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The Journal of Immunology, 1998, 160: 163-170.
Copyright © 1998 by The American Association of Immunologists

Regulation of TCR Signal Transduction in Murine Thymocytes by Multiple TCR {zeta}-Chain Signaling Motifs1

Nicolai S. C. van Oers2,{dagger}, Paul E. Love{ddagger}, Elizabeth W. Shores{ddagger} and Arthur Weiss3,{dagger},*

Departments of * Medicine, and Microbiology and Immunology, and {dagger} Howard Hughes Medical Institute, University of California, San Francisco, CA 94143; and {ddagger} Laboratory of Mammalian Genes and Development, National Institutes of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The {alpha}ß TCR is a multimeric protein complex comprising ligand-binding and signal-transducing subunits. The signal transduction processes are mediated by the immunoreceptor tyrosine-based activation motifs (ITAMs), and up to 10 ITAMs are present within a single TCR complex. This multiplicity may allow for signal amplification and/or the formation of qualitatively distinct intracellular signals. Notably, the TCR-{zeta} subunit contains three ITAMs, and exists as a disulfide-linked homodimer in the TCR complex. In normal murine thymocytes and peripheral T cells, a proportion of TCR-{zeta} molecules is constitutively tyrosine phosphorylated and associated with the ZAP-70 protein tyrosine kinase. We examined the contribution of the different TCR-{zeta} ITAMs in regulating the constitutive phosphorylation of the TCR-{zeta} subunit in thymocytes by analyzing TCR-{zeta}-deficient mice that had been reconstituted with either full-length or single ITAM-containing TCR-{zeta} subunits. We report in this work that in the absence of a full-length TCR-{zeta} subunit, there is no apparent constitutive phosphorylation of the remaining TCR/CD3 ITAMs. Following TCR ligation, all of the CD3 ITAMs become inducibly phosphorylated and associate with the ZAP-70 protein tyrosine kinase. Regardless of the number of TCR-{zeta} ITAMs present in the TCR complex, we report that a number of molecules involved in downstream signaling events, such as ZAP-70, SLP-76, and pp36, are all inducibly tyrosine phosphorylated following TCR ligation. These results support the notion that the different TCR ITAMs function in a quantitative rather than qualitative manner.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The {alpha}ß TCR is assembled as an oligomeric complex consisting of six different transmembrane proteins (1). The six proteins include the Ag-binding subunits (TCR-{alpha}ß) that are noncovalently associated with the signal-transducing chains (CD3-{gamma}, CD3-{delta}, CD3-{epsilon}, and TCR-{zeta} and/or TCR-{eta}). These signaling chains all contain a common sequence (YxxLx(6, 7, 8)YxxL), termed the immunoreceptor tyrosine-based activation motif (ITAM)4 (2). The ITAM motif is present as a single copy in the CD3-{gamma}, CD3-{delta}, and CD3-{epsilon} subunits, and as three copies in the TCR-{zeta} subunit (3). In T cell lines and clones, one of the initial events following TCR engagement is the phosphorylation of tyrosine residues in the ITAMs, a process regulated by the Src-family PTKs, Lck or Fyn (4, 5, 6, 7). The phosphorylation of the two tyrosines within an ITAM promotes a high affinity interaction with a second family of PTKs, the Syk/ZAP-70 family. This interaction is mediated by the two SH2 domains of Syk/ZAP-70 and the two phosphotyrosines in the ITAM (8, 9, 10). The recruited Syk/ZAP-70 molecules are activated by phosphorylation and contribute to a cascade of downstream signals that are crucial for the initiation of cellular responses.

The most widely held structural model of the TCR is one comprising a {zeta} dimer and two CD3 pairs ({gamma}{epsilon}, {delta}{epsilon}). Therefore, 10 ITAMs may be present within a single TCR complex. It remains to be determined how 10 ITAMs contribute to Ag receptor signal transduction. The presence of multiple ITAMs within the TCR complex may allow for signal amplification and/or the formation of qualitatively distinct intracellular signals. These questions have, in part, been addressed in both cell lines and in mice by the use of chimeric proteins containing the cytoplasmic domains of one or more TCR ITAMs. In the Jurkat T cell line, stimulation of such chimeric receptor complexes comprising one, two, or three copies of a single TCR-{zeta} ITAM resulted in the induction of identical patterns of tyrosine-phosphorylated proteins, with the triplication of one ITAM resulting in enhanced signaling (11). In transgenic mice expressing chimeric constructs encoding the cytoplasmic domain of either the TCR-{zeta} or CD3-{epsilon} subunit, cross-linking the chimeric receptor expressed on thymocytes induced a greater degree of proliferation with TCR-{zeta} than CD3-{epsilon} (12). These results are consistent with the idea that three ITAMs (TCR-{zeta}) provide quantitatively stronger signals than a single ITAM (CD3-{epsilon}). However, one cannot rule out the possibility in these systems that TCR-{zeta} ITAMs activated qualitatively distinct pathways that enhanced proliferation relative to CD3-{epsilon}.

