©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
A Comparison of the Interaction of Shc and the Tyrosine Kinase ZAP-70 with the T Cell Antigen Receptor Chain Tyrosine-based Activation Motif (*)

Narin Osman , Susan C. Lucas , Helen Turner , Doreen Cantrell (§)

From the (1) Lymphocyte Activation Laboratory, Imperial Cancer Research Fund, London WC2A 3PX, United Kingdom

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Tyrosine-based activation motifs (TAMs) define a conserved signaling sequence, EXYXL/IXYXL/I, that couples the T cell antigen receptor to protein tyrosine kinases and adapter molecules. The present study shows that phosphorylation of both tyrosines within the motif is required for high affinity binding of the tyrosine kinase ZAP-70 whereas phosphorylation of the single COOH-terminal tyrosine within the motif is optimal for the binding of the adapter Shc. There were also quantitative differences in the ZAP-70 and Shc association with the 1-TAM since nM concentrations of the doubly phosphorylated 1-TAM are sufficient for ZAP-70 recruitment whereas micromolar levels of singly phosphorylated TAMs are necessary for Shc binding. Shc is tyrosine phosphorylated in antigen receptor-activated T cells and can potentially form a complex with the adapter molecule Grb2 and could thus recruit the Ras guanine nucleotide exchange protein Sos into the antigen receptor complex. The present data show that Grb2 can bind to the phosphorylated TAM, but this binding is independent of Shc and there is no formation of 1-TAMShcGrb2Sos complexes in antigen receptor-activated cells. Accordingly, Shc function should not be considered in the context of Grb2/Sos recruitment to the T cell antigen receptor complex.


INTRODUCTION

T cell activation is controlled by the antigen receptor (TCR)() CD3 complex which comprises the polymorphic TCR and subunits, the invariant polypeptide chains of the CD3 complex (, , and ), and the non-covalently associated chains. The capacity of the TCR to transduce signals across the T cell membrane is mediated by the cytoplasmic domains of these invariant chains of the TCR complex (1, 2, 3) . The intracellular tails of the CD3 and molecules contain a common motif, EXYXL/IXYXL/I, which is present in a single copy in each of the CD3 chains and triplicated in the subunit (4) . This activation motif is termed the tyrosine-based activation motif (TAM) and is crucial for TCR coupling to intracellular protein tyrosine kinases and hence absolutely required for all subsequent T cell responses (2, 3, 5) . TAM motifs are also found in the Ig and Ig subunits of the B cell antigen receptor and in the and subunits of the FcRI and FcRIIIA receptors (4, 6, 7). As well, two viral proteins, the gp130 envelope protein of bovine leukemia virus and the latent membrane protein 2A of Epstein-Barr virus (8) may use this evolutionarily conserved signaling motif to activate lymphocytes.

The earliest biochemical response elicited by the TCR is the activation of protein tyrosine kinases (9) . One essential protein tyrosine kinase controlled signaling pathway to originate from the TCR involves the guanine nucleotide-binding proteins p21ras (10) . A recent report has described that the adapter protein Shc, which is a substrate for TCR-activated protein tyrosine kinases, can bind to the TCR chain (11). By analogy with the role of Shc in fibroblasts, the coupling of Shc and the subunit suggests a possible mechanism for linking the TCR to the Ras guanine nucleotide binding cycle. Thus T cells express a Ras guanine nucleotide exchange protein Sos, the homologue of the Drosophila ``son of sevenless'' gene product (12) . Sos complexes to the adapter protein Grb2/Sem5 (13) which is composed of one SH2 domain and two SH3 domains. The SH3 domains of Grb2 bind to Sos whereas the interaction between the Grb2 SH2 domain and tyrosine phosphorylated molecules such as Shc may regulate the function-cell localization of Sos (13, 14, 15, 16) .

