°C for 10 min, and were then either left unstimulated or were stimulated with human anti-CD3 antibody (UCHT1-Pharmingen) for 2 min at 37°C. Stimulations were halted by addition of ice cold phosphatase inhibitor buffer (PBS pH 7.4, 10 mM Na4P2O7, 100 mM NaF, 1 mM Na3 VO4), cells were collected by centrifugation and lysed in PLC lysis buffer (50 mM HEPES pH 7.5, 150 mM NaC1, 10% glycerol, 1% Triton X-100, 1.5 mM MgC12, 1 mM EDTA, 10 mM Na4,P2O7, 100 mM NaF, 1 mM Na3 VO4) containing protease inhibitors. Lysates were clarified by centrifugation at 14 000 r.p.m. for 10 min at 4°C. Clarified lysates were then incubated for 90 min with protein A-sepharose or protein G-sepharose (Sigma) with appropriate antibody as described above. Beads were then washed five times with 1 ml NP-40 lysis buffer and protein complexes were released by boiling sepharose beads in SDS-Laemmli sample buffer for 5 min. Eluted proteins were resolved by SDS-PAGE and transferred to PVDF membranes for further analysis. All membranes were blocked with Tris-buffered saline containing 1% Tween (TBST) and 5% milk powder, or in the case of phosphotyrosine blotting, TBST containing 1% BSA. Following 1 h of blocking at room temperature, membranes were then incubated for 1 h at room temperature in blocking buffer containing primary antibody as described above. Following incubation with primary antibody, membranes were washed with TBST and incubated for 1 h at room temperature with appropriate secondary antibodies conjugated to horseradish peroxidase. Membranes were then washed thoroughly and antibodies were visualized by enhanced chemiluminescence and autoradiography.
In vitro cleavage assays
For in vitro transcription and translation reactions, 1 g of pcDNA3.1 wild type or D235A Gads was incubated with rabbit reticulocyte lysate and reaction components, including S35 Methionine for 90 min at 30°C according to the manufactuer's instructions (Promega). Fifteen l of each reaction was then immunoprecipitated with affinity-purified anti-Gads antibody for 90 min and then washed 3´ with NP-40 lysis buffer. Immunoprecipitations were then incubated in 50 l of caspase reaction buffer (10 mM HEPES [pH 7.4], 100 mM NaC1, 10 mM DTT, 1 mM EDTA, 0.1% CHAPS) with or without 150 ng of recombinant caspase-3 or caspase-7 (Pharmingen). Protein samples were then separated by SDS-PAGE and exposed to film for 2 h at room temperature.
The authors thank Dr Stan Liu for lively discussions, Dr Gary Koretzky for anti-SLP-76 and anti-SLAP-130 antiserum and Dr Ian Clarke for isolating primary lymphocytes. This work was funded by operating grants from the Cancer Research Society and from the Medical Research Council of Canada to CJ McGlade. SJ Benn is supported by a Natural Science and Engineering Research Council Fellowship. CJ McGlade is a Research Scientist of the National Cancer Institute of Canada supported with funds from the Canadian Cancer Society.