The suggestion that the 10 TCR ITAMs function in a quantitative manner is also consistent with studies on mice expressing TCR complexes lacking one or more ITAMs. Mice deficient in the TCR {zeta}-chain (TCR-{zeta}-/-) have substantially reduced numbers of both CD4+CD8+ thymocytes and mature single-positive T cells (13, 14, 15, 16). Reconstituting these TCR-{zeta}-/- mice with transgenes encoding TCR-{zeta} subunits lacking all three ITAMs restored T cell development (17, 18). This indicates that the developmental defects in the {zeta}-deficient mice are primarily a consequence of impaired TCR surface expression, with the TCR-{zeta} ITAMs primarily augmenting the efficiency of T cell development (17). This is especially evident in TCR-{alpha}ß transgenic mice bearing TCRs of known specificity, in which a reduction in the number of TCR-{zeta} ITAMs can dramatically reduce the efficiency of both positive and negative selection (18). Second, T cells isolated from TCR-{zeta}-deficient mice, when reconstituted with the TCR-{zeta} subunit, exhibited a significant level of self-reactivity in vitro (19). These data suggest that multiple {zeta} ITAMs are involved in signal amplification and can influence T cell repertoire selection.

In addition to their contribution to signal amplification, the 10 ITAMs in the TCR complex may also have qualitatively distinct functions. For example, in certain cell lines, stimulation of chimeric proteins comprising the cytoplasmic domains of either TCR-{zeta} or CD3-{epsilon} can result in qualitatively different patterns of induced phosphoproteins (20). Different TCR ITAMs differ both qualitatively and quantitatively in their ability to couple to programmed cell death, with the first ITAM of TCR-{zeta} being particularly effective in inducing apoptosis (21). Finally, the third ITAM of TCR-{zeta} and the CD3-{epsilon} ITAM may also be required for activation-dependent interactions with the actin cytoskeleton (22, 23). These qualitative differences may be explained by the sequence heterogeneity in the different ITAMs (24). Thus, although all doubly phosphorylated ITAMs can interact with the ZAP-70 PTK, the phosphorylated ITAMs have differential abilities to interact with distinct SH2-containing proteins such as Fyn, Shc, Grb2, and SHIP (SH2-containing inositol phosphatase) (25, 26, 27). For example, Shc has been reported to interact with phosphorylated TCR-{zeta}, but not CD3-{epsilon} (28). Moreover, in in vitro binding studies, Shc appears to complex a doubly phosphorylated CD3-{gamma} or CD3-{delta} ITAM, or the third ITAM of {zeta} ({zeta}3) much more effectively than the first or second {zeta} ITAMs ({zeta}1 or {zeta}2), while Grb2 appears to bind more effectively to the phosphorylated {zeta}1, CD3-{gamma}, and CD3-{delta} ITAMs than the other TCR ITAMs.

It is interesting to note that the TCR {zeta}-chain is constitutively tyrosine phosphorylated and associates with the ZAP-70 PTK when isolated from normal murine thymocytes and peripheral lymph node T cells (29). The constitutive tyrosine phosphorylation appears specific to TCR-{zeta}, since the other CD3 ITAMs are not easily detected in a constitutively phosphorylated form, but can be inducibly phosphorylated (29, 30). The constitutive phosphorylation of the TCR {zeta}-chain in murine thymocytes is regulated primarily by the Lck PTK (7, 29). It remains unclear which TCR-{zeta} ITAMs are required for this constitutive tyrosine phosphorylation and how this influences TCR signal transduction.