Transfection of chimeric receptors where the receptor cytoplasmic tail comprises a single TAM which contains 2 tyrosine residues is sufficient to activate TCR signal transduction pathways (2, 3, 5) . Initial studies suggested that mutation of either tyrosine within a TAM would destroy distal signaling functions. The 2 tyrosine residues within the TAM are necessary for the SH2 domain-mediated association of the protein tyrosine kinase ZAP-70 with the TCR complex (5, 17) . ZAP-70 has two SH2 domains both of which seem necessary for interaction with the chain (17, 18). The binding of Shc to the chain requires TAM phosphorylation, but the relative importance of phosphorylation of the 2 tyrosine residues within a TAM for Shc binding have not been determined. Similarly, it has been described that ZAP-70 binds to nanomolar levels of phosphorylated TAMs, but similar quantitation of the Shc interaction with the phosphorylated TAM has not been explored. Accordingly, the object of the present study was to compare the phosphorylation requirements for ZAP-70 and Shc binding to the 1-TAM. The ability of TAM-associated Shc to form a complex with the adapter Grb2 and the Ras exchange protein Sos was also examined.


EXPERIMENTAL PROCEDURES

Cells, Antibodies, and Synthetic Oligopeptides

Human peripheral blood-derived T cells were prepared as described previously (19). The following antibodies were used in this study: CD3 mAb UCHT1 kindly provided by Prof. Peter Beverley (University College, London, United Kingdom), Grb2 mAb was purchased from Affiniti (Nottingham, UK), Sos and Shc polyclonal antisera and phosphotyrosine (pTyr) mAb 4G10 were purchased from Upstate Biotechnology Inc., and ZAP-70 polyclonal antisera was generously given by Dr. Joseph Bolen (Bristol Myers Squibb). Peptides corresponding to the sequence of the membrane proximal TAM of the human TCR chain (1, residues 49-65, QLYNELNLGRREEYDVL) were synthesized by Nicola O'Reilly (ICRF, London UK), with either both tyrosines unphosphorylated (1-YY), both tyrosines phosphorylated (1-pYpY) or with a single tyrosine phosphorylated i.e. residue 51 phosphorylated and residue 62 unphosphorylated (1-pYY), residue 51 unphosphorylated and residue 62 phosphorylated (1-YpY). Control phosphopeptides corresponded to tyrosine phosphopeptides with defined SH2-domain binding capacity: EGFR-Y1068 autophosphorylation site of the human EGFR (PVPEpYINQS); TRK-Y490 the binding site for Shc (IENPQpYFSDA); PDGFR-Y751-binding site for p85 subunit of phosphatidylinositol 3`-kinase and Nck (SVDpYVPMLDMK) and PDGFR-Y1009-binding site for Syp (PVPEpYINQS). GST fusion proteins of the proline-rich carboxyl-terminal region of Sos (GST Sos) and wild type Grb2 (GST Grb2) were generated and purified as described previously (13, 14) .

T cells (2 10/ml or as indicated) were either unstimulated or were activated via the TCRCD3 complex using the anti-CD3 antibody UCHT1 (10 µg/ml) or with 20 ng/ml of IL2 for 3 min, or as indicated, at 37 °C. Cells were pelleted, lysed for 20 min on ice in 1 ml of immunoprecipitation buffer containing 1% Nonidet P-40, 150 mM NaCl, 50 mM HEPES, pH 7.5, 10 mM iodoacetamide, 1 mM phenylmethylsulfonyl fluoride, 1 mM sodium orthovanadate, and 1 mg/ml each of antipain, chymostatin, leupeptin, and pepstatin and immunoprecipitated as described previously (20) . Precipitations using synthetic peptides were performed using 75 nM-25 µM peptide as indicated coupled to Affi-Gel 10 affinity matrix (Bio-Rad). Peptides were coupled according to the manufacturer's recommendations. Precipitations with GST fusion proteins used 5 µg of fusion protein immobilized on glutathione-agarose beads.

Western Blotting

Peptide precipitates or acetone-precipitated protein from whole cell lysates were resolved by 10% SDS-polyacrylamide gel electrophoresis and electroblotted at 0.35 A, 60 V for 5 h onto polyvinylidene difluoride membranes (Millipore, UK). Filters were blocked overnight at 4 °C with 5% non-fat milk protein in phosphate-buffered saline, washed in phosphate-buffered saline, 0.05% Tween 20 and probed with primary antibody as indicated. Horseradish peroxidase-coupled anti-rabbit or anti-mouse IgG were used as second stage reagents as required to allow detection by enhanced chemiluminescence (ECL, Amersham, UK). Quantitation of ECL bands was by densitometry of autorads as recommended by the manufacturer.


