©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
SH2 Domain Function Is Essential for the Role of the Lck Tyrosine Kinase in T Cell Receptor Signal Transduction (*)

(Received for publication, July 5, 1995; and in revised form, January 9, 1996)

David B. Straus (1)(§) Andrew C. Chan (2)(¶) Barbara Patai (1) Arthur Weiss (2) (3)

From the  (1)Department of Medicine, University of Chicago, Chicago, Illinois 60637 and the (2)Department of Medicine, (3)Howard Hughes Medical Institute, University of California, San Francisco, California 94143

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Tyrosine kinase activity is required for signal transduction through the T cell antigen receptor (TCR). The Src family tyrosine kinase Lck appears to play a key role in the initiation of TCR signaling events. We have investigated the role of the phosphotyrosine-binding Src homology-2 (SH2), domain of Lck in TCR signaling. Lck containing a mutation in the phosphotyrosine binding pocket of the SH2 domain was expressed in an Lck-deficient cell line. We found that, in contrast to wild-type Lck, the SH2 domain mutant was unable to restore even the earliest TCR-mediated signaling events. To investigate the role of the Lck SH2 domain, we examined the association of tyrosine phosphoproteins with Lck. The predominant associated phosphoprotein was the ZAP-70 tyrosine kinase, which has also been implicated in the initiation of TCR signaling. In addition, the subunit of the T cell receptor was found to weakly associate with Lck. Further analysis indicated that the SH2 domain of Lck can directly recognize both ZAP-70 and in immunoprecipitates from TCR-stimulated cells. Our findings demonstrate that the SH2 domain of Lck is essential for the initiation of signaling events following TCR stimulation probably as a result of its ability to mediate an interaction between Lck and the ZAP-70 tyrosine kinase and/or the subunit of the T cell receptor.


INTRODUCTION

Stimulation of the T cell antigen receptor (TCR) (^1)initiates intracellular signaling events that lead to T cell differentiation and proliferation. Several pathways have been implicated in this activation process, notably the induction of tyrosine phosphorylation and the phosphatidylinositol and Ras signaling pathways(1, 2) . Cross-linking of the TCR results in the recruitment and apparent activation of protein-tyrosine kinases, which then leads to the phosphorylation of downstream substrates. The increase in tyrosine phosphorylation of cellular proteins is a crucial event in TCR signaling(3) . Tyrosine phosphorylation of phospholipase C-1 is increased following TCR stimulation(4, 5) , which leads to an induction of enzymatic activity and an increase in the second messengers of the phosphatidylinositol pathway. Activation of the Ras pathway also requires protein-tyrosine kinase activity (6) . Ras may become activated through phosphotyrosine-dependent recruitment of a guanine nucleotide exchange factor to the membrane (7) or through the tyrosine phosphorylation and activation of Vav(8) .

At least two distinct types of protein-tyrosine kinases have been implicated in TCR signal transduction. The ZAP-70 tyrosine kinase, homologous to Syk, associates with the tyrosine-phosphorylated cytoplasmic domains of the TCR subunits following receptor stimulation (9, 10) . Patients with a rare form of severe combined immunodeficiency have been identified whose T cells fail to express ZAP-70. These patients exhibit a defect in the development of CD8+ T cells, and their peripheral CD4+ T cells fail to respond to stimulation through the TCR (11, 12, 13) . In addition to ZAP-70, the Src family of tyrosine kinases has been implicated in the initiation of signaling through the TCR. Expression of the activated forms of either the Fyn or Lck tyrosine kinases leads to enhanced tyrosine phosphorylation following TCR stimulation(14, 15, 16) . Fyn can be found associated with the TCR(17) , and mature thymocytes that are homozygous for null alleles of Fyn show defects in TCR-mediated signal transduction(18, 19) . Mice that lack a functional Lyk gene show a severe defect in thymocyte development(20) . This may be related to a deficit in TCR function since thymocytes that carry null alleles at the TCR beta locus are blocked at the same stage of development(21) . Direct evidence for the role of Lck in TCR signaling has come from the analysis of cell lines that are deficient in Lck function(22, 23) . These mutant cell lines fail to respond to stimulation through the TCR. Previously we have shown that signaling events are blocked at the earliest stages following TCR stimulation in an Lck kinase-deficient derivative of the Jurkat cell line(22) . Subsequent work using a COS cell reconstitution system has indicated that one function of Lck in the initiation of TCR signaling is to mediate the association of ZAP-70 with the receptor through the phosphorylation of the cytoplasmic domains of the TCR(24) .

To further examine the action of Lck in the initiation of TCR signaling events, we have investigated the role of the Lck Src homology 2 (SH2) domain. SH2 domains can mediate protein interactions by virtue of their ability to bind phosphotyrosine residues, and they are present in a large number of signal-transducing proteins(25) . The SH2 domains of the Src family kinases are thought to participate in the control of kinase activity through an intramolecular interaction with a carboxyl-terminal phosphotyrosine residue(26) . Consistent with this, deletion of the Lck SH2 domain enhances its oncogenic potential in fibroblasts(27) . However, such a deletion also inhibits the ability of an activated form of Lck to increase tyrosine phosphorylation in T cells, suggesting that the SH2 domain may also have a positive role in Lck function(28) . We have taken advantage of the Lck-deficient cell line JCaM1 to examine how the phosphotyrosine binding function of the SH2 domain of Lck might contribute to the initiation of TCR signal transduction.


MATERIALS AND METHODS

Cells and Plasmids

The Jurkat leukemic T cell line and its Lck kinase-deficient derivative, JCaM1(22