To address the contribution of the TCR-{zeta} ITAMs in regulating the constitutive phosphorylation of the TCR-{zeta} subunit, and to examine qualitative signaling differences with the various ITAMs, we analyzed mice deficient in the TCR-{zeta} subunit that had been reconstituted with full-length {zeta}-chain or TCR {zeta}-chain subunits containing single ITAMs. We report in this work that in the absence of a full-length TCR-{zeta} subunit containing three ITAMs, there is no apparent constitutive phosphorylation of the remaining TCR/CD3 ITAMs. Following TCR ligation, all of the CD3 ITAMs become inducibly phosphorylated and associate with the ZAP-70 PTK. Regardless of the number of TCR-{zeta} ITAMs present in the TCR complex, we report that various substrates, including ZAP-70, SLP-76, and pp36, were all inducibly tyrosine phosphorylated. These results support the notion that the different TCR ITAMs function in a quantitative manner.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell lines and animals

C57BL/6 mice were purchased from The Jackson Laboratory (Bar Harbor, ME) and maintained in Animal Care Facility at University of California, San Francisco (UCSF). The TCR-{zeta}-deficient mice reconstituted with full-length TCR-{zeta}, the third ITAM of {zeta} (D66–114), the first ITAM of {zeta} (D108–150), or TCR-{zeta} molecules lacking all three ITAMs (D67–150) have been described in detail elsewhere (13, 17, 18). All of the mice are maintained in microisolator cages at UCSF. Murine thymocytes, lymph node T cells, and spleen cells were isolated as previously described (29).

Abs and antisera

The Abs used for flow cytometry, immunoprecipitations, and Western blotting are as follows: fluorescein-conjugated anti-CD8{alpha}, phycoerythrin-conjugated anti-CD4, and rabbit anti-p44 erk were purchased from Caltag (South San Francisco, CA). 145-2C11, CD3-{epsilon} (American Type Culture Collection, Rockville, MD); 4G10, phosphotyrosine (Upstate Biotechnology, Lake Placid, NY); 6B10.2, TCR-{zeta} (31) were used as indicated. Anti-ZAP-70 antisera were generated against a peptide corresponding to the human sequence from amino acids 282–307. Anti-SLP-76 was kindly provided by Dr. G. Koretzky (University of Iowa, Iowa City, IA). Horseradish peroxidase-conjugated goat anti-mouse IgG and goat anti-rabbit IgG were obtained from Southern Biotechnology (Birmingham, AL); alkaline phosphatase-conjugated goat anti-rabbit Ig and goat anti-mouse Ig were purchased from Bio-Rad (Hercules, CA).

Stimulation, precipitation, and immunoblotting

Thymocyte and lymph node T cells were washed several times in PBS and were resuspended in PBS at a concentration of 1 to 2 x 108 cells/ml. The stimulations, precipitations, and Western blotting was performed as described in detail elsewhere (29).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The constitutive and inducible phosphorylation of the TCR-{zeta} subunit requires more than the first or third TCR-{zeta} ITAMs

We have reported previously that in both murine thymocytes and lymph node T cells, a proportion of TCR-{zeta} subunits is constitutively tyrosine phosphorylated and associated with the ZAP-70 PTK (29). Since the TCR-{zeta} subunit comprises three ITAMs, we assessed the contribution of the different TCR-{zeta} ITAMs in regulating the constitutive and inducible phosphorylation of the TCR/CD3 subunits and the inducible phosphorylation of ZAP-70. For this purpose, we analyzed TCR-{zeta}-deficient mice that had been reconstituted with TCR-{zeta} transgenes lacking one or more {zeta} ITAMs (13, 17). Thymocytes were isolated from normal mice and TCR-{zeta}-deficient mice reconstituted with transgenes encoding TCR-{zeta} molecules without any ITAMs (D67–150), the third ITAM of {zeta} (D66–114), the first ITAM of {zeta} (D108–150), or the full-length TCR-{zeta} subunit. As described in detail elsewhere, and shown in this study for comparative purposes, thymocytes from the D67–150 and D66–114 mice have relatively normal numbers of CD4+CD8+ thymocytes that are capable of maturing into both the CD4-CD8+ and CD4+CD8- single-positive populations (Fig. 1Go) (17). It should be noted that as a consequence of the transgene expression, the TCR density is elevated relative to wild-type mice (Fig. 1Go).



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  		FIGURE 1. Flow-cytometric analysis of thymocytes from wild-type and TCR-{zeta}-deficient mice reconstituted with different TCR-{zeta} ITAMs. Thymocytes from normal C57BL, or TCR-{zeta}-deficient mice reconstituted with TCR-{zeta} molecules lacking all ITAMs (D67–150), with TCR-{zeta} molecules containing the third ITAM of {zeta} (D66–114), or full-length {zeta} molecules (4–6 wk of age) were stained with directly labeled mAbs for CD4, CD8, or TCR/CD3, and analyzed by two- or one-color flow cytometry. For two-color plots, the percentage of cells in each quadrant is listed. For one-color histograms, the dotted line represents staining with a control phycoerythrin-conjugated mAb.