RESULTS

ZAP-70 and Shc Bind to Tyrosine-phosphorylated 1-TAM

In initial experiments we compared the binding of Shc and ZAP-70 to the tyrosine-phosphorylated 1-TAM. To determine the relative importance of phosphorylation of the different tyrosine residues in the protein interactions of TAMs, we used 1-TAM peptides (see ``Experimental Procedures'') that were unphosphorylated (1-YY) or singly phosphorylated on either the NH-terminal (1-pYY) or COOH-terminal (1-YpY) tyrosine or phosphorylated on both tyrosines (1-pYpY). We selected the 1-TAM for these analyses on the basis that when expressed as a receptor chimera the 1-TAM is sufficient for the induction of early and late T cell activation events (5) . Equivalent amounts of each of the 1-TAM peptides coupled to agarose matrix were used to precipitate proteins from Nonidet P-40 lysates of peripheral blood-derived T cells. Bound proteins were eluted and resolved by SDS-polyacrylamide gel electrophoresis, transferred electrophoretically to PVDF membranes, and subjected to Western blot analysis with anti-ZAP-70 or anti-Shc antibodies. The data in Fig. 1a show that the doubly phosphorylated -TAM peptide precipitates ZAP-70 and Shc from T cell lysates. ZAP-70 and Shc did not bind to the non-phosphorylated 1-YY peptide or to any of the control tyrosine phosphopeptides used: the EGFR-Y1068, the PDGFR-Y751, or the PDGFR-Y1009 (Fig. 1a). Densitometry measurements showed that relative to whole cell lysate the 1-pYpY peptide precipitated a major fraction of the total cellular ZAP-70 (approximately 70%) and the 1-pYY peptide somewhat less (approximately 30%), but the 1-YpY peptide was very inefficient in binding ZAP-70 precipitating approximately 5% of the total (Fig. 1a). There was no detectable ZAP-70 associated with the non-phosphorylated 1-YY peptide. The most efficient peptide for Shc binding was the singly phosphorylated 1-YpY peptide (2% of total Shc), the doubly phosphorylated peptide 1-pYpY was less efficient, and Shc bound very poorly to the 1-pYY peptide and did not bind to non-phosphorylated peptide 1-YY (Fig. 1a). Thus, ZAP-70 and Shc show a preference for binding to different tyrosine residues when only a single tyrosine within the 1-TAM is phosphorylated. Anti-Shc immunoblots of total cell lysates show that T cells express the 46- and 52-kDa isoforms of Shc (Fig. 1). Both forms of Shc bind to the phosphorylated 1-TAM although there was a clear preferential binding of the 52-kDa Shc molecule (Fig. 1a). The 1-pYpY peptides precipitated a major fraction (approximately 70%) of the total cellular ZAP-70 but were much less efficient in binding Shc, only precipitating approximately 1% of the total Shc protein (Fig. 1a). The optimal TAM peptide for Shc binding, 1-YpY, was also relatively inefficient for Shc binding, preclearing 2% of total Shc from cell lysate.


Figure 1: ZAP-70 and Shc associate with the 1-TAM. T cells (2 10/point) were lysed, and proteins were precipitated as follows: a, with 25 µM 1-YY, 1-pYY, 1-YpY, 1-pYpY or EGFR-Y1068, TRK-Y490, PDGFR-Y751 or PDGFR-Y1009 phosphopeptide or were acetone-precipitated (Lysate). Lysate in anti-ZAP-70 immunoblot is from 5 10 cells. b, T cells (2 10/point) were lysed and precipitated with 25 µM 1-YpY peptide in the presence of increasing concentrations of TRK-Y490 peptide as shown. Western blots were probed with anti-ZAP-70 or anti-Shc antibodies as indicated.



