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[Cancer Research 59, 1422-1427, April 1, 1999]
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[Cancer Research 59, 1422-1427, April 1, 1999]
© 1999 American Association for Cancer Research

Advances in Brief

Caspase-mediated Degradation of T-Cell Receptor {zeta}-Chain1

Brian R. Gastman, Daniel E. Johnson, Theresa L. Whiteside and Hannah Rabinowich2

Departments of Pathology [T. L. W., H. R.], Pharmacology [D. E. J.], Medicine [D. E. J.], and Otolaryngology [B. R. G., T. L. W.], University of Pittsburgh School of Medicine, and University of Pittsburgh Cancer Institute [D. E. J., T. L. W., H. R.], Pittsburgh, Pennsylvania 15213


   ABSTRACT
Top
 ABSTRACT
Introduction
 Materials and Methods
 Results
 Discussion
 REFERENCES
 
We recently reported an association between loss in T-cell receptor (TcR) {zeta}-chain expression and tumor-induced apoptosis of T lymphocytes. In this study, the possibility that {zeta}-chain serves as a direct substrate for activated caspases was investigated. Here, we report that two DXXD motifs, which are putative recognition sequences for caspase-3-related proteases and are present in the amino acid sequence of the {zeta}-chain, are cleaved in apoptotic Jurkat T lymphocytes. Cleavage of {zeta}-chain in Jurkat cells ligated by agonistic anti-Fas antibody was inhibited in the presence of peptide inhibitors of caspases, including the pan-caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone and N-benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethyl ketone, an inhibitor of caspase-3-like activity. Fas-induced cleavage of {zeta}-chain was also inhibited in Jurkat cells overexpressing the intracellular inhibitors of caspase activity, Bcl-2 or cytokine response-modifier A. In vitro translated {zeta}-chain was cleaved in a similar fashion by recombinant caspase-3 or caspase-7 in a dose-dependent manner. In the presence of N-benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethyl ketone, no cleavage of in vitro translated {zeta}-chain was observed. These results suggest that the loss of TcR {zeta}-chain, previously associated with tumor-induced immune dysfunction and more recently associated with tumor-induced apoptosis of T lymphocytes, is mediated by a direct degradation of the {zeta}-chain by activated caspases. This is the first report of involvement of caspases in degradation of the {zeta} protein.


   Introduction
Top
 ABSTRACT
 Introduction
Materials and Methods
 Results
 Discussion
 REFERENCES
 
Tumor-infiltrating and, to a lesser extent, peripheral T and natural killer lymphocytes from cancer patients have been reported to exhibit decreased expression of TcR3 {zeta} protein (1, 2, 3, 4, 5, 6, 7, 8) . Decreased expression of this signaling protein has been correlated with immune dysfunction in cancer patients, including reduced proliferative responses following antigenic or mitogenic stimulation, reduced cytotoxic effector function, and reduced production of Th1 cytokines (1 , 4 , 9) . In addition, a correlation has been demonstrated between tumor progression and decreased levels of TcR {zeta}-chain expression (2 , 6) . The mechanisms responsible for the decreased expression of {zeta}-chain in T lymphocytes of patients with cancer are unknown. It was reported that splenic macrophages from tumor-bearing mice or normal spleen macrophages activated with zymosan A and lipopolysaccharide were able to induce size-related changes in CD3-{zeta}, -{gamma}, -{delta}, and -{epsilon} by contact-dependent interactions (10) . Another study has demonstrated that hydrogen peroxide secreted by tumor-derived macrophages down-modulated expression of the {zeta}-chain (11) . However, the molecular mechanisms involved in macrophage-mediated loss in expression of {zeta}-chain are unknown. We have recently reported that loss in expression of {zeta}-chain is a common feature in T-cell apoptosis, regardless of the nature of the apoptotic stimuli. Reduction in the levels of expression of {zeta}-chain was observed in T lymphocytes induced to undergo apoptosis by either FasL-expressing ovarian carcinoma cells, recombinant FasL, agonistic anti-Fas Ab, or etoposide (12) . We have also demonstrated that both T-cell apoptosis and the associated loss in expression of {zeta}-chain were inhibited by peptide inhibitors of caspases (12) . These findings suggested that activity of caspases was responsible for the two related phenomena, i.e., T-cell apoptosis and loss in {zeta}-chain expression. However, the nature of the relationship between caspase activity and loss of {zeta}-chain expression has not yet been elucidated. In this study, we investigated the possibility that the {zeta}-chain serves as a substrate for activated caspases. In reviewing the protein sequence of the {zeta}-chain (13) , we identified two DXXD sequences, which may serve as potential cleavage sites for caspase-3-like protease activity (14 , 15) . We present evidence that {zeta}-chain is cleaved at these sites in Fas-ligated Jurkat cells or when coincubated with recombinant caspase-3 or caspase-7. This study is the first to link a molecular mechanism to the loss of {zeta}-chain observed at tumor sites.