 
The thymocytes were lysed and the TCR/CD3 complex was immunoprecipitated with mAbs directed against the CD3 {epsilon}-chain under conditions that maintain the association of the TCR-{zeta} subunit. The precipitates were resolved on SDS-PAGE and subsequently immunoblotted with anti-phosphotyrosine Abs. As shown in Figure 2GoA, a proportion of TCR-{zeta} molecules is constitutively tyrosine phosphorylated (21 kDa) when isolated from control thymocytes (Fig. 2GoA, lane 1). TCR ligation resulted in the tyrosine phosphorylation of CD3-{epsilon} (lane 2) and a weak induction of ZAP-70 phosphorylation that was revealed following more prolonged enhanced chemoluminescence exposures. In mice reconstituted with TCR-{zeta} molecules lacking any TCR-{zeta} ITAMs (D67–150), there was no detectable phosphoprotein migrating near 10 kDa, a size corresponding to the mutated {zeta} subunit (lane 3). Moreover, none of the CD3 chains demonstrated any appreciable constitutive phosphorylation in these mice (D67–150). Following TCR ligation, there was a marked increase in the tyrosine phosphorylation of CD3-{gamma}, CD3-{delta}, and CD3-{epsilon}, and ZAP-70 in the thymocytes from the D67–150 mice (lane 4 vs lane 3) (32). In the {zeta}-/- mice reconstituted with the third ITAM of TCR-{zeta} (D66–114), the TCR {zeta}-chain was also not constitutively tyrosine phosphorylated since no phosphoprotein was detected migrating near 12 kDa (lane 5). TCR stimulation resulted in the tyrosine phosphorylation of the CD3 subunits and ZAP-70 (lane 6). However, the third ITAM of {zeta} was not easily detected as a phosphorylated protein even after TCR stimulation. Only prolonged enhanced chemoluminescence exposures revealed the presence of some phosphorylated {zeta} from the D66–114 mice (data not shown). We have also determined that thymocytes from the TCR-{zeta}-/- mice reconstituted with the first ITAM of TCR-{zeta} (D108–150) also exhibited very little constitutive or inducible phosphorylation of TCR-{zeta} (data not shown). TCR-{zeta}-/- mice reconstituted with the full-length TCR-{zeta} responded in a manner similar to wild-type mice (lanes 7–8 vs lanes 1–2).



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  		FIGURE 2. The ZAP-70 PTK is inducibly tyrosine phosphorylated and associates with the CD3 ITAMs in the absence of TCR-{zeta} ITAMs. Murine thymocytes were from normal (lanes 1 and 2), TCR-{zeta} ITAM-deficient (lanes 3 and 4; D67–150), TCR-{zeta} single ITAM (lanes 5 and 6; D66–114), or TCR-{zeta} full-length mice (lanes 7 and 8; {zeta} Tg) were either unstimulated (lanes 1, 3, 5, and 7) or stimulated with anti-CD3-{epsilon} mAbs for 3 min (lanes 2, 4, 6, and 8) and subsequently lysed in 0.5% Triton X-100-containing lysis buffers. The TCR/CD3 complex was immunoprecipitated with anti-CD3-{epsilon} mAbs (A) or anti-ZAP-70-specific antisera (B). The precipitates were resolved on 12.5% SDS-PAGE, and the gels were transferred to PVDF membranes and immunoblotted with an anti-phosphotyrosine mAb (4G10).