As a positive control for these Shc binding experiments, the Shc interaction with the -TAM was compared to the binding of Shc to a tyrosine phosphopeptide, TRK-Y490, which has been previously described as a high affinity binding site for the Shc SH2 domain (21) . The data in Fig. 1a show that TRK-Y490 binds Shc more efficiently than 1-YpY. Hence 25 µM of TRK-Y490 precleared 95% of the Shc from a cell lysate whereas an equivalent amount of 1-YpY removed only 2% of Shc (Fig. 1a). Moreover, there was a clear preferential binding of the 52-kDa Shc molecule to 1-TAM peptides whereas the TRK-Y490 peptide is less discriminatory for the different isoforms of Shc. The TRK-Y490 peptide did not bind ZAP-70 (Fig. 1a).

The data in Fig. 1a show that Shc binding to 25 µM 1-YpY was completely inhibited by the TRK-Y490 peptide. TRK-Y490 did not compete ZAP-70 binding to 1-YpY (Fig. 1b). We have also examined the binding of ZAP-70 and Shc to tyrosine-phosphorylated peptides corresponding to the two other -TAMs, 2 and 3. These peptides also associated efficiently with ZAP-70 but much more weakly with Shc, exactly analogous to the low efficiency 1-TAM/Shc association (data not shown).

To examine further the interactions of phosphorylated 1-TAM peptides with ZAP-70 and Shc, we precipitated proteins from T cell lysates with a titration of concentrations of the TAM peptides (25 µM to 75 nM). The data in Fig. 2 show titrations of the optimal peptides for ZAP-70-binding 1-pYpY and 1-pYY and the optimal peptide for Shc-binding 1-YpY. These data (Fig. 2) show that the doubly phosphorylated 1-pYpY peptide binds ZAP-70 at concentrations as low as 75 nM. In contrast, equivalent binding of ZAP-70 to the singly phosphorylated peptide 1-pYY required 1.5 µM of peptide (Fig. 2b). Thus ZAP-70 binds with approximately a 20-fold higher affinity to a double phosphorylated TAM peptide than to a single phosphorylated TAM peptide. To analyze the interaction of Shc with 1-YpY, we precipitated T cell lysates with a titration of 1-YpY peptide (25 µM to 150 nM) and Western blotted with anti-Shc antibody (Fig. 2c). The Shc association with 1-YpY was only detected with concentrations >7.5 µM of peptide (Fig. 2c). The differences in the peptide concentrations required for ZAP-70 versus Shc binding show that the 1-TAM peptides bound Shc with low efficiency compared to 1-TAM/ZAP-70 binding.


Figure 2: Quantitation of ZAP-70 and Shc binding to phosphorylated 1-TAM. Proteins from Nonidet P-40 lysates of T cells (2 10/point) were precipitated with the following phosphopeptides coupled to Affi-Gel 10 (a) 15 µM, 1.5 µM, 150 nM or 75 nM 1-pYpY peptide or (b) 15 µM, 1.5 µM, 150 nM, or 75 nM 1-pYY peptide or (c) 25 µM, 15 µM, 7.5 µM, 1.5 µM, 150 nM 1-YpY peptide. Western blots were probed with anti-ZAP-70 or anti-Shc antibodies as indicated.



The effectiveness of the TAMs in affinity purifying Shc and ZAP-70 from T cell lysates is determined by the relative affinity of Shc or ZAP-70 for the TAMs and the intracellular concentrations of the two proteins. Thus the low efficiency of the 1-TAM-Shc interaction compared to the 1-TAM/ZAP-70 binding could reflect differences in the affinity of Shc and ZAP-70 for the 1-TAM or it could reflect that cellular concentrations of Shc are lower than ZAP-70 levels. The present data do not resolve these two possibilities. However, as a comparison we carried out peptide competition experiments with the TRK-Y490 peptide and the phosphorylated 1-TAM peptides. These studies indicated that nM levels of TRK-Y490 were sufficient for Shc binding compared to the µM 1-YpY peptide concentrations required for Shc binding (data not shown). The high affinity of TRK-Y490 for Shc compared to the 1-TAMs is supported by the data in Fig. 1that show that TRK-Y490 can efficiently preclear >95% of Shc from T cell lysates whereas the 1-TAMs could purify a maximum of 1-2% of cellular Shc.