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Figure 1 Induction of apoptosis in T cells results in proteolytic cleavage of Gads and SLP-76 (a) Primary lymphocytes were isolated and cultured for 24 h. Gads was immunoprecipitated and Western blotted with anti-Gads anti-sera. The immunoreactive bands corresponding to Gads and a 26 kDa product are indicated by arrowheads. (b) Gads was immunoprecipitated from lysates of Jurkat T cells stimulated with FasL for the indicated times. The Gads immunoprecipitates were blotted with anti-Gads (upper panel). The arrows indicate the immunoreactive full length Gads and the putative cleavage product. To determine if the Gads associated molecule SLP-76 was also cleaved during apoptosis, the membrane was stripped and blotted with SLP-76 anti-sera (middle panel). The arrows indicate full length SLP-76 and a faster migrating immunoreactive species. Total cell lysates from the same FasL treated samples were blotted with anti-PARP (lower panel). Arrows indicate the full length and cleavage product of PARP. (c) Jurkat T or K562 cells were treated with etoposide or staurosporine to induce cell death. Gads was immunoprecipitated from cell lysates and membranes were blotted with anti-Gads. The arrows indicate the immunoreactive full length Gads and the putative cleavage product
Figure 2 Cleavage of Gads associated molecules following FasL stimulation. Jurkat T cells were treated with FasL for the indicated times. Protein lysates of treated cells were separated by SDS-PAGE and blotted with the indicated anti-sera as described in Materials and methods. Arrowheads indicate the full length protein and putative cleavage product in each panel
Figure 3 The unique linker region of Gads contains a potential caspase cleavage site. (a) Schematic representation of Gads protein indicating the location of the potential caspase 3 cleavage site (DIND235) and the region of Gads recognized by the anti-Gads anti-sera. (b) Jurkat T cells were pretreated with the caspase inhibitors DEVD (caspase 3), VAD (caspase 8) or YVAD (ICE) for 30 min and then stimulated with FasL. Gads and its cleavage product were detected by immunoprecipitation and Western blotting with anti-Gads anti-sera
Figure 4 Mutation of aspartate residue 235 in the Gads linker region inhibits cleavage by caspase 3. (a) 35S-methionine labeled wild type Gads (WT) or a mutant form of Gads in which aspartate residue 235 was changed to alanine (D235A) was produced by in vitro translation and incubated with recombinant caspase 3 or caspase 7. Cleavage reactions were separated by SDS-PAGE and 35S-methionine labeled Gads products were detected by autoradiography. (b) To confirm that D235 is also the caspase cleavage site in vivo, Jurkat T cells were electroporated with Flag epitope tagged wild type (WT) or mutant Gads (D235A) expression plasmids. Following FasL treatment Gads protein was immunoprecipitated using anti-FLAG, separated by SDS-PAGE and membranes were blotted with anti-Gads
Figure 5 Gads caspase cleavage products retain binding specificity. Jurkat T cells were transfected with Flag-tagged constructs corresponding to the N-terminal (CL-N) or C-terminal (CL-C) cleavage products of Gads, or Flag-tagged full-length Gads. Twenty-four hours after transfection, cells were stimulated for 2 min with anti-CD3. Anti-Flag immunoprecipitations were performed on clarified lysates to identify proteins bound to the cleavage products. Western membranes were blotted with anti-LAT (top panel), anti-SLP-76 (middle panel) or anti-phosphotyrosine (bottom panel). The Gads cleavage products retain binding specificity for LAT and SLP-76. The anti-phosphotyrosine blot did not reveal any new proteins co-precipitating with the cleavage products when compared to full length Gads. Arrows indicate LAT (top), SLP-76 (middle), or phospho-SLP-76 or phospho-LAT (bottom panel)
Figure 6 Gads caspase cleavage products inhibit activation of NFAT. (a) Gads cleavage products inhibit the enhanced NFAT activity induced by SLP-76 and Gads expression. Jurkat T cells were transiently transfected with a reporter plasmid containing the luciferase cDNA driven by the NFAT-binding site from the IL2 promoter and either empty pEF-FLAG vector or FLAG-tagged SLP-76, wild-type Gads, and/or Gads cleavage constructs (CL-N or CL-C). The cells were stimulated 24 h after transfection with anti-CD3, anti-CD3+PMA or ionomycin+PMA (maximum stimulation) for 16 h, lysed and luciferase activity was measured with a luminometer. The results (mean of duplicates) are expressed as the percentage of arbitrary luciferase units induced by ionomycin plus PMA treatment (percent of maximum). No difference in the maximum luciferase units induced by ionomycin and PMA was observed between transfectants. (b) Gads cleavage products, when combined, inhibit the enhanced NFAT activation induced by SLP-76 in a dose-dependent manner. Jurkat T cells were electroporated with pEF-FLAG-SLP-76 and increasing amounts of the Gads CL-N and CL-C. The cells were stimulated 24 h after transfection with anti-CD3, anti-CD3+PMA or ionomycin+PMA (maximum stimulation) for 16 h, lysed and luciferase activity was measured with a luminometer. The results (mean of duplicates) are expressed as the percentage of arbitrary luciferase units induced by ionomycin plus PMA treatment (per cent of maximum)
|Received 10 October 2000; revised 15 December 2000; accepted 3 January 2001|
|8 March 2001, Volume 20, Number 10, Pages 1203-1211|
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