 
Since a single {zeta} ITAM (D66–114, D108–150) subunit exhibited limited constitutive or inducible phosphorylation when prepared from murine thymocytes, it was possible that the truncated TCR-{zeta} subunits were not coprecipitating with the TCR complex and/or were not preferred substrates following TCR ligation. We did not have any anti-TCR-{zeta} antisera or mAbs that could precipitate all of the truncated TCR-{zeta} construct. Instead, we directly immunoprecipitated ZAP-70, as ZAP-70 can associate with any of the doubly phosphorylated ITAMs (9). As reported elsewhere and shown in this study for comparative purposes, ZAP-70 is constitutively associated with the tyrosine-phosphorylated TCR-{zeta} subunit (21 kDa) when isolated from control unstimulated thymocytes (Fig. 2GoB, lane 1). In mice reconstituted with TCR-{zeta} molecules lacking any TCR-{zeta} ITAMs (D67–150), there was no detectable 10-kDa phosphoprotein that coprecipitated with ZAP-70. In addition, none of the CD3 subunits appeared to be associated with ZAP-70, consistent with our results with the anti-CD3-{epsilon} immunoprecipitates (Fig. 2Go, A and B). Following TCR ligation, there was a marked increase in the tyrosine phosphorylation of CD3-{gamma}, CD3-{delta}, and CD3-{epsilon}, which coprecipitated with tyrosine-phosphorylated ZAP-70 (lane 4 vs lane 3) (32). It also should be noted that we were unable to detect any constitutively or inducibly tyrosine-phosphorylated 12-kDa TCR-{zeta} (D66–114) molecules coprecipitating with ZAP-70 before or after TCR ligation in the {zeta}-/- mice reconstituted with the third ITAM of TCR-{zeta} (D66–114) (lanes 5 and 6). TCR-{zeta}-/- mice reconstituted with the full-length TCR-{zeta} responded in a manner similar to wild-type mice (lanes 7–8 vs lanes 1–2). Taken together, the aforementioned results suggested that the single {zeta} ITAM (D66–114) subunit was not a preferred substrate following TCR ligation.

To further examine the possibility that the D66–114 TCR-{zeta} subunit was not normally phosphorylated following receptor engagement, thymocytes from the different mice were treated with the protein tyrosine phosphatase inhibitor pervanadate. As shown in Figure 3Go, pervanadate treatment of normal thymocytes leads to a significant increase in {zeta} phosphorylation (lane 2). In the absence of any {zeta} ITAMs, pervanadate treatment resulted in a substantial increase in the phosphorylation of the CD3 subunits (lane 4 vs lane 3). Importantly, when thymocytes from the D66–114 mice were stimulated in this manner, a large increase in the phosphorylation of the third ITAM of TCR-{zeta} that coprecipitated with CD3-{epsilon} was found (lane 6). These results suggest that TCR-{zeta} containing only the third ITAM of TCR-{zeta} is stably associated with the TCR complex, but is not a preferred substrate in vivo or in vitro following anti-TCR stimulation. We have also confirmed these findings by directly precipitating the TCR-{zeta} subunit with anti-phosphotyrosine mAbs, and found very little constitutive or inducible phosphorylation of the truncated TCR-{zeta} subunits (data not shown). In summary, the results demonstrate that even in the absence of any constitutive or inducible phosphorylation of the truncated TCR-{zeta} constructs, the CD3-{gamma}, CD3-{delta}, and CD3-{epsilon} subunits are effectively tyrosine phosphorylated and can associate with ZAP-70 following TCR engagement. In fact, the degree of tyrosine phosphorylation of the CD3 subunits appears much more pronounced in mice reconstituted with {zeta}-chains lacking ITAMs than in wild-type mice. However, the variability noted in the TCR density for the various mice precludes a conclusive statement regarding this possibility. Second, the constitutive and inducible tyrosine phosphorylation of the TCR {zeta}-chain detected in murine thymocytes requires more than the first or third ITAM of TCR-{zeta}.



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  		FIGURE 3. The TCR-{zeta} subunit consisting of a single TCR-{zeta} ITAM is inducibly phosphorylated following pervanadate stimulation. Murine thymocytes were either unstimulated (lanes 1, 3, 5, and 7) or stimulated with pervanadate for 10 min (lanes 2, 4, 6, and 8) and subsequently lysed in 0.5% Triton X-100-containing lysis buffers. Normal (lanes 1 and 2), TCR-{zeta} ITAM-deficient (lanes 3 and 4; D67–150), TCR-{zeta} single ITAM (lanes 5 and 6; D66–114), or TCR-{zeta} full-length mice (lanes 7 and 8; {zeta} Tg) were analyzed as in Figure 2Go. The TCR/CD3 complex was immunoprecipitated with anti-CD3-{epsilon} mAbs and blotted with anti-phosphotyrosine Abs, as described in Figure 2Go.