The Adapter Proteins Shc and Grb2 Associate with Tyrosine-phosphorylated 1-TAM but No 1-TAMShcGrb2Sos Complexes Are Detected

Shc is tyrosine phosphorylated in TCR-activated T cells (11) . Anti-phosphotyrosine Western blots of pTyr precipitates and acetone precipitates of whole cell lysates showed a marked increase in tyrosine phosphoproteins following TCR stimulation (Fig. 3a). Tyrosine-phosphorylated proteins were not seen in 1-TAM peptide precipitates from unstimulated cells (Fig. 3a). ZAP-70 is tyrosine phosphorylated in TCR-activated cells, and a major tyrosine-phosphorylated 70 kDa band coprecipitated with the 1-pYpY peptide following stimulation as well as several weaker bands between 80 and 90 kDa. A very small amount of the tyrosine-phosphorylated 70 kDa band was also found to precipitate with the 1-pYY peptide in stimulated cells (5-10% of that seen with the 1-pYpY peptide). No 46- or 52-kDa tyrosine phosphoproteins corresponding to tyrosine-phosphorylated Shc were seen in anti-pTyr Western blots of 1-TAM precipitates from unstimulated or TCR-activated cells (Fig. 3a). As a positive control, Western blot analysis of Shc precipitates with anti-pTyr antibodies confirmed the tyrosine phosphorylation of Shc in TCR-activated cells (data not shown).


Figure 3: Association of pTyr proteins with the 1-TAM. a, T cells (2 10/point) were either unstimulated or stimulated with CD3 mAb UCHT1 (10 µg/ml) for 3 min and were then lysed and proteins precipitated with 25 µM of the following phosphopeptides coupled to Affi-Gel 10: 1-YY, 1-pYY, 1-YpY, 1-pYpY, EGFR-Y1068 as indicated or pTyr mAb coupled to protein A-Sepharose or were acetone-precipitated (Lysate, 5 10 cells). Western blots were probed with anti-pTyr mAb. Molecular mass markers are in kDa. b, T cells (2 10/point) were left unstimulated or were stimulated with CD3 antibody UCHT1 (10 µg/ml) for the times indicated. Cells were lysed, and proteins were precipitated with GSTGrb2 fusion protein. Acetone-precipitated whole cell lysates (Lysate, 5 10 cells) were run in an adjacent lane. Western blots were probed with anti-Shc antibody.



Tyrosine-phosphorylated Shc has the potential to form a complex with the adapter protein Grb2 (15, 22) which has been shown to couple the guanine nucleotide exchange factor Sos to activated growth factor receptors (13, 15) . Therefore, we examined in T cells whether Grb2, Shc, and the 1-TAM are associated. First, and consistent with the TCR-induced tyrosine phosphorylation of Shc, the data show that a Grb2 GST fusion protein can bind in vitro to the 52-kDa isoform of Shc in T cell lysates prepared from TCR-activated but not quiescent T cells (Fig. 3b). The data (Fig. 4a) show also that Grb2 binds to tyrosine-phosphorylated 1-TAM peptides interacting preferentially with a 1-pYpY peptide and binding less efficiently to the 1-pYY peptide. Grb2 did not bind to the 1-YY or 1-YpY peptides. Grb2 binds to the 1-TAM via its SH2 domain as judged by competition experiment where proteins from T cell lysates were precipitated with 1-pYpY in the presence of increasing concentrations of a peptide that binds with high affinity to the Grb2 SH2 domain, EGFR Y1068. (15, 23, 24) . The data (Fig. 4a) show a high level of Grb2 binding in the EGFR-Y1068 peptide precipitates.Fig. 4b shows that 2.5 µM EGFR-Y1068 peptide effectively competed for Grb2 binding to 25 µM of 1-pYpY but did not compete ZAP-70 binding to 1-pYpY. In Fig. 4c we precipitated unactivated T cell lysates with a titration of concentrations of the 1-pYpY peptide coupled to agarose (15 µM to 75 nM). Western blot analyses (Fig. 4c) showed that Grb2 binding to 1-pYpY was detected only with peptide concentrations higher than 1.5 µM compared to ZAP-70 binding which was readily observed with 75 nM of peptide (Fig. 4c, cf.Fig. 2a).