   Materials and Methods
Top
 ABSTRACT
 Introduction
 Materials and Methods
Results
 Discussion
 REFERENCES
 
Reagents.
Agonistic anti-Fas Ab (CH-11, IgM) was purchased from Upstate Biotechnology (Lake Placid, NY). NH2 terminus-specific anti-{zeta} mAb (6B10.2) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA), and COOH terminus-specific anti-{zeta} mAb (8D3) was purchased from PharMingen (San Diego, CA). Inhibitors of apoptosis, including Z-VAD-FMK, Z-DEVD-FMK, and a control peptide, Z-FA-FMK, were purchased from Enzyme Systems (Livermore, CA). Recombinant caspase-3 and caspase-7 were purchased from PharMingen. Apoptosis TUNEL kits were purchased from Boehringer Mannheim (Indianapolis, IN), and PhiPhiLux-G2D2 DEVD substrate was obtained from OncoImmunin (College Park, MD).

Cell Lines.
Jurkat T leukemic cell line was obtained from American Type Culture Collection (Manassas, VA). Fas-resistant Jurkat cells were obtained by multiple cycles of treatment of Jurkat cells with agonistic anti-Fas Ab (CH-11; 200 ng/ml) followed by selection for Fas-positive cells by fluorescence-activated cell sorting. To generate stable cell lines expressing epitope-tagged CrmA or Bcl-2 proteins, the CMV/Neo/CrmA-KT3 or CMV/Neo/Bcl-2-KT3 expression constructs, prepared as described previously (16) , were introduced into Jurkat cells by electroporation (250 V, 960 µF). As a control, the CMV/Neo vector alone was electroporated into Jurkat cells. Independent clonal cell lines were isolated by limiting dilution in selection media containing 0.5 mg/ml G418. Expression of the CrmA/KT3 or Bcl-2/KT3 proteins in isolated clonal cell lines was assessed by Western blotting using anti-KT3 mAb (16) .

Assessment of Apoptosis.
To identify fragmented DNA in Jurkat cells, we performed a flow cytometry-based TUNEL assay (Boehringer-Mannheim, Indianapolis, IN; 17 ). The cells (106/sample) were washed in PBS and fixed in 2% paraformaldehyde for 30 min at room temperature. After fixation, the cells were washed twice in PBS containing 0.01% BSA and resuspended in TUNEL reaction mixture containing fluorescein dUTP and terminal deoxynucleotidyl transferase. Control cells were resuspended in TUNEL reaction mixture containing fluorescein dUTP without terminal deoxynucleotidyl transferase. Fluorescein labels incorporated in DNA strand breaks were detected by flow cytometry.

To assess intracellular caspase activity following treatment with agonistic anti-Fas Ab, Jurkat cells (5 x 105) were resuspended in 50 µl of 10 µM PhiPhiLux-G2D2 substrate solution (OncoImmunin) in RPMI 1640 supplemented with 10% FCS. After incubation for 1 h at 37°C avoiding direct light, the sample was diluted with 0.5 ml of ice-cold flow cytometry dilution buffer (OncoImmunin). Flow cytometric analysis was performed within 60 min of the end of the incubation period.