 
The induction of multiple tyrosine-phosphorylated substrates does not require the TCR-{zeta} ITAMs

Several reports have indicated that both quantitative and qualitative differences exist between the various TCR/CD3 ITAMs and their ability to couple to cytosolic effector molecules and promote T cell apoptosis (20, 21). Since differences in the constitutive and inducible phosphorylation of the TCR/CD3 ITAMs were noted between the normal and the D67–150 and D66–114 transgenic mice, it was possible that the various effector molecules could be differentially regulated. To examine this issue, we initially compared the patterns of tyrosine phosphoproteins induced following TCR ligation. Ligation of the TCR expressed on thymocytes from normal mice results in the induced or increased tyrosine phosphorylation of a number of proteins with m.w. of 110, 95, 80, 70, 36, and 21 kDa (Fig. 4Go). Notably, with the exception of the TCR-{zeta} subunit, most of these phosphoproteins were also induced in the cells from D67–150, D66–114, and {zeta} transgenic mice (Fig. 4Go, lanes 4, 6, and 8). Differences in the intensity of the phosphoproteins shown in the blots may reflect the variability seen in the TCR density and were not a consistent finding (Fig. 1Go). Kinetic analyses from 1 to 30 min after TCR stimulation failed to reveal any significant differences (data not shown).



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  		FIGURE 4. Similar phosphoproteins are induced in thymocytes containing variable numbers of TCR-{zeta} ITAMs. Murine thymocytes were from normal (lanes 1 and 2), TCR-{zeta} ITAM-deficient (lanes 3 and 4; D67–150), TCR-{zeta} single ITAM (lanes 5 and 6; D66–114), or TCR-{zeta} full-length mice (lanes 7 and 8; {zeta} Tg) were either unstimulated (lanes 1, 3, 5, and 7) or stimulated with anti-CD3-{epsilon} mAbs for 3 min (lanes 2, 4, 6, and 8) and subsequently lysed in 0.5% Triton X-100- containing lysis buffers. The lysates were loaded onto 12.5% SDS-PAGE gels, subsequently transferred to PVDF membranes, and immunoblotted with an anti-phosphotyrosine mAb (4G10).

 
Although similar phosphoproteins were found to be inducibly tyrosine phosphorylated in all of the mice examined, we also directly compared specific regulators of TCR signaling. SLP-76 is a recently cloned hemopoeitic-specific 76-kDa protein that is expressed abundantly in thymocytes and becomes readily tyrosine phosphorylated upon Ag receptor stimulation (33). In addition, SLP-76 also interacts with additional effector molecules, including p130 and pp36. As shown in Figure 5Go, TCR ligation of thymocytes from normal mice results in the phosphorylation of SLP-76 (Fig. 5Go, lane 2 vs lane 1). Notably, two phosphoproteins of 36 and 130 kDa are also coprecipitated with SLP-76 in the thymocytes. In the absence of any functional TCR-{zeta} ITAMs, or in mice expressing a single TCR-{zeta} ITAM, SLP-76 is inducibly phosphorylated and coprecipitates with p36 and p130, as detected by anti-phosphotyrosine immunoblotting (Fig. 5Go, lanes 2, 4, and 6). Precipitates formed with normal rabbit sera served as negative controls in these experiments (lanes 7 and 8). To further confirm that no differences in the patterns of substrates that are inducibly phosphorylated exist between the various mice, we also used GST-Grb2 fusion proteins to coprecipitate inducibly phosphorylated proteins. As shown in Figure 6Go, in normal mice, a number of phosphoproteins can be precipitated with GST-Grb2 fusion following TCR ligation (lane 2 vs lane 1). Regardless of whether the TCR complex lacks all three TCR-{zeta} ITAMs, or comprises one TCR-{zeta} ITAM, similar phosphoproteins are found to precipitate with GST-Grb2 (Fig. 6Go, lanes 3–8). We have also directly immunoprecipitated specific signaling molecules, including vav, Pyk2, and erk, and found that these proteins are inducibly tyrosine phosphorylated following TCR stimulation in all of the TCR-{zeta} ITAM mice examined (data not shown). In summary, these results demonstrate that a number of TCR-induced substrates, including SLP-76, p36, and p130, are induced even in situations in which the TCR has no TCR-{zeta} ITAMs.



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  		FIGURE 5. The SLP-76 protein is inducibly phosphorylated in mice regardless of the number of TCR-{zeta} ITAMs. A, Murine thymocytes were from normal (lanes 1, 2, 7, and 8), TCR-{zeta} ITAM-deficient (lanes 3 and 4; D67–150), or TCR-{zeta} single ITAM (lanes 5 and 6; D66–114) mice were either unstimulated (lanes 1, 3, 5, and 7) or stimulated with anti-CD3-{epsilon} mAbs for 3 min (lanes 2, 4, 6, and 8) and subsequently lysed in 0.5% Triton X-100-containing lysis buffers. Proteins from the lysates were immunoprecipitated with anti-SLP-76 antisera (lanes 1–6) or normal rabbit sera (lanes 7 and 8). The precipitates were resolved on 12.5% SDS-PAGE, and the gels were transferred to PVDF membranes and immunoblotted with an anti-phosphotyrosine mAb (4G10). B, The blots in A were stripped and subsequently reprobed with anti-SLP 76 antisera.