Figure 4: A comparison of the Grb2/Shc association with the 1-TAM. Anti-Shc, anti-Grb2, or anti-Sos Western blots of (a) proteins precipitated from unstimulated or UCHT1-stimulated T cell (2 10) lysates with 25 µM peptides coupled to Affi-Gel 10: 1-YY, 1-pYY, 1-YpY, 1-pYpY, EGFR-Y1068 as indicated or acetone-precipitated (Lysate 5 10 cells). b, proteins precipitated from unstimulated T cell lysates (2 10/point) with 25 µM 1-pYpY peptide in the presence of increasing concentrations of EGFR-Y1068 peptide as indicated. c, proteins precipitated from T cell lysates (2 10/point) with concentrations of 15 µM, 1.5 µM, 150 nM, or 75 nM 1-pYpY peptide as shown.



These experiments show that Grb2 can bind to the 1-TAM. However, the ability of the 1-TAM peptides to bind to Grb2 in lysates of quiescent T cells was apparently not mediated by Shc as judged by the completely different pattern of reactivity of these two proteins with the different 1-TAM tyrosine phosphopeptides (Fig. 4a). In particular Shc bound preferentially to the 1-YpY peptide which did not bind Grb2. In TCR-activated cells the pattern of Shc reactivity with the 1-TAM was very similar to the pattern from unactivated cells (Fig. 4a). Moreover, the reactivity pattern of Grb2 with the 1-TAM peptides did not change upon T cell activation (Fig. 4a) whereas the ability of tyrosine phosphorylated Shc to bind to the Grb2 fusion protein was TCR induced (Fig. 3b).

The SH3 domains of Grb2 bind to effector proteins that include the Ras exchange protein Sos. Two other Grb2 SH3-binding proteins of 75 and 116 kDa, that are both substrates for TCR activated protein tyrosine kinases, have also been described in T cells (25, 26) . Shc interactions with the TCR chain were proposed as a mechanism to recruit Grb2 and hence the Ras exchange protein Sos into the TCR complex although Grb2 or Shc/Grb2 could equally recruit p75 or p116 into the TCR complex. The data in Fig. 3a show that no 75- or 116-kDa tyrosine phosphoproteins were detected in the 1-TAM precipitates. Moreover, Western blot analysis failed to detect Sos in the 1-TAM precipitates isolated from TCR-activated or quiescent T cells (Fig. 4a). The EGFR-Y1068 peptide can affinity purify the total cellular pool of Grb2 from T cells and hence the subpopulation of Grb2Sos complexes. As a positive control, the data show that our Western blot analyses could detect Sos in EGFR-Y1068 peptide precipitates.

In further experiments to explore whether 1-TAMShcGrb2Sos complexes form, we analyzed the tyrosine phosphoproteins associated with the SH2 domains of endogenous Grb2 in TCR-activated cells. It is well established that IL2 induces formation of ShcGrb2 complexes in T lymphocytes (27) . Therefore, as a positive control, Grb2 complexes from TCR-activated cells were compared with the Grb2 complexes isolated in parallel from IL2-activated cells. The data in Fig. 5show that in IL2-activated T cells a 52-kDa tyrosine phosphoprotein corresponding to Shc can be detected in the endogenous Grb2 complexes. The identity of this 52-kDa protein as Shc was confirmed by Western blot analyses with specific Shc antisera (data not shown). The data in Fig. 5show also that a TCR-induced 36-kDa tyrosine phosphoprotein is detected in the endogenous Grb2 complexes, but no 52-kDa tyrosine phosphoprotein corresponding to Shc was observed. Thus in TCR-activated cells, Shc binding to GST fusion proteins of Grb2 can be observed (Fig. 3b), but in vivo ShcGrb2 complexes are not detected (Fig. 5). Similarly, no 16-21-kDa tyrosine phosphoproteins corresponding to the TCR chain could be detected in the endogenous Grb2 complexes (Fig. 5). In this context, in repeated experiments we have failed to demonstrate coprecipitation of endogenous chainGrb2 or chainShc complexes in peripheral blood T cells (Fig. 5, data not shown).


Figure 5: A comparison of the TCR- and IL2-induced Grb2-binding tyrosine phosphoproteins. Antiphosphotyrosine Western blots of the endogenous Grb2 complexes precipitated from unstimulated, UCHT1, or IL2-activated T cells (2 10/point) with the GST Sos fusion protein. GST Sos binds to the SH3 domains of Grb2 and efficiently purifies the total cellular pool of Grb2 and any associated Grb2 SH2-binding proteins.




DISCUSSION

The present data show that the phosphorylation requirements for the binding of the 1-TAM to ZAP-70 and Shc were quite distinct. ZAP-70 bound preferentially to the doubly phosphorylated motif (1-pYpY) whereas Shc bound preferentially to a TAM with a single phosphorylation of the COOH-terminal tyrosine residue (1-YpY). ZAP-70 could also bind to a singly phosphorylated TAM but had a marked preference for a TAM in which the NH-terminal tyrosine was phosphorylated (1-pYY), binding extremely poorly to the COOH terminally phosphorylated motif and not at all to the non-phosphorylated TAM. The presence of the 2 tyrosines within a TAM is proposed as a prerequisite for optimal ZAP-70 association (2, 5, 6, 7, 28) . The present data are in accord with this model since the affinity of ZAP-70 for the doubly phosphorylated TAM was 20-fold higher than its estimated affinity for the singly phosphorylated 1-pYY. The pattern of reactivity of ZAP-70 and Shc with the different phosphoforms of the 1-TAM highlights the specificity of these interactions as does the control experiments carried out with a range of tyrosine phosphopeptides. Interestingly, Shc clearly binds with less efficiency to the doubly phosphorylated 1-TAM than to the motif containing a single COOH-terminal phosphotyrosine despite identical sites being present in both TAMs. This suggests that phosphorylation of the NH-terminal tyrosine in the TAM may result in allosteric effects which interfere with Shc binding to the COOH-terminal tyrosine. Moreover, the hierarchy of reactivity of ZAP-70 and Shc with the different phosphoforms of the 1-TAM and the preferential binding of Shc and ZAP-70 to different phosphotyrosine residues within the 1-TAM may allow the binding of two molecules to a doubly phosphorylated TAM, thereby facilitating their interaction.

Recently, the interaction of Shc and the TCR chain was proposed to couple the TCR to the Ras signaling pathway by recruiting Grb2Sos into the TCR complex (11) . The present data confirm that Shc is capable of binding to a phosphorylated 1-TAM but show that this association is relatively inefficient. The best in vitro binding data showed that only a small subpopulation of predominantly the 52-kDa isoform Shc (<2%) bound to the 1-TAM, and since no 46- or 52-kDa tyrosine phosphoproteins bound to the TAM peptides it seems that tyrosine-phosphorylated Shc does not bind to the TAM peptides. Shc isolated from TCR-activated cell lysates could bind to a Grb2 GST fusion protein confirming the potential for Shc and Grb2 to form a complex in T cells. However, TCR-induced endogenous TAMShcGrb2 complexes were not detected. Nevertheless, Grb2 could bind to the phosphorylated 1-TAM, but this association was not influenced by TCR activation. The independence of Shc/Grb2 association with the 1-TAM is also indicated by the different preference of the two molecules for different phosphorylated forms of the 1-TAM. Thus, Grb2 binds best to the doubly phosphorylated TAM and to the peptide singly phosphorylated on the NH-terminal phosphotyrosine whereas Shc bound poorly to the doubly phosphorylated TAM and of the singly phosphorylated peptides had a preference for the COOH-terminal phosphotyrosine. Grb2 can bind via its SH3 domains to the Ras guanine nucleotide exchange protein Sos, but no Sos was ever detected in 1-TAM precipitates and it is unlikely that Shc or Grb2 binding to the chain recruits Sos to the TCR complex. In particular, although ShcGrb2 complexes can be readily detected in IL2-activated T cells they cannot be detected in cell lysates isolated from TCR-activated cells. Rather, an alternative model for Sos/p21ras regulation in T cells is suggested by observations that Sos-associated Grb2 forms a complex predominantly with a 36-kDa tyrosine phosphoprotein in TCR-activated cells (26) .

The failure of TAM-associated Shc or Grb2 to recruit Sos does not mean that the Shc and Grb2 binding to the chain is irrelevant because it is unlikely that the cellular function of Shc and Grb2 is limited solely to a role in Sos cellular localization-p21ras regulation. For example, Grb2 SH3 domains do not only bind Sos but are also constitutively associated with 75- and 116-kDa proteins of unknown function (25, 26) . We also failed to detect p75 or p116 in the TAM complexes, but the principal still applies that there are multiple Grb2 effector complexes in T cells and Grb2 function should not just be considered in the context of the Grb2Sos interaction. Similarly, it has been described that Shc can associate with a 150-kDa tyrosine phosphoprotein in TCR-activated cells (29) . Accordingly, Shc function in T cells should not only be considered in the context of Grb2Sosp21ras regulation.

The present data confirm that Shc can specifically bind a tyrosine-phosphorylated TAM and that this binding is inhibited by a tyrosine phosphopeptide Trk Y490 previously described as a high affinity binding site for the Shc SH2 domain. Recently, a second phosphotyrosine-binding domain in Shc has been described that is distinct to the SH2 domain (30) . The TAMs were predicted as binding sites for the SH2 domains of Shc (31) , and a GST fusion protein of the Shc SH2 domains will bind to a tyrosine-phosphorylated TAM. However, whether TrkY490 and the TAM recognize the SH2 domain or the other phosphotyrosine-binding site on Shc is not yet known. In the present study, competition and peptide titration experiments indicated that Shc will only bind to micromolar levels of 1-YpY. Grb2 binding to the phosphorylated 1-pYpY peptide was also only observed when micromolar concentrations of peptides were used compared to the nanomolar range affinity of ZAP-70 for this doubly phosphorylated TAM. Thus there are clear quantitative differences between the ZAP-70 and Shc interaction with the phosphorylated 1-TAM. The relatively high efficiency of ZAP-70/TAM binding would enable ZAP-70 to form stable complexes with the tyrosine-phosphorylated chain whereas the much lower efficiency of Shc and Grb2 interaction with the TAM may be insufficient for stable recruitment of these adapters to the subunits in endogenous TCR complexes. ZAP70 binding to the TCR is readily detectable in coprecipitation experiments which is consistent with the high efficiency of ZAP70/TAM binding shown herein. In contrast, we found that endogenous TCRShc complexes or TCRGrb2 complexes were not detectable, but this was also predictable from the current binding data showing that the Shc and Grb2 interactions with the TAMs are inefficient. The efficiency of the Shc or Grb2/TAM binding may thus only allow transient associations that are difficult to maintain during cell lysis and purification of receptor complexes. Nevertheless, these interactions might be sufficient in vivo to facilitate phosphorylation of Shc or Grb2-associated proteins by protein tyrosine kinases such as ZAP-70 that are stably associated with high affinity within the TCR complex.


FOOTNOTES

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

§
To whom correspondence should be addressed. Tel.: +44-71-269-3307; Fax: +44-71-269-3417.

The abbreviations used are: TCR, T cell antigen receptor; EGFR, epidermal growth factor receptor; PDGFR, platelet-derived growth factor; Sos, Son of Sevenless; TAM, tyrosine-based activation motif; ZAP 70, -associated protein kinase; mAb, monoclonal antibody; GST, glutathione S-transferase; IL-2, interleukin 2.


ACKNOWLEDGEMENTS

We thank Nicola Reilly and Elizabeth Li for the synthesis and purification of the tyrosine phosphorylated peptides.


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