Western Blot Analysis.
Following treatment with agonistic anti-Fas Ab, the cells were lysed in 1% NP40, 20 mM Tris, 137 mM NaCl, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, and 10 µg/ml aprotinin. Proteins were separated by SDS-PAGE using 15% polyacrylamide gels and transferred to polyvinylidene difluoride membranes, as described previously (12) . Following probing with a specific primary Ab and horseradish peroxidase-conjugated secondary Ab, the protein bands were detected by enhanced chemiluminescence (Pierce, Rockford, IL).

In Vitro Translation.
Human CD3-{zeta} cDNA cloned into HindIII-XhoI sites of pCDM8 vector was a generous gift from Dr. Lewis Lanier (DNAX, Palo Alto, CA; Ref. 18 ). Plasmid DNA was translated in the TNT T7 transcription-translation-coupled reticulocyte lysate system (Promega). Each coupled transcription-translation reaction contained 1 µg of plasmid DNA in a final volume of 50 µl in a methionine-free amino acid mixture supplemented with 35S-labeled methionine, according to the manufacturer’s instructions. After incubation at 30°C for 90 min, the reaction products were stored at -70°C.

In Vitro Cleavage Reaction.
In vitro cleavage reactions were performed in a buffer containing 20 mM HEPES (pH 7.4), 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 20% glycerol, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, and 10 µg/ml aprotinin for 4 h at 37°C with 2 µl of 35S-{zeta}-chain, in the presence or absence of recombinant caspases and peptide inhibitors of caspases. Reactions were terminated by the addition of SDS loading buffer and boiling for 5 min. Products of the cleavage reactions were separated by 15% SDS-PAGE, fixed in 20% methanol-10% acetic acid, vacuum-dried, and detected by autoradiography. Alternatively, the reaction products were detected by Western blotting with anti-{zeta} mAb.


   Results
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
Discussion
 REFERENCES
 
Fas-mediated Cleavage of {zeta}-Chain.
To investigate the possibility that {zeta}-chain is a direct substrate of activated caspases in apoptotic T lymphocytes, we induced apoptosis by treatment of Fas-sensitive Jurkat cells with agonistic anti-Fas Ab (200 ng/ml) for 10 or 24 h. As assessed by flow cytometry-based TUNEL, DNA degradation was demonstrated in 50% of the Fas-sensitive cells following 10-h treatment and in 90% of the cells after 24-h coincubation with cross-linking Ab (Fig. 1A)Citation . No DNA degradation was detected in Fas-resistant Jurkat cells treated with the agonistic Ab (Fig. 1A)Citation or in Fas-sensitive cells treated with IgM isotype control (data not shown). Using Jurkat cells pretreated with PhiPhiLux, a cell-permeable, DEVD-containing protease substrate, which emits fluorescence upon cleavage (19) , we demonstrated caspase-3-like activity in 38% of cells treated with agonistic anti-Fas Ab for 12 h (Fig. 1B)Citation . Following treatment with agonistic anti-Fas Ab, Jurkat cells were lysed and tested by immunoblotting for the presence of proteins detected by either NH2 terminus-specific or COOH terminus-specific anti-{zeta} mAb. The amino acid sequences, which include the recognition sites for these mAbs, are illustrated in Fig. 2Citation . In Fas-sensitive Jurkat cells but not in Fas-resistant Jurkat cells, treatment with agonistic anti-Fas Ab for 10 h resulted in cleavage of {zeta}-chain into two fragments (Fig. 3)Citation . One of these fragments was detected by the NH2 terminus-specific anti-{zeta} mAb (Mr 10,000; Fig. 3Citation , left), whereas the other was detected by the COOH terminus-specific mAb (Mr 8,000; Fig. 3Citation , middle). As demonstrated in Fig. 2Citation , the COOH terminus Ab recognition sequence of {zeta}-chain encompasses the DTYD153 cleavage motif. Therefore, the detection of a fragment of {zeta}-chain by COOH terminus-specific anti-{zeta} mAb suggests that, in cells treated by anti-Fas Ab for 10 h, cleavage of the COOH-terminal region may not have proceeded to completion. In Jurkat cells treated with anti-Fas Ab for 24 h, two fragments of the {zeta}-chain were detected by the NH2 terminus-specific Ab, including the Mr 10,000 fragment, detected 10 h after Fas cross-linking, and an additional fragment of Mr 7,000 (Fig. 4)Citation . Cleavage fragments were also detected in normal T cells stimulated by either etoposide or staurosporin.4 As judged by the {zeta}-chain protein sequence (Fig. 2)Citation , the additional Mr 7,000 fragment could have been generated by further cleavage of the Mr 10,000 fragment at either GLLD28 or YLLD36 sequences. These cleavage sequences have not yet been described or matched with any specific caspase.



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  		Fig. 1. Assessment of apoptosis in Fas-ligated Jurkat cells. In A, TUNEL staining was assessed by flow cytometry. Fas-sensitive (left panel) or Fas-resistant (right panel) Jurkat cells were treated with agonistic anti-Fas Ab (CH-11, 200 ng/ml) for 24 h. The cells were then fixed and stained for DNA breaks by TUNEL. Top right (both panels), percentage TUNEL-positive cells. In B, caspase activity was assessed by flow cytometry in Jurkat cells treated with anti-Fas Ab (CH-11, 200 ng/ml) for 12 h at 37°C. The treated cells were collected, washed, and incubated with 10 µM PhiPhiLux-G2D2 substrate for 1 h. Controls indicate baseline fluorescence of substrate-loaded cells in the absence of apoptotic stimulation.

 


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  		Fig. 2. Amino acid sequence of TcR {zeta} protein.

 


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  		Fig. 3. Detection of {zeta}-chain cleavage products by NH2 terminus- or COOH terminus-specific anti-{zeta} mAbs. Fas-sensitive (Fas-S) and Fas-resistant (Fas-R) Jurkat cells were coincubated in the presence of agonistic anti-Fas Ab (CH-11, 200 ng/ml, 10 h). At the end of the incubation period, the cells were lysed and analyzed by immunoblotting with NH2 terminus or COOH terminus anti-{zeta} mAbs.

 


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  		Fig. 4. Kinetics of {zeta}-chain cleavage in Jurkat cells treated with agonistic anti-Fas Ab. Cleavage products were detected by Western blotting with NH2 terminus-specific anti-{zeta}-chain mAb. Whereas one cleavage product was detected following a 10-h incubation with anti-Fas Ab, two fragments were observed after 24 h.

 
To confirm the involvement of caspases in {zeta}-chain cleavage in Fas-ligated T cells, treatment with anti-Fas Ab was performed in the presence of peptide inhibitors of caspases. The presence of Z-VAD-FMK or Z-DEVD-FMK but not control peptide Z-FA-FMK during 24 h incubation of Jurkat cells with agonistic anti-Fas Ab inhibited {zeta}-chain cleavage (Fig. 5)Citation . No cleavage products were detected by the COOH terminus-specific Ab in Jurkat cells treated for 24 h with anti-Fas Ab in the presence of the control peptide (Fig. 5)Citation . These results suggest that, under the apoptotic conditions generated, the COOH terminus Ab recognition site was modified by a complete cleavage at the DTYD153 site.



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  		Fig. 5. Inhibition of Fas-mediated cleavage of {zeta}-chain in Jurkat cells treated with peptide inhibitors of caspases. Jurkat cells were treated with Z-VAD-FMK, Z-DEVD-FMK, or control Z-FA-FMK (50 µM) for 2 h prior to the addition of anti-Fas Ab (CH-11, 200 ng/ml). Following 24 h anti-Fas treatment, the cells were lysed and analyzed by immunoblotting with NH2 terminus-specific anti-{zeta} mAb. The filters were stripped and reprobed with COOH terminus-specific anti-{zeta}-chain mAb. The cleavage products did not contain COOH terminus Ab recognition site because only full-length {zeta}-chain was detected upon stripping and reprobing with COOH terminus-specific mAb.