 


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  		FIGURE 6. Similar phosphoproteins are associated with Grb2 in mice lacking one or more TCR-{zeta} ITAMs. Murine thymocytes were from normal (lanes 1 and 2), TCR-{zeta} ITAM-deficient (lanes 3 and 4; D67–150), TCR-{zeta} single ITAM (lanes 5 and 6; D66–114), or TCR-{zeta} full-length mice (lanes 7 and 8; {zeta} Tg) were either unstimulated (lanes 1, 3, 5, and 7) or stimulated with anti-CD3-{epsilon} mAbs for 3 min (lanes 2, 4, 6, and 8) and subsequently lysed in 1% Triton X-100-containing lysis buffers. Proteins from the lysates were immunoprecipitated with a GST-Grb2 fusion protein (lanes 1–8). The precipitates were resolved on 12.5% SDS-PAGE, and the gels were transferred to PVDF membranes and immunoblotted with an anti-phosphotyrosine mAb (4G10).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
To examine the contribution of the TCR-{zeta} ITAMs on TCR-mediated signal transduction in thymocytes and peripheral lymph node T cells, we analyzed TCR-{zeta}-deficient mice that had been reconstituted with TCR-{zeta} constructs lacking one or more TCR-{zeta} ITAMs.

We have reported previously that in thymocytes and peripheral T cells, the TCR-{zeta} subunit is constitutively tyrosine phosphorylated and associated with the ZAP-70 PTK (29). The constitutive phosphorylation of TCR-{zeta} is, in part, but not entirely a consequence of TCR interactions with MHC molecules in both the thymus and peripheral lymphoid organs (29). In this study, we show that there is no apparent constitutive phosphorylation of TCR-{zeta} subunits containing only the first or third {zeta} ITAM. These results suggest that the constitutive phosphorylation of the TCR-{zeta} subunit requires at least two, and perhaps all three, ITAMs. In fact, it is proposed that one of the roles of tandem-phosphorylated ITAMs is to facilitate the binding and autophosphorylation of ZAP-70 (34). The binding of ZAP-70 to multiple phosphorylated ITAMs may also protect the ITAMs from protein tyrosine phosphatases (35). Alternatively, the constitutive phosphorylation of TCR-{zeta} may reside in the second ITAM. It is also possible that the truncation of the TCR {zeta}-chains in the transgenic mice decreases the accessibility of the Lck PTK, since Lck regulates the constitutive TCR {zeta}-chain phosphorylation in murine thymocytes (7). This seems less likely since any of the three TCR-{zeta} ITAMs, when expressed independently as chimeric proteins, are readily phosphorylated following chimera receptor cross-linking (11). Additional TCR-{zeta} transgenic mice would be needed to address these issues.

The importance of the constitutive tyrosine phosphorylation of the full-length TCR-{zeta} subunit remains unclear at present. Since the ZAP-70 PTK is already complexed to the pool of tyrosine-phosphorylated TCR-{zeta}, the population of ZAP-70/phosphorylated TCR-{zeta} complexes may represent cells undergoing signaling events (29). Alternatively, the TCR complex may be poised or "armed" to respond to TCR interactions, leading to activation of ZAP-70, which is already localized at the plasma membrane. It also has been suggested that the phosphorylated TCR-{zeta} molecules may be blocked in their capacity to respond to TCR stimulations, unless the CD4--associated Lck PTK is co-engaged with the TCR (36, 37). Regardless of which interpretation is correct, our results suggest that a composite of TCR-{zeta} ITAMs is required for the constitutive tyrosine phosphorylation of TCR-{zeta}.

In this study, we also show that none of the CD3 subunits appears to undergo any constitutive tyrosine phosphorylation even in the absence of any TCR-{zeta} ITAMs. However, following TCR ligation, there is a dramatic increase in the induction of CD3 subunit phosphorylation, as well as the recruitment and phosphorylation of ZAP-70. This occurs with all of the CD3 ITAMs, {gamma}, {delta}, and {epsilon} (32). This finding is consistent with the findings of Malissen and colleagues, who showed that the CD3-{gamma}, CD3-{delta}, and CD3-{epsilon} can form an independent signaling module (38). Even in the situation in which a single ITAM of TCR-{zeta} is present as part of the TCR complex, the ITAMs from the CD3 subunits are preferentially phosphorylated relative to TCR-{zeta} following TCR cross-linking. The simplest interpretation of these results is that in the absence of full-length TCR-{zeta}, the CD3 subunits become more accessible to Lck.

The TCR-{zeta} subunit is important for T cell development by promoting surface expression of the TCR (17). Notably, the capacity of TCR-{zeta} to facilitate thymopoeisis does not require {zeta} ITAM-mediated signals (17). This suggests that the remaining CD3 ITAMs (4/TCR complex) in TCR-{zeta}-/- mice reconstituted with ITAM-less TCR-{zeta} transgenes are either compensating for and/or functioning in the absence of TCR-{zeta}. Our results are consistent with these interpretations since, in the absence of TCR-{zeta} ITAMs, the CD3 subunits are inducibly phosphorylated and associated with the ZAP-70 PTK. Moreover, thymocytes expressing low avidity TCRs require more TCR-{zeta} ITAMs for their selection (18). Taken together, the aforementioned data support the concept that the TCR-{zeta} ITAMs function primarily as signal amplifiers during signaling and repertoire selection in the thymus.

One unresolved issue concerning the different TCR ITAMs is whether they couple to distinct signaling pathways or substrates. For example, previous studies have revealed that the TCR-{zeta} ITAMs and the CD3-{epsilon} ITAMs, when expressed as chimeric molecules in cell lines and cross-linked, resulted in the induction of distinct phosphoproteins (20). Yet, the same chimeric constructs, when expressed as transgenes in mice, mediated the phosphorylation of identical substrates (12). In a related signaling system, the ITAM of the Fc{epsilon}RIß serves as a signal amplifier by recruiting the Lyn PTK, which in turn phosphorylates the ITAM of the Fc{epsilon}RI{gamma} (39). Importantly, Lyn and not Syk appears to interact with the phosphorylated Fc{epsilon}RIß ITAM, suggesting some SH2 specificity. However, it should be noted that the distance between the YxxL sequences is only six amino acids, a distance that may not be adequate for Syk-SH2 binding. The idea that different SH2-containing signaling molecules can couple differentially to different phosphorylated ITAMs has been reported in studies mainly involving in vitro binding assays (25, 26, 27, 40). However, the affinities between these SH2-containing proteins and phosphorylated ITAMs are 10- to 100-fold less than that between ZAP-70/Syk and the doubly phosphorylated ITAMs. In our experiments, the induction of a number of phosphoproteins, including SLP-76, vav, Pyk2, ZAP-70, p36, and p130, occurs with TCR complexes comprising only CD3 ITAMs or complexes, including the third ITAM of TCR-{zeta}. These results would indicate a substantial amount of redundancy exists between the different ITAMs. This is also supported by the reports that both CD3 {epsilon}- and TCR {zeta}-chain promote programmed cell death in thymocytes, and induce double-positive development in a Rag- mice (12). The implication of these findings is that the ITAMs function primarily to couple to the ZAP-70/Syk family of PTKs. It is possible that the different ITAMs may still interact with distinct substrates; however, the importance of such interactions will require further study.


   Acknowledgments
 
We thank members of the Weiss laboratory for helpful discussions and suggestions. A. Weiss is a member of Howard Hughes Medical Institute.


   Footnotes
 
1 This work was supported in part by a grant from Human Frontier Science Program (LT-505/93 to N.S.C.v.O.), and a National Institutes of Health grant (GM-39553 to A.W.). Back

2 Current address: Department of Microbiology, The University of Texas Southwestern Medical Center, 6000 Harry Hines Blvd., Dallas, TX 75235-9140. Back

3 Address correspondence and reprint requests to Dr. A. Weiss, Howard Hughes Medical Institute, U-330, 3rd and Parnassus Avenues, UCSF, San Francisco, CA 94143-0724. Back

4 Abbreviations used in this paper: ITAM, immunotyrosine-based activation motif; GST, glutathione-S-transferase; PTK, protein tyrosine kinase; PVDF, polyvinylidene fluoride; SH2, Src homology 2. Back

Received for publication June 16, 1997. Accepted for publication September 23, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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