Qualitatively differential regulation of T cell activation and apoptosis by T cell receptor {zeta} chain ITAMs and their tyrosine residues

Wook-Jin Chae1, Heung-Kyu Lee1, Jin-Hwan Han1, Sang-Won Vincent Kim1,2, Alfred L.M. Bothwell2, Tomohiro Morio3 and Sang-Kyou Lee1

1 Department of Biotechnology, Yonsei University, Seodaemun-Gu Shinchon-Dong 134 120-749, Republic of Korea
2 Section of Immunobiology, Yale University, School of Medicine, New Haven, CT 06520, USA
3 Department of Pediatrics, Tokyo Medical and Dental University, School of Medicine, Tokyo 113-8519, Japan

Correspondence to: S.-K. Lee; E-mail: sjrlee{at}yonsei.ac.kr


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Supplementary data
 References
 
The issue of whether three ITAMs in the TCR {zeta} chain can transmit qualitatively distinct signals or redundantly amplify TCR-mediated activation signals was extensively investigated using stable hCD8-{zeta} Jurkat transfectants which contain stepwise deletions of each ITAM or mutations of tyrosine residues in each ITAM of TCR {zeta} chain. The influence of mutations of each tyrosine residue on reduction of the amount and species of tyrosine phosphorylated proteins recruited to {zeta} chain was quite distinctive, but they were roughly proportional to the number of functionally intact ITAMs. However, the first N-terminal ITAM had a signaling potential to trigger most intracellular signaling events for T cell activation and apoptosis similar to wild-type CD8-{zeta}, but this level was substantially reduced in the presence of the first and second N-terminal ITAM together. Mutations of tyrosine residues in first and second N-terminal ITAM significantly impaired most signaling events leading to T cell activation and activation-induced cell death, but phosphorylation of mitogen-activated protein kinases (MAPKs) was differentially impaired in each mutant. The mutation of the first tyrosine residue in C-terminal ITAM did not show any impairment in induction of surface antigens and cell death, but rather increased IL-2 secretion and MAPK phosphorylation. Therefore, in this study we demonstrated that the ITAMs and their tyrosine residues of TCR {zeta} chain can transmit qualitatively differential intracellular signals upon TCR stimulation through distinctive regulation of recruitment of tyrosine phosphorylated proteins to {zeta} chain and activation of various MAPKs.

Keywords: apoptosis, cellular activation, signal transduction, T cell receptors


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Supplementary data
 References
 
T cells control the diverse immune responses through their highly organized antigen recognizing T cell receptor (TCR) complexes. The TCR complex is composed of 10 signal transducing subunits termed the immunoreceptor tyrosine-based activation motif (ITAM) whose consensus amino acid sequence is D/ExxYxxL/Ix 6–8 YxxL/I (1,2). A dimeric form of the TCR {zeta} chain containing six ITAMs can trigger intracellular signals leading to proliferation, induction of cytolytic activity and cytokine production (3,4). Following TCR stimulation, phosphorylation of tyrosine residues in three ITAMs of the TCR {zeta} chain plays a crucial role in initiating diverse TCR-mediated signaling events. This signaling process is coupled with activation of Src-family PTKs, Lck and Fyn, and subsequent sequential activation of SLP-76 and Vav (57) coupled with the activation and phosphorylation of mitogen-activated protein kinase (MAPK) family members such as extracellular signal regulated kinase (ERK) and c-jun N-terminal kinase (JNK) (8,9). Among the outcomes of T cell signaling events, the upregulated expression of IL-2 and FasL is one of the essential events. IL-2 secretion is the hallmark of activated T cells and critical for proliferation, clonal expansion and differentiation. It is also involved in activation induced cell death (AICD) by suppressing FLICE-like inhibitory protein (FLIP) and enhancing Fas-mediated apoptosis. Upregulation of FasL triggers caspase-mediated cell death with the loss of mitochondrial membrane potential, thereby leading to AICD for maintaining immunological homeostasis (10,11).

There have been a number of studies in which the functional importance of tyrosine residues in each ITAM of TCR {zeta} chain was analyzed in in vivo and in vitro model systems (1012). In most studies using CD3 {zeta} –/–TCR transgenic mice reconstituted with various mutant forms of CD3 {zeta} chain, the different ITAMs of CD3 {zeta} ITAMs function quantitatively during T cell development, thereby being involved in amplification of signals from external stimuli and selection of T cell repertoire. However, selection of T cells with a specific TCR is altered and autoreactive T cells can be found in these mice. The substantial defects in the absence of the TCR {zeta} chain resulted in substantial defects in TCR-mediated responses, clearly indicating that specific signals through TCR {zeta} chain play an important role in shaping the TCR repertoire and regulation of immune reactions, and that each of the ITAMs in the TCR complex serves distinctive signaling functions for T cell activation (1315).

The three ITAMs denoted as A, B, C (containing two tyrosines in each ITAM denoted as A1, A2, B1, B2, C1, C2) in the TCR {zeta} chain could yield more than 60 different phospho-species upon TCR stimulation. Two major tyrosine-phosphorylated forms of TCR {zeta} chain, p21 and p23, have been consistently detected in resting and activated T cells, respectively. The progressive activation model hypothesizes that ordered {zeta} chain phosphorylation is dependent on the nature of the TCR ligand and that each of the intermediate phospho-species of the {zeta} chain accumulated in response to antagonistic ligands may confer the ability to trigger distinct intracellular signals to TCR complexes (16). Based on this hypothesis, TCR antagonism was a consequence of an inhibitory signal generated by the constitutively phosphorylated form of TCR {zeta} chain, p21 in resting T cells. In contrast to this hypothesis, the identity of these two forms of TCR {zeta} chain revealed that p21 is generated by phosphorylation of tyrosine residues in the second and third ITAM (B1, B2, C1 and C2), whereas p23 is formed by additional phosphorylation of tyrosines in the first ITAM (17). This model proposed that TCR {zeta} undergoes a stepwise phosphorylation that may be initiated at the carboxy-terminal ITAM.

In determining whether individual CD3 {zeta} ITAMs exert unique or redundant functions, somewhat disparate results have been obtained depending upon activation conditions, or the use of T cells from different origins or different developmental circumstances. Examination of the functional influences of the mutations of tyrosine residues of TCR {zeta} chain ITAM on modulation of intracellular signaling have been restricted to several representative signaling events such as IL-2 secretion, apoptosis induction and protein tyrosine phosphorylation. Therefore, it is necessary to determine the functional contribution of each tyrosine residue in the TCR {zeta} chain ITAMs to regulate diverse and independent intracellular signaling events.

To resolve the above issue of whether each tyrosine residue and ITAMs of TCR {zeta} chain have a signaling potential with qualitative specificity or quantitative redundancy we utilized a set of engineered point mutations and deletions for CD3 {zeta} ITAMs which is composed of extracellular and transmembrane region of human CD8{alpha}. The influence of mutations in the cytoplasmic domain of TCR {zeta} chain on activation of ZAP-70, MAPKs (ERK-1/2 and SAPK/JNK) and their activation kinetics, the inducible expression of CD69 and CD25 on the cell surface, the amount of IL-2, sFasL production, apoptosis induction and mitochondrial membrane potential depolarization was analyzed in the absence or presence of CD28 costimulation.

We provide evidence that the three ITAMs of CD3{zeta} and their tyrosine residues induce qualitatively distinctive signals for T cell activation and death events via the differential phosphorylation of CD3{zeta} and its associated proteins and MAPKs.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Supplementary data
 References
 
CD8-{zeta} chimera construction, mutagenesis and transfection
The vector encoding the CD8-{zeta} chimeric molecule (ptfß-neo CD8-{zeta}) containing the extracellular and transmembrane domain of human CD8{alpha} and the intracellular portion of human CD3-{zeta} was generously provided by Dr A. Weiss (UCSF, San Francisco, CA). The CD8-{zeta} chimeric DNA was subcloned into pcDNA3 (–) Myc-His A (Invitrogen, Carlsbad, CA). The tyrosine (Y) residues were replaced by phenylalanine (F) at the indicated residue of the TCR {zeta} molecule by using the Quik Change Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA). The double point mutations for Y51F-Y62F (A1A2), Y89F-Y101F (B1B2) and Y120F-Y131F (C1C2) were made from Y71F (A1), Y89F (B1) and Y120F(C1) point mutant plasmids as a template, respectively. The primers for these mutants are as follows: for Y51F (A1): sense 5'-gaaccagctctttaacgagc-3', antisense 5'-tgagctcgttaaagagctgg-3', for Y62F(A2): sense 5'-gagaggagttcgatgttttgg-3', antisense 3'-ctctcctcaagctacaaaacc-5', for Y89F(B1): sense 5'-aggcctgttcaatgaactgc-3', antisense 5'-tgcagttcattgaacaggcc-3', for Y101F (B2): sense 5'-cggaggccttcagtgagattg-3', antisense 3'-gcctccggaagtcactctaac-5', for Y120F (C1): sense 5'-cgagttccttttccagggtctc-3', antisense 5'-gagacccgttaaaaggccatcg-3', for Y131F (C2): sense 5'-ccaccaagcacaccttcgacgcccttc-3', antisense 5'-gaagggcgtcgaaggtgtccttggtgg-3'. The truncated forms of CD3-{zeta} were generated using ptfß-neo CD8-{zeta} as a template and cloned into the EcoRI and BglII site of pcDNA3.1 (–) Myc-His A expression vector, which contains the external and transmembrane domain of human CD8. The primers used for the first ITAM were: 5'-gaaccagctctttaacgagc-3' and 5'-tggggggaaagccgagaagg-3' and those for the first and the second ITAM were: 5'-cgaagaagatctttcctgagagtgaagttcagc-3' and 5'-ccgtcggaattcttaccccatctcagggtcccggcc-3'. Nucleotide sequences of all mutated or truncated vectors were confirmed. To generate stable Jurkat transfectants, 40 µg of wild-type CD8-{zeta} or its mutant forms were transfected into human leukemia Jurkat T cells by electroporation (250 V/960 µF). Stable transfectants were selected four times independently in the presence of 2.2 mg/ml of G418 (Invitrogen, Carlsbard, CA) for 2 weeks. Four independent clones were selected and the expression level of CD8-{zeta} chimera on one representative clone was measured by flow cytometry with FITC-conjugated anti-human CD8 antibody.

Cells and antibodies
Human T cell leukemia cell line Jurkat (clone E6.1) was obtained from American Type Culture Collection (ATCC, Manassas, VA). Cells were cultured in RPMI 1640 with 2 mM L-glutamine, 1% (v/v) penicillin/streptomycin and 10% (v/v) heat-inactivated fetal bovine serum (Invitrogen, Carlsbad, CA) and maintained at 37°C in a humidified incubator containing 5% CO2. The anti-ZAP-70 antibody and anti-phosphoZAP-70 Tyr319, anti-phospho ERK-1/2, anti-phospho SAPK/JNK, anti-phospho p38, anti-phospho I{kappa}B alpha and anti-p38 antibodies were purchased from Cell Signaling Technology (Beverly, MA). Anti-TCR {zeta} antibody was obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). OKT3 or OKT8 mAb was purified from culture supernatant from OKT3 and OKT8 hybridoma (American Type Culture Collection) by affinity purification. FITC-conjugated anti-human CD25, PE-conjugated anti-human CD69, FITC-conjugated anti-human CD8 and anti-human CD28 and isotype control antibodies for each antibody were purchased from BD Pharmingen (San Diego, CA). Horseradish peroxidase conjugated anti-mouse and anti-rabbit IgG antibodies were purchased from Sigma (St Louis, MO).

Cell stimulation and lysis, immunoprecipitation and immunoblotting
Cells were harvested, washed twice and resuspended in medium at a density of 1 x 107 cells/ml. An equal volume of medium containing 5 µg/ml of OKT8, anti-CD28 antibody and goat anti-mouse IgG was added to cells and cells were incubated for 15 min at 4°C. Stimulation was performed for indicated time points at 37°C and cells were then lysed with ice-cold lysis buffer (1% Triton X-100, 150 mM NaCl, 10 mM Tris–HCl pH 8.0, 400 µM EDTA, 1 mM Na3VO4, 10 mM NaF, 1 mM PMSF, 10 µg/ml aprotinin and 10 µg/ml leupeptin). After 30 min incubation on ice, postnuclear lysates were centrifuged at 4°C for 30 min. For detecting TCR {zeta} chain-associated proteins, cell lysates were incubated with 2 µg/ml OKT8 mAb for 1 h followed by incubation with protein G–agarose (Gibco/BRL, Gaithersburg, MD) for 2 h at 4°C. After washing three times with the lysis buffer, immunoprecipitates were boiled with 2x SDS–PAGE sample buffer. To detect the phosphorylated ZAP70 upon OKT8 and anti-CD28 antibody costimulation, lysates were incubated with anti-phosphotyrosine antibody-conjugated agarose beads (Upstate Biotechnology, Lake Placid, NY) for 2 h at 4°C. Immunoprecipitates were washed three times with the lysis buffer, and whole-cell lysates or immunoprecipitates were resolved by 10% SDS–PAGE and transferred to polyvinylidene difluoride (PVDF) membranes according to the manufacturer's instructions (Millipore, Bedford, MA). The blots were visualized by using the ECL system (PharmaciaBiotech, Buckinghamshire, UK).

Measurement of cell death induction and mitochondrial membrane depolarization
5 x 105 cells were stimulated with 1 µg/ml OKT8 and 1 µg/ml CD28 antibodies in a 24-well plate which was coated with 2 µg/ml of rabbit affinity purified mouse IgG 1 day before cell stimulation. After 48 h incubation, the cell death induction was analyzed by annexin-V/propidium iodide (BD Pharmingen) staining.

For detection of mitochondrial membrane potential depolarization, a lipophilic cation JC-1 was used (18). Cells were stimulated for 48 h as above, washed three times with PBS, and incubated in the presence of JC-1 (Molecular Probes, Eugene, OR) at a concentration of 10 µg/ml for 10 min at room temperature. After three washes with PBS, cells were analyzed by flow cytometry.

Flow cytometry
Cells were stimulated using OKT8 and anti-CD28 mAb as described above. After 16 h of stimulation, cells were washed three times with ice cold PBS, and resuspended in FACS staining buffer (PBS + 0.5% BSA) containing PE-conjugated anti-CD69 antibody. After 20 min of incubation on ice, cells were washed three times with FACS staining buffer and the level of early activation-induced surface marker CD69 was determined using a FACSCalibur flow cytometer (Becton Dickinson). For detection of CD25 expression on the surface, cells were stimulated for 48 h and analyzed by a FACScan flow cytometer using FITC-conjugated antihuman CD25 antibody. Flow cytometric data were collected in a logarithmic mode on light scatter-gated live cells using CellQuest software (Becton Dickinson).

Measurement of IL-2 and sFasL secretion
1 x 105 cells were stimulated with 0.5 µg/ml OKT8 and 0.5 µg/ml anti-CD28 antibody or 0.5 µg/ml OKT8 alone in 96-well plates and cross-linking was performed using 1 µg/ml of rabbit affinity purified mouse IgG. For measuring secreted IL-2 and soluble Fas ligand, supernatants were harvested and assayed according to the manufacturer's protocol (BD Pharmingen and Oncogene, Boston, MA, respectively) and the absorbance was measured at 450 nm.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Supplementary data
 References
 
Generation of stable human Jurkat CD8-{zeta} transfectant and mutants
To investigate the specificity of each ITAM on TCR-mediated downstream signaling events, we designed TCR {zeta} chain ITAM mutants as chimeric proteins with human CD8. These chimeric molecules can be utilized to quantitate expression and activate cells via antibody-mediated crosslinking. The constructs with stepwise deletions from the C-terminal ITAM were designated as CD8-{zeta} AB or CD8-{zeta} A, respectively, and those with substitution of one or two tyrosine residues in each ITAM with phenylalanine as A1, A1A2, B1, B1B2, C1 and C1C2 (Fig. 1A). These mutant constructs were introduced into Jurkat T cells and stable transfectants for each construct were identified by flow cytometry (Fig. 1B). Stable transfectants from four independent transfections were chosen which showed similar levels of chimeric protein expression. CD8-T transfectants expressing CD8{alpha} lacking the cytoplasmic tail of CD3{zeta} chain were also generated and used as a control in all of our experiments. All tested transfectants retained the ability to produce IL-2 in response to the OKT3, confirming that signaling pathways through their endogenous TCR was intact after the transfection procedure. The expression of TCR, Fas and CD28 on the cell surface of these transfectants was equivalent to that of parental Jurkat T cells (data not shown). As previously shown, stimulation of wild-type CD8-{zeta} with anti-CD8 antibody OKT8 induced ZAP-70 phosphorylation and downstream activation signaling events whereas activation was not observed in CD8-T (data not shown) (4). One representative transfectant for each chimeric construct was used in all of our experiments and the results were experimentally confirmed in another transfectant.



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Fig. 1. Structure of human CD8-{zeta} chimeras with deletion or point mutations in the cytoplasmic domain of TCR {zeta}chain, and their stable expression on Jurkat T cells. (A) Structure of human CD8-{zeta} chimeras with deletion or point mutations. Deletion mutants CD8-{zeta}A and CD8-{zeta}AB contain amino acids of 31–76 and 31–110 of the human CD3 {zeta} chain, respectively. Closed circles indicate the mutated tyrosine residue(s). EC, extracellular domain; TM, transmembrane domain. CD8-T contains the extracellular and transmembrane domain of human CD8{alpha} only. (B) Stable surface expression of human CD8-{zeta} or its mutants on Jurkat T cells.

 
Differential binding of tyrosine phosphorylated protein complex to mutant forms of TCR {zeta} chain
The recruitment of phosphorylated proteins to TCR {zeta} chain is known to be a prominent marker for early activation events in TCR-mediated signaling. To examine the level of phosphorylation induction of TCR {zeta} chain-binding proteins upon TCR stimulation, we immunoprecipitated proteins bound to the TCR {zeta} chain and tyrosine phosphorylated proteins were detected by phosphotyrosine immunoblot (Fig. 2A). As expected, wild-type CD8-{zeta} became bound with many phosphorylated proteins when CD8-{zeta} chain was crosslinked. Interestingly, the quantity and quality of phosphorylated proteins bound to the {zeta} chain were altered as the number of ITAMs was reduced in the CD8-{zeta} A and CD8-{zeta} AB, indicating the possibility of differential regulation of TCR {zeta} chain-mediated signaling events by each ITAM. More importantly, the differential effects on binding of phosphorylated proteins to CD8-{zeta} chain by substitution mutations of tyrosine residues in each ITAM were observed. The single substitution mutation of the first tyrosine residue in each ITAM (A1, B1 and C1 mutants) significantly reduced the amount of the phosphorylated proteins recruited to the TCR {zeta} chain, which was most prominent in the B1 mutant. However, the species of phosphorylated proteins bound to CD8-{zeta} chain clearly differed among these mutants. The additional substitution mutation of the A2 or C2 tyrosine residue in A and C ITAM significantly affected the amount and species of phosphoproteins recruited to the TCR {zeta} chain. Stimulation of TCR did not induce recruitment of tyrosine phosphorylated proteins to the CD8-{zeta} chain in the A1A2 mutant at all. The effect of the additional B2 tyrosine mutation in B ITAM was not substantial. These results suggested that each ITAM and their tyrosine residues are functionally distinct for the formation of activation-induced phosphoprotein complex in the TCR {zeta} chain. When the phosphorylation status of tyrosine residues of CD8-{zeta} chain was examined, two forms of phosphorylated {zeta} chain were clearly detected (Fig. 2B). The level of TCR {zeta} chain phosphorylation induction by TCR stimulation was consistent with that of total phosphoproteins bound to TCR {zeta} chain in each transfectant, as shown in Fig. 2(A).



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Fig. 2. Differential tyrosine phosphorylation of the TCR {zeta} chain and its associated protein complex upon activation in CD8-{zeta} chimera. Each CD8-{zeta} chimera was stimulated for 3 min with OKT8, and {zeta} chain binding proteins were immunoprecipitated. The immunoprecipitates and lysates were resolved by SDS–PAGE. (A) The tyrosine-phosphorylated proteins interacting with the TCR {zeta} chain were immunoblotted with anti-phosphotyrosine antibody. (B) The level of tyrosine phosphorylation of the {zeta} chain was detected by immunoblotting with anti-phosphotyrosine antibody. (C) ZAP-70 or phospho-ZAP-70Tyr319 associated with the {zeta} chain was examined by immunoblotting with anti-ZAP-70 mAb or anti-phospho-ZAP-70Tyr319 mAb. (D) Each CD8-{zeta} chimera was stimulated for 5 and 15 min with OKT8 and CD28. The phosphorylated proteins were immunoprecipitated with anti-phosphotyrosine antibody-conjugated beads and immunoblotted with anti-ZAP-70 antibody. An equal amount of protein was loaded in each lane, which was confirmed by Ponceau S staining and immunoblotting with anti-ZAP-70 antibody. The result shown here is one representative of three experiments.

 
Within the stimulation-induced phosphoprotein complex bound to TCR {zeta} chain, ZAP-70 is a critical component for transmitting T cell activation. To trigger downstream signaling events for T cell activation, the tyrosine 319 residue of ZAP-70 should be phosphorylated (1921). To examine the amount of total ZAP-70 protein and its functionally active form, the protein complex associated with CD8-{zeta} chimeric protein was immunoprecipitated and the ZAP-70 protein was detected by immunoblot using anti-ZAP-70 mAb or anti-phospho ZAP70Tyr319 mAb (Fig. 2C). While deletion of the C ITAM did not influence the level of ZAP-70 binding to {zeta} chain, additional deletion of the B ITAM significantly reduced the amount of ZAP-70 recruited to CD8-{zeta} chain upon TCR stimulation. Consistent with the results in Fig. 2(B), mutation of A1 and C1 tyrosine residues did not affect the binding of ZAP-70 to the {zeta} chain. However, mutation of the B1 or C2 tyrosine residue considerably inhibited the recruitment of ZAP-70 protein to {zeta} chain, and mutation of both tyrosine residues in the A or B ITAM completely blocked the recruitment of ZAP-70 protein to {zeta} chain.

A substantial portion of ZAP-70 protein associated with {zeta} chain upon TCR stimulation in CD8-{zeta}A, CD8-{zeta}AB, A1, C1, C1C2 was phospho ZAP-70Tyr319. The A1A2, B1 and B1B2 mutants did not have this phosphorylated form of ZAP-70 in the activation-induced protein complex bound to the {zeta} chain. We then examined the time-dependent phosphorylation of ZAP-70 upon stimulation of chimeric proteins by OKT8 with anti-CD28 mAb, considering that not only the phosphorylation induction of ZAP-70 but also its maintenance may be modulated by the status of {zeta} chain phosphorylation. ZAP-70 phosphorylation was strongly induced 5 min after CD8/CD28 costimulation in wild-type CD8-{zeta} and all mutants (Fig. 2D). The induced phosphorylation of ZAP-70 was sustained with a gradual decrease in wild-type CD8-{zeta}, CD8-{zeta}A, CD8-{zeta}AB, C1 and C1C2 mutants. The A1A2, B1 and B1B2 mutants did not show the sustained phosphorylation of ZAP-70 and a very low level of sustained ZAP-70 phosphorylation was detected in the A1 mutant. These results indicated that all three ITAMs, especially A and C ITAM, are required for optimal recruitment of the tyrosine phosphorylated protein complex to {zeta} chain, and B ITAM is important for activation-induced ZAP-70 binding to {zeta} chain.

A2 and B1 tyrosine residues are most critical not only for {zeta} chain phosphorylation but also for recruitment of ZAP-70 proteins including its phosphorylated form at tyrosine 319 upon TCR stimulation. B2 and C2 tyrosine residues are important for ZAP-70 recruitment to {zeta} chain, and binding of phosphorylated form of ZAP-70 to {zeta} chain was normal in the C2 mutant. While recruitment of tyrosine phosphorylated proteins to {zeta} chain was decreased in the absence of A1 tyrosine, {zeta} chain phosphorylation and binding of ZAP-70 and its phosphorylated form at tyrosine 319 to {zeta} chain was not affected in the A1 mutant. All tyrosine residues in the full context of three ITAMs are dispensable for the initial induction of total cellular ZAP-70 phosphorylation, but A1, A2, B1 and B2 tyrosines play important roles for its sustained phosphorylation. Collectively, these results clearly demonstrated that each tyrosine residue and ITAM of TCR {zeta} chain has differential roles in phosphorylation of {zeta} chain and recruitment of ZAP-70 to the {zeta} chain.

Phosphorylation of ERK and JNK is differentially regulated by tyrosine residues in TCR {zeta} chain ITAMs
MAPKs are involved in diverse signaling pathways including T cell proliferation, differentiation and death events, but the specific regulatory roles of each tyrosine residue within TCR {zeta} chain ITAMs on MAPK phosphorylation have not been extensively studied. To determine the role of tyrosine residues of TCR {zeta} chain ITAMs and CD28 in MAPK activation, phosphorylation of ERK-1/2 and JNK was examined in each mutant. As shown in Fig. 3(A), stimulation of wild-type and CD8-{zeta}A with OKT8 and CD28 antibodies induced phosphorylation of ERK-1/2 up to 10 min and the phosphorylated ERK-1/2 disappeared 20 min after stimulation. The CD8-{zeta}AB transfectant showed a rapid decrease in ERK-1/2 phosphorylation, suggesting the possibility that B ITAM of the TCR {zeta} chain may play a negative regulatory role in the sustained phosphorylation of ERK-1/2.



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Fig. 3. Each ITAM or tyrosine residue of CD8-{zeta} differentially regulates the phosphorylation of MAPKs. Cells were stimulated with OKT8 mAb plus anti-CD28 antibody (A), or with OKT8 mAb alone (B) for the indicated times at 37°C. Cells were lysed, and the cell lysate was analyzed by 10% SDS–PAGE, and immunoblotted with anti-phospho-ERK-1/2, anti-phospho-SAPK/JNK and anti-phospho-p38 antibody. An equal amount of protein was loaded in each lane, which was confirmed by Ponceau S staining and immunoblotted with the anti-p38 mAb as lysate control. The result shown here is one representative of three experiments.

 
Although mutation of the A1 tyrosine did not influence the induction kinetics of ERK-1/2 phosphorylation, the phosphorylation of ERK-1/2 was completely abolished in the A1A2 mutant, indicating an important role of the A2 tyrosine in activation of ERK-1/2. While the B1 mutant showed reduction of overall ERK-1/2 phosphorylation, sustained phosphorylation of ERK-1/2 was not observed in the B1B2 mutant, suggesting qualitatively different signaling contributions of the B1 or B2 tyrosine residue to ERK-1/2 phosphorylation. In the C1 mutant, enhanced and sustained phosphorylation of ERK-1/2 was observed at 20 min and later time points. The induction of ERK-1/2 phosphorylation was delayed and reduced in the C1C2 mutant. These results demonstrated that the phosphorylation and subsequent activation of ERK-1/2 is differentially regulated by the phosphorylation status of tyrosine residues in the TCR {zeta} chain ITAMs. When ERK-1/2 phosphorylation induced by OKT8 stimulation was examined, a similar pattern of induction kinetics of ERK-1/2 phosphorylation was observed, indicating that the influence of CD28 costimulation on ERK-1/2 activation is marginal (Fig. 3B).

We next examined the regulatory effect of TCR {zeta} chain ITAMs on SAPK/JNK activation (Fig. 3A and B). Strong and sustained p54 SAPK/JNK phosphorylation was induced in wild-type CD8-{zeta} up to 20 min after CD8 ligation, but this was gradually decreased in the presence of CD28 costimulation. With CD8 and CD28 coligation, all truncated and substituted mutants except the A1A2 and C1C2 mutants showed significantly enhanced phosphorylation of p54 SAPK/JNK comparable to that of wild-type CD8-{zeta}. Delayed and weak induction of phosphorylation of p54 SAPK/JNK was observed in the A1A2 and C1C2 mutants, indicating that A2 and C2 tyrosines can play critical role for initial induction of p54 SAPK/JNK phosphorylation.

As seen in wild-type CD8-{zeta}, phosphorylation of p46 SAPK/JNK was induced 5 min after stimulation, but rapidly decreased in the CD8-{zeta}AB and C1 mutants. However, stimulation of CD8-{zeta}A, B1 or C1C2 mutants showed sustained, but gradually decreased phosphorylation of p46 SAPK/JNK. Mutation of A1, A1A2 or B1B2 tyrosine residues completely inhibited the induction of phosphorylation of p46 SAPK/JNK in the presence of CD28 costimulation. These results demonstrate the importance of the A1, A2 and B2 tyrosines for the initial phosphorylation of p46 SAPK/JNK. It has been shown that ERK and SAPK phosphorylation correlated with its enzymatic activity (22). Stimulation of CD8 chimeric molecules with OKT8 only induced the phosphorylation of p38 MAPK, and its phosphorylation was sustained until 20 min after stimulation in wild-type, CD8-{zeta} A, A1, B1B2, C1 mutants, and the level of phosphorylation induction was weak in the other CD8-{zeta} mutants (Fig. 3B). The signaling through CD28 in combination with CD8-{zeta} stimulation markedly enhanced the phosphorylation of p38 MAPK in all CD8-{zeta} mutants that recovered the defect in OKT8 stimulation. This recovery of phosphorylation was similarly shown in phosphorylation of I{kappa}B{alpha} (see supplementary fig. 1, available at International Immunology Online). Taken together, our observations suggested that each tyrosine residue in the three ITAMs of TCR {zeta} chain plays distinctive roles in induction and maintenance of ERK-1/2, SAPK/JNK phosphorylation, thereby contributing to the fine tuning of diverse signaling cascades from the TCR {zeta} chain.

Induction of IL-2 secretion and activation-induced cell surface antigen expression are differentially regulated by TCR {zeta} chain ITAMs
Although upregulation of CD69, CD25 and IL-2 secretion upon TCR-mediated activation are known to be critical markers of T cell activation, the regulatory roles of TCR {zeta} ITAMs or their tyrosines residues for these activation events are still controversial. Our observations in Figs 2 and 3 suggested the possibility of differential regulation of activation-induced cell surface antigens and IL-2 secretion by different TCR {zeta} chain ITAMs. To test this possibility, we measured the amount of IL-2 secretion from these mutants following CD8 and CD28 costimulation (Fig. 4A). While wild-type CD8-{zeta} and CD8-{zeta}A produced a comparable amount of IL-2, IL-2 secretion was substantially reduced in the CD8-{zeta}AB and C1C2 mutants. The C1 mutant secreted higher levels of IL-2 than wild-type CD8-{zeta}. These results are consistent with the pattern of ERK-1/2 phosphorylation shown in Fig. 3. They strongly support the possibility that the B ITAM may contain negative regulatory elements, and the C1 tyrosine may play a negative role in T cell activation. Mutation of A1, A1A2, B1 or B1B2 tyrosine residues completely abolished the potential to secrete IL-2 upon stimulation.



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Fig. 4. Differential regulation of activation-induced cytokine secretion and cell surface antigen expression in CD8-{zeta} chimera. (A) CD8-{zeta} wild-type and mutants were stimulated with OKT8 plus anti-CD28 antibody or OKT8 antibody alone. After stimulation for 20 h, supernatants were harvested and subjected to IL-2 ELISA. Open bar: no stimulation; closed bar: OKT8 plus CD28 stimulation; hatched bar: OKT8 stimulation alone. The results express the mean ± SD of three different experiments. (B) Cells were stimulated with OKT8 plus anti-CD28 antibodies or OKT8 antibody alone for 16 h at 37°C, and analyzed by flow cytometry with PE-conjugated anti-human CD69. (C) Cells were stimulated for 48 h, and the surface expression level of CD25 was analyzed by flow cytometry using FITC-conjugated anti-human CD25. The result is a representative of three independent experiments. Shaded curve: unstimulated cells; bold curve: cells stimulated with OKT8 plus anti-CD28 antibody; dotted curve: cells stimulated with OKT8 alone.

 
Interestingly, OKT8 stimulation alone did not induce IL-2 production in the wild-type CD8-{zeta} transfectant, and this was restored to normal levels by additional anti-CD28 costimulation. Furthermore, the A ITAM by itself has the potential to trigger normal levels of IL-2 production in the absence of CD28 costimulation. Together, these results demonstrated that phosphorylation of tyrosine residues in the A and B ITAMs are required for induction of IL-2 production and C1 and C2 tyrosine residues may play quite different roles in induction of T cell activation.

We next investigated the regulatory role of each ITAM of TCR {zeta} chain on activation-dependent induction of CD69 or CD25 on the cell surface in the presence of OKT8 or OKT8/CD28 stimulation. As shown in Fig. 4(B), CD8-{zeta}A, CD8-{zeta}AB and the C1 mutants showed induction of CD69 cell surface expression comparable to that of wild-type CD8-{zeta} with CD8 and CD28 costimulation. Interestingly, CD69 induction reached maximal levels without CD28 costimulation in CD8-{zeta}A and CD8-{zeta}AB mutants, however CD28 costimulation was required for IL-2 production in the CD8-{zeta}AB mutant. While the single A1 mutation slightly reduced CD69 induction, inhibitory effects of the B1 mutation on CD69 induction was quite noticeable. The double mutation of two tyrosine residues in the A and B ITAMs completely abolished the potential of the TCR {zeta} chain to induce CD69 expression. However, mutation of one or two tyrosine residues in the C ITAM did not abrogate the CD69 induction on the surface. These results suggested that the A ITAM of the TCR {zeta} chain itself transmits strong activation signals for full induction of CD69 even in the absence of CD28-mediated signals. Two tyrosine residues in the A and B ITAMs are critical for CD69 induction. Functional contribution of A1 and A2 tyrosine residues to CD69 induction was quantitative, but this quantitative effect was not apparent in B1 and B2 tyrosine residues. However, the C1 and/or C2 mutations did not influence the induced expression of CD69.

Mutation of any tyrosine residue in the A and B ITAM completely abolished the activation-dependent induction of CD25 (Fig. 4C). In contrast to quantitative functional effect of each tyrosine residue in A and B ITAM on CD69 induction, this effect was not observed for CD25 induction. However, CD25 induction was not impaired in the C1 and C1C2 mutants. Therefore, each tyrosine residue and ITAM plays a unique regulatory role in CD69 and CD25 induction on the surface.

Distinct regulation of activation-induced cell death by each ITAM of TCR {zeta} chain through modulation of sFasL production and mitochondrial membrane potential
To test the death-inducing capability of each ITAM and tyrosine residue of TCR {zeta} chain, the level of cell death, depolarization of mitochondrial membrane potential ({Delta}{psi}mn) and sFasL production was examined in wild-type and mutant CD8-{zeta} transfectants when stimulated with OKT8 in the absence or presence of CD28 costimulation. While wild-type CD8-{zeta} or CD8-{zeta}A showed similar levels of cell death induction as evaluated by annexin-V exposure after 48 h of stimulation, deletion of C-terminal ITAM (CD8-{zeta} AB) substantially reduced the cell death (Fig. 5A). This is consistent with the pattern of IL-2 induction supporting the possibility of the inhibitory roles of B ITAM on activation-induced apoptosis. Complete inhibition of TCR-mediated apoptosis induction was observed in A1, A1A2, B1 and B1B2 mutants, suggesting that tyrosine residues in the A and B ITAM are also critical for inducing apoptotic process. In agreement with previous findings (23), treatment of the pan caspase inhibitor (z-VAD-fmk) partially inhibited the exposure of phosphatidylserine in activated T cells, indicating both Fas-mediated cell death pathway and caspase-independent pathways are involved in induction of TCR-mediated apoptosis (data not shown). These results suggested that four tyrosine residues in the A and B ITAMs are key elements for activation-induced cell death and two tyrosine residues in C-terminal ITAM are functionally dispensable for this event.



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Fig. 5. Signaling from each ITAM or tyrosine residue of TCR {zeta} chain differentially regulates apoptotic T cell death. CD8-{zeta} wild-type and mutants were stimulated with OKT8 plus anti-CD28 antibody or OKT8 antibody alone for 48 h. (A) Induction of apoptosis was evaluated by FACS analysis using annexin-V and the results were expressed as a percentage of specific apoptosis. (B) Cells were stimulated with OKT8 plus anti-CD28 antibody or OKT8 antibody alone for 18 h. Supernatants were harvested, centrifuged and the level of soluble FasL secretion was analyzed by ELISA. (C) The level of mitochondrial membrane depolarization was analyzed by flow cytometry after staining cells with JC-1. The results were expressed as a percentage of a change of JC-1 color from green to greenish orange. Closed bar: stimulation with OKT8 plus anti-CD28 antibodies; hatched bar: stimulation with OKT8 alone. All results represent the mean ±SD of three different experiments.

 
Consistent with these results, the production of sFasL was significantly reduced in the CD8-{zeta}AB mutant and the C1 or C2 tyrosine was dispensable for sFasL secretion. In addition, the mutation of any tyrosine residue in the A or B ITAM largely abolished the capability of CD8-{zeta} to secrete sFasL (Fig. 5B).

Next, we investigated whether the mitochondrial membrane potential loss also can be regulated in a similar manner by functional tyrosine residues or ITAMs of {zeta} chain. Notably, CD8-{zeta}A showed a greater loss of mitochondrial membrane potential upon stimulation when compared to that of wild-type CD8-{zeta}, C1 or the C1C2 mutant (Fig. 5C), demonstrating that a significant portion of cell death triggered by A ITAM of TCR {zeta} chain is mitochondria-mediated. The loss of mitochondrial membrane potential was barely detected in the CD8-{zeta}AB, A1, A1A2, B1 or B1B2 mutants, indicating that tyrosine residues in the A and B ITAM are important for depolarization of mitochondrial membrane potential leading to the induction of mitochondria-mediated AICD. The C1 or C2 tyrosine is dispensable for this alternative pathway to cell death. As the apoptotic events are accompanied by a decrease in mitochondrial enzyme activity, we extended our study into examination of the mitochondrial dehydrogenase activity as an indicator of cell viability. Consistent with the loss of mitochondrial membrane potential, the CD8-{zeta}A mutant showed a marked decrease in mitochondrial dehydrogenase activity upon stimulation, whereas the activity of this enzyme was not changed in the CD8-{zeta}AB, A1, A1A2, B1 or B1B2 mutants (data not shown). Collectively, these results suggested that, as with its capacity to regulate IL-2 secretion, the A ITAM of TCR {zeta} chain by itself has the signaling potential to trigger the early phase of cell death and induce rapid mitochondrial membrane potential loss upon TCR {zeta} chain-mediated activation.


    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Supplementary data
 References
 
Whether individual TCR {zeta} chain ITAMs and their tyrosine residues exert qualitatively distinctive or quantitatively redundant functions, or their roles are dispensable in regulation of various intracellular signals leading to T cell activation and apoptosis, is one of the central issues. Two models for TCR signal transmission were proposed. The first one is the ITAM multiplicity model, in which there is functional redundancy within the ITAMs of the TCR {zeta} and CD3 signaling modules. In the differential signaling model, distinct functions for the TCR {zeta} and CD3 signaling modules are proposed (24,25). However, these two models are not mutually exclusive. The relative contributions of one model versus the other could be dependent on differentiation or developmental status of T cells, the interaction strength between TCR and its antigenic complex and/or the microenvironmental context.

Interestingly, as demonstrated in the CD8-{zeta} A mutant, an isolated N-terminal ITAM of {zeta} chain can trigger most T cell signaling events without CD28 costimulation and induce a remarkably higher level of mitochondrial membrane potential loss than wild-type CD8-{zeta}. It can be suggested that these results demonstrated that the strong activation and apoptotic signals from the A ITAM of TCR {zeta} chain appear to be modulated into the fine intracellular signals with appropriate intensity and specificity in the presence of the other two ITAMs and CD28 costimulation. And each ITAM of TCR {zeta} chain has distinctive signaling potential rather than functional redundancy for triggering various signaling events for T cell activation and apoptosis.

In our results, it is apparent that each tyrosine residue makes a unique functional contribution to the amount and species of tyrosine phosphorylated to the {zeta} chain. Also the level of tyrosine phosphorylated protein recruitment to the {zeta} chain correlated with the degree of overall tyrosine phosphorylation of the {zeta} chain, but tyrosine residues in each ITAM make a differential contribution to the recruitment of ZAP-70. However, normal levels of initial induction of ZAP-70 phosphorylation was observed in all tyrosine mutants of CD8-{zeta} mutants. This is consistent with previous evidence in which phosphorylated ZAP-70 was detected in all doubly mutated ITAMs of TCR {zeta} chain (26).

Consistent with our findings about TCR-proximal signaling events, functional roles of each tyrosine in induction and maintenance of MAPK activation was also quite distinctive.

However, this distinctive impairment of activation-induced phosphorylation of these kinases in each {zeta} chain mutant was not fully reflected in the functional outcome of T cell activation signaling. IL-2 induction, apoptosis induction by Fas-dependent and mitochondria-dependent pathway, and CD69 and CD25 induction on the surface were completely abolished in the A1, A1A2, B1 and B1B2 mutants. The effect of mutation of tyrosine residues of A and B ITAM on upregulation of CD69 was cumulative. Substantially high level of IL-2 secretion, and normal level of apoptosis induction and upregulation of CD69 and CD25 was detected in the C1 mutant, which can be explained by normal or high levels of induction of ZAP-70 and MAPK phosphorylation. These results suggested functional requirements of all tyrosine residues in the A and B ITAM for these signaling events and strongly supported the possibility that the C1 tyrosine may have a negative role in regulation of signaling events for T cell activation.

From our results that IL-2 secretion, apoptosis induction and maintenance of ERK1/2 phosphorylation were inhibited in CD8-{zeta}AB, it can be suggested that B ITAM may contain the elements for recruitment of negative regulators for these signaling events. This hypothesis is consistent with our results in CD8-{zeta}A which showed comparable or higher signaling capacity to CD8-{zeta} wild type. Therefore, in wild-type CD8-{zeta} the presence of all three ITAMs may block the association of this negative regulatory factor to the B ITAM. Alternatively, binding of positive regulatory factors including ZAP-70 to {zeta} chain with three ITAMs may functionally offset the negative effects of a factor bound to the B ITAM. The negative regulatory role of C1 tyrosine residue makes a functional contribution to the phenotype of CD8-{zeta}A. However, the capacity of {zeta} chain to trigger intracellular signaling events for T cell activation was significantly abolished in B1 or B1B2 mutants, suggesting that the B ITAM has a possibility to contain two functionally distinctive submotifs. Two tyrosine residues in the B ITAM have a stimulatory effect on T cell activation and other amino acid residues in the B ITAM might be involved in negative regulation of {zeta} chain-mediated signaling. This idea is supported by recent findings which have identified several new proteins that specifically associate with {zeta} chain (2732).

The possibility of functional involvement of non-tyrosine residues in TCR {zeta} chain was further supported by two previous studies showing that the +3 amino acid in the ITAM is important for TCR {zeta} chain function (33,34), and the N-terminal leucine residue in the A ITAM is important for induction of Ca2+ mobilization and ERK activation (35). Our observations can provide an explanation regarding a previous study, in which the IL-2 response to the TCR ligand was inhibited in mutants, 4F (A1A2B2C1) and 3F (A1B2C1) (36). It is possible that the strong negative effects of A1A2B2 or A1B2 mutations may overwhelm the positive effects of the C1 mutation in these mutants, thereby making these TCR {zeta} chain mutants act as dominant negative forms. Previous observations of the 2F mutant (B2C1) suggested an inhibitory role of the C1 tyrosine residue in T cell activation (36).

Somewhat disparate results were reported in another previous study where A1 or A2 mutants prevented induction of ZAP-70 and ERK phosphorylation (35). Our results with CD8-{zeta}A and CD8-{zeta}AB mutants shows that the B and C ITAMs play an important role in modulation of TCR {zeta} chain-mediated signaling. In this sense, functions of tyrosine residues of TCR {zeta} chain cannot be analyzed properly in the context of an isolated N-terminal ITAM only. It is quite possible that the functional and structural conformation induced by phosphorylation of tyrosine residues in the B and C ITAM influences the regulatory activity of A1 and A2 tyrosine. Therefore, our observation clearly supports the model that each ITAM and their tyrosine residues of TCR {zeta} chain have diverse regulatory potentials for qualitatively differential translation of external stimuli through the TCR complex into different intracellular signal events for T cell activation and apoptosis induction. Furthermore, our hypothesis was supported by recent studies, revealing that not only the spatio-temporal relationship of proximal signaling molecules in immunological synapse upon TCR {zeta} chain phosphorylation but also the newly found signaling molecules such as CARMA1 or targeting of signaling complexes like NEMO/IKK{gamma}, are important in fine tuning of TCR {zeta} chain-mediated signaling outcomes (37,38). Moreover, as suggested by several reports, various types of phosphatases and adaptor molecules which interact with TCR {zeta} chain upon T cell activation may play a role in specification of intracellular signals initiated by distinct status of TCR {zeta} chain phosphorylation (39,40).

The qualitatively different contributions by individual TCR {zeta} chain ITAMs to signaling events leading to apoptosis induction observed in this report are consistent with a previous study in which the ability of the TCR {zeta} chain to promote apoptosis induction was examined using single chain chimeras between the extracellular domain of IL-2R{alpha} and the intracytoplasmic portions of TCR {zeta} chain, except that the B1B2 mutant was still able to trigger apoptosis. A distinctive functional and structural conformation induced by crosslinking of monomeric (IL-2R{alpha}) in BW5147 and dimeric (CD8) chimera in Jurkat T cells may be ascribed to this differential finding (17). Alternatively, this different result may be due to the presence of CD28 costimulation in our study, which created the intracellular milieu. In conclusion, we have shown that the functional elements comprising tyrosine residues in TCR {zeta} chain ITAM and possibly its neighboring residues are important for transmitting qualitatively differential signals upon TCR stimulation. This provides insight for fine signaling specificity of TCR {zeta} chain toward induction of different functional outcomes in T cell activation and apoptosis. Identification of distinctive functional/structural signaling modules and their binding proteins to specify distinctive states of TCR {zeta} chain is being investigated.


    Supplementary data
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Supplementary data
 References
 
Supplementary data are available at International Immunology Online.
Table 1. Summary of differential regulation of T cell activation and apoptosis events by TCR {zeta} chain ITAM and their tyrosine residues


Events mutants

ZAP-70


IL-2

Phosphorylation


CD 69

CD 25

Ax-V

{Delta}{psi}m

sFasL


Ba

Pb

Sc



ERK-1/2

JNK p54

JNK p46

p38












WT ++ ++ + ++ ++ ++ +++/ {downarrow} ++ ++ ++ +++ ++ ++
A1 ++ + + - ++ +++ + + +
A1A2 +/{uparrow} ++
B1 + + +++ + ++
B1B2 ++/{downarrow} +++ ++
C1 ++ ++ + +++ +++ +++ +++/{downarrow} ++ ++ ++ +++ ++ ++
C1C2 + ++ + + ++/{uparrow} +/{uparrow} ++ ++ + ++ +++ ++ ++
A + +++ + ++ ++ +++ +++ ++ ++ ++ +++ +++ ++
AB

++

+++

+

+

++/{downarrow}

+++

+++/{downarrow}

+/–

++

++

+

+

+

Ba, the relative amount of ZAP-70 bound to {zeta} chain (OKT8 stimulation); Pb, the relative intensity of phosphorylation for ZAP-70Tyr319 for each mutant (OKT8 stimulation); Sc, the maintenance of ZAP-70 phosphorylation (OKT8 plus anti-CD28 mAb stimulation) in each mutant were indicated as compared with that of wild-type CD8- {zeta} transfectant. The arrow {downarrow} means decrease and {downarrow}{downarrow} means rapid decrease, respectively. The arrow {uparrow} means delayed but sustained phosphorylation. The activation and apoptosis events were evaluated when wild-type or mutant forms of CD8- {zeta} was stimulated with OKT8 plus anti-CD28 mAb.


    Acknowledgements
 
This work was supported in part by grants for The Functional Proteomics Center from the Ministry of Science and Technology (M102KM010001-02K1301-01100), the Korea Science and Engineering Foundation (199-1-212-001-5) and the Biochallenge project of Ministry of Korea Science and Technology (M1031040003-03B4640-00310).


    Abbreviations
 
AICD   activation induced cell death
CARMA1   caspase recruitment domain/membrane associated guanylate kinase1
ERK   extracellular signal regulated kinase
FLIP   FLICE-like inhibitory protein
IKK{gamma}   I{kappa}B kinase {gamma}
ITAM   immunoreceptor tyrosine-based activation motif
JNK SAPK   c-Jun N-terminal kinase/stress-activated protein kinase
MAPK   mitogen-activated protein kinase
PTK   protein tyrosine kinase
SH2 domain   Src-homology 2 domain

    Notes
 
Transmitting editor: C. Terhorst

Received 30 January 2004, accepted 31 May 2004.


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 Discussion
 Supplementary data
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