 
The effects of intracellular antiapoptotic molecules on Fas-mediated {zeta}-chain cleavage was investigated in Jurkat cells overexpressing Bcl-2 or CrmA. We have recently evaluated the protective effects of Bcl-2 and CrmA on Fas-induced activation of caspase-3. Although Fas ligation of Neo-Jurkat cells resulted in full processing of pro-caspase-3 into active subunits, partial inhibition of caspase-3 processing was observed in Bcl-2-Jurkat cells and complete inhibition was observed in CrmA-Jurkat cells.5 CrmA is a potent inhibitor of caspase-1 and caspase-8 and its inhibitory effect on caspase-3 activation is mainly indirect, mediated via inhibition of caspase-8 function, upstream of caspase-3 (14 , 20) . Protective effects of CrmA or Bcl-2 against {zeta}-chain cleavage were similar to those observed by us previously5 against Fas-induced activation of caspase-3. Thus, as shown in Fig. 6Citation , cleavage of {zeta}-chain, as assessed by NH2 terminus-specific anti-{zeta} mAb, was partially inhibited in Bcl-2-Jurkat cells and completely inhibited in CrmA Jurkat. These findings further suggest the involvement of caspase-3-like activity in {zeta}-chain cleavage.



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  		Fig. 6. Inhibition of Fas-mediated {zeta}-chain cleavage in Jurkat cells transfected with CrmA or Bcl-2. Neo-, CrmA-, or Bcl-2-transfected Jurkat cells were treated with agonistic anti-Fas Ab for 24 h. At the end of the incubation period, the cells were lysed and analyzed by immunoblotting with NH2 terminus-specific anti-{zeta}-chain mAb.

 
Cleavage of {zeta}-Chain by Recombinant Caspases.
To identify the caspases involved in {zeta}-chain cleavage, we prepared in vitro translated 35S-{zeta}-chain and incubated it with various concentrations of recombinant caspase-3, caspase-7, or caspase-6. The in vitro translated product was resolved by SDS-PAGE and detected as an Mr 18,000 protein by Western blotting with either NH2 terminus- or COOH terminus-specific anti-{zeta} mAb or by autoradiography (Figs. 7Citation and 8)Citation . In vitro translated 35S-{zeta}-chain incubated with increasing doses of recombinant caspase-3 (0–3 µg) was cleaved into at least two fragments, of which the Mr 10,000, which does not contain any methionine residue, was recognized by the NH2 terminus-specific anti-{zeta}-chain mAb (Fig. 7Citation , top). The COOH terminus-specific anti-{zeta} mAb detected only uncleaved {zeta}-chain, suggesting that recombinant caspase-3 cleaves the DYTD153 site, precluding the recognition of the COOH terminus {zeta}-chain fragments. However, the use of either NH2- or COOH terminus-specific anti-{zeta} mAb demonstrated a caspase dose-dependent loss of the Mr 18,000 {zeta}-chain. As illustrated in Fig. 2Citation , all methionine residues in the {zeta}-chain sequence are located within the COOH terminus fragments, which, thereby, could be detected by autoradiography. Thus, autoradiography was used to examine 35S-{zeta}-chain cleavage in the presence of increasing doses of recombinant caspase-3 or recombinant caspase-7 and the presence or absence of Z-DEVD-FMK. The 35S-radioactive signal of the dried SDS gels demonstrated a caspase-3 and caspase-7 dose-dependent generation of a Mr 7,000 fragment of the in vitro translated 35S-{zeta}-chain (Fig. 8, A and B)Citation . In addition, a caspase dose-dependent loss of the Mr 18,000 {zeta}-chain was observed. The presence of Z-DEVD-FMK during incubation with either caspase-3 or caspase-7, inhibited the cleavage of the 35S-{zeta}-chain (Fig. 8, A and B)Citation . As a control, the activity of recombinant caspase-6 and its specific inhibitor, Z-VEID-FMK, on cleavage of 35S-{zeta}-chain was examined. No cleavage of 35S-{zeta}-chain by caspase-6 was detected even in the absence of Z-VEID-FMK (Fig, 8C)Citation . Taken together, our results suggest that both caspase-3 and caspase-7 are capable of cleaving the {zeta}-chain at DXXD sites.



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  		Fig. 7. Cleavage of in vitro translated {zeta}-chain by recombinant caspase-3. In vitro translated 35S-{zeta}-chain was incubated with recombinant caspase-3 (0–3 µg) for 4 h. The reaction products were resolved by SDS-PAGE, transferred to PVDF membrane, and detected by blotting with NH2 terminus-specific anti-{zeta} mAb (top), or COOH terminus-specific anti-{zeta} mAb (bottom). Caspase-3 dose-dependent loss of the Mr 18,000 full-length {zeta}-chain was detected in both panels. A fragment of {zeta}-chain was detected in the presence of recombinant caspase-3 by NH2 terminus-specific anti-{zeta} mAb.

 


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  		Fig. 8. Inhibition by Z-DEVD-FMK of cleavage of 35S-{zeta}-chain by recombinant caspase-3 or caspase-7. In A and B, in vitro translated 35S-{zeta}-chain was treated for 4 h with recombinant caspase-3 or recombinant caspase-7 (0–3 µg), respectively, in the presence or absence of Z-DEVD-FMK (50 µM). In C, in vitro translated 35S-{zeta}-chain was treated with recombinant caspase-6 (0–3 µg), in the presence or absence of Z-VEID-FMK (50 µM). The reaction products were resolved by SDS-PAGE and detected by autoradiography. The [35S]methionine-containing Mr 7000 fragment was detected in the presence of 1 or 3 µg caspase-3 or caspase-7 in the absence of Z-DEVD-FMK. (In A, the weak band in the empty space between Lanes 3 and 4 represents an overflow from Lane 4.)

 

   Discussion
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
REFERENCES
 
Here, we demonstrated that TcR-{zeta} chain is cleaved by a caspase protease activity in cells undergoing apoptosis. The involvement of caspase-3-like activity in {zeta}-chain degradation was indicated by inhibition of {zeta}-chain cleavage by Z-DEVD-FMK, an inhibitor of caspase-3-like proteases. Fas-induced cleavage of {zeta}-chain was also inhibited in CrmA-overexpressing Jurkat cells, and to a lesser extent, in Bcl-2-Jurkat cells. Caspase-3 and caspase-7 were identified as the specific members of the caspase family involved in {zeta}-chain cleavage using a cell-free system composed of recombinant caspases and in vitro translated 35S-{zeta}-chain. This study demonstrates for the first time that caspases are involved in altered expression of TcR {zeta} subunit.

The TcR is a protein complex receptor composed of the CD3 {gamma}-, {delta}-, {epsilon}-, and {zeta}-chains. The {zeta}-chain is a 163-amino acid protein that contains a short extracellular domain, a single transmembrane domain (amino acids 31–51; Refs. 13 ), and a long cytoplasmic domain. The CD3 {gamma}-, {delta}-, {epsilon}-, and {zeta}-chains contain signaling motifs called ITAMs with the consensus sequence TXX(L/I)X6-8YXX(L/I) (21) . The {gamma}-, {delta}-, and {epsilon}-chains each contain one ITAM, which upon phosphorylation is sufficient to transduce signals from the TcR (21) . In contrast, the {zeta}-chain contains three ITAMs located between amino acids 72–86, 110–125, and 141–155. The multiple ITAMs are thought to amplify signals from the TcR. Recently, it has been reported that the {zeta}-chain undergoes a series of ordered phosphorylation events upon TcR engagement (22) . The level of {zeta}-chain phosphorylation is dependent on the nature of the T-cell ligand, and therefore, phosphorylation steps may establish thresholds for T-cell activation (22) .

Altered expression of {zeta}-chain in lymphocytes from cancer patients as well as in autoimmune or infectious diseases has been demonstrated by several groups (3 , 5 , 23, 24, 25) . In contrast to what is seen in human patients, evidence for reduced expression of {zeta}-chain in murine experimental models of tumor growth has been conflicting (26) . However, to date, no molecular mechanism for the loss in expression of this critical signaling component of TcR has been elucidated. Our recent ex vivo studies demonstrated that coincubation of ovarian carcinoma cells with activated lymphocytes induced a substantial decrease in {zeta}-chain expression (12) . The observed loss in {zeta}-chain expression was linked to the process of apoptosis because it was partially inhibited by neutralizing Ab to Fas or FasL,5 Fas-Fc fusion protein, or peptide inhibitors of apoptosis (12) . These in vitro results strongly suggested that the loss in {zeta}-chain expression was related to apoptotic proteolytic pathways in the immune cells. However, the exact proteolytic mechanism of {zeta}-chain degradation has not been resolved. Activation-induced proteolysis of the {zeta}-chain was observed following CD3 cross-linking of T-cell hybridoma (27) , stimulation of T-cell clones by presentation of specific peptide on antigen-presenting cells (28) , and mitogenic activation of human peripheral blood T cells by concanavalin A, phytohemagglutinin, or phorbol 12-myristate 13-acetate (29) . Although, each of these means of T-cell activation potentially could have resulted in activation-induced cell death of the stimulated lymphocytes (30) , {zeta}-chain was reported to undergo either ubiquitination (27) or lysosomal degradation (28) in response to TcR engagement. Because it is well established that ubiquitinated proteins are not only recognized by the proteasomal complex but may also be subjected to lysosomal degradation (31 , 32) , there is no apparent contradiction between the two reported mechanisms of {zeta}-chain degradation. However, the relationship between the previously reported ubiquitination of {zeta}-chain and the present findings of caspase-mediated degradation of {zeta}-chain is unclear. As ubiquitination is a mechanism for tagging damaged proteins for degradation by either lysosomes or proteasomes, it may be a manifestation of the proteolytic activity taking place subsequent to {zeta}-chain cleavage by caspases.

Although analysis of nucleotide sequence demonstrates high degree of identity (63%) between the human and mouse {zeta}-chain cDNA, it is interesting to note that, in mouse {zeta}-chain, the aspartic acid residue at D87 site is replaced by glutamine (13) . This difference in a single amino acid might have contributed to the conflicting observations of altered {zeta}-chain expression reported for human and mouse (26) . Due to this Asp-to-Glu change, the mouse {zeta}-chain might demonstrate a reduced susceptibility to caspase-mediated degradation.

Several signaling proteins have been reported to be cleaved by caspases in apoptotic cells (33) . In particular, the activity of certain kinases is regulated by caspase cleavage. Caspase-dependent cleavage of enzymes, can either inactivate or activate the substrate proteins. Examples of inactivation include poly(ADP-ribose) polymerase, DNA protein kinase catalytic subunit, and focal adhesion kinase (34, 35, 36) . Cleavage of poly(ADP-ribose) polymerase or DNA protein kinase catalytic subunit abrogates their ability to participate in DNA repair, and cleavage of focal adhesion kinase impairs the ability of cells to maintain matrix adherence. In contrast, certain enzymes are activated when cleaved by caspases. Examples of such substrates include mitogen-activated protein kinase kinase kinase 1 (37) , p21-activated kinase (38) , protein kinase C{delta} (39) , protein kinase C{theta} (40) , and protein kinase C-related kinase-2 (41) . When cleaved, these enzymes produce constitutively active kinases which promote apoptosis. Other caspase cleavage products are also reported to enhance apoptosis. In particular, antiapoptotic Bcl-2 protein, is cleaved by caspase-3 to a product containing the BH3 and the transmembrane domains. This cleaved Bcl-2 fragment provides a positive feedback to the cascade of caspase activation (42) . In view of the role of caspase products in the apoptotic process, it remains to be determined whether caspase-mediated cleavage of {zeta}-chain is a cause or consequence of T-cell death. Thus, future studies should determine whether the cleaved fragments of {zeta}-chain play a role in the apoptotic process.

Certain caspases have been shown to function outside of apoptosis. In particular, ICE/caspase-1, the IL-1-converting enzyme, is responsible for the processing of precursor IL-1ß into its mature form (43) . Additional proinflammatory cytokines, including IL-16 and IL-18, were recently reported to be processed and activated by caspases (44 , 45) . Although pro-IL-18 is activated by caspase-1 and inactivated by caspase-3 (44) , cleavage of the IL-16 COOH-terminal region by caspase-3 releases bioactive IL-16 (45) . The activity of caspase-3-like enzymes in nonapoptotic T cells has been suggested (46) , but remains controversial (47) . Caspase-mediated processing of an element of the DNA-replication complex (RFC-140; Ref. 47 ) and of Rb (48) might suggest a role for caspases in control of the cell cycle. Cell survival despite the presence of active caspases in the cytoplasm has been reported for mitogenic stimulated T cells (46) as well as for terminally differentiated cells, such as eunucleated lens cells (49) . In light of the possibility that caspases may function outside apoptosis, studies should also be conducted to determine whether {zeta}-chain degradation might represent a function of caspases in activated, nonapoptotic T lymphocytes. Furthermore, cleavage of {zeta}-chain at the DVLD87 site appears to generate a fragment, which includes the transmembrane domain (amino acids 31–51) and one intact ITAM motif (amino acids 72–86). Therefore, it is also important to determine whether caspase-cleaved fragments of the {zeta}-chain have any signaling capability.

Although it is currently unclear whether all T cells demonstrating reduced expression of {zeta}-chain are apoptotic, our findings strongly suggest that the activation of intracellular caspases plays a role in {zeta}-chain degradation. Caspase activity may represent a molecular mechanism for altered expression of {zeta}-chain in patients with cancer or infectious diseases, in whom chronic activation or other apoptotic stimuli may induce the activities of these enzymes.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 The work was supported by The Pittsburgh Foundation (to H. R.), The Wendy Will Case Cancer Fund, Inc. (to H. R.), American Cancer Association Grant RPG-98-288-01-CIM (to H. R.), NIH Grant R29CA66044 (to D. E. J.), and National Cancer Institute Grant PO1DE 12321-01 (to T. L. W. and H. R.). Back

2 To whom requests for reprints should be addressed, at University of Pittsburgh Cancer Institute, W952 Biomedical Science Tower, 200 Lothrop Street, Pittsburgh, PA 15213. Phone: (412) 624-0289; Fax: (412) 624-7737; E-mail: rabinow+{at}pitt.edu Back

3 The abbreviations used are: TcR, T-cell receptor; FasL, Fas ligand; Ab, antibody; mAb, monoclonal Ab; Z-VAD-FMK, Z-Val-Ala-Asp-fluoromethyl ketone; Z-DEVD-FMK, Z-Asp-Glu-Val-Asp-fluoromethyl ketone; Z-FA-FMK, Z-Phe-Ala-fluoromethyl ketone; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling; CrmA, cytokine response-modifier A; CMV, cytomegalovirus; ITAM, immune receptor tyrosine-based activation motif; Z-VEID-FMK, Z-Val-Glu-Ile-Asp-fluoromethyl ketone; IL, interleukin; Z, N-benzyloxycarbonyl. Back

4 Unpublished data. Back

5 B. R. Gastman, D. E. Johnson, T. L. Whiteside, and H. Rabinowich. Activation of caspase-3 and cleavage of Bcl-2 in tumor-induced apoptosis of T lymphocytes, submitted for publication. Back

Received 11/ 4/98. Accepted 2/15/99.


   REFERENCES
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
 REFERENCES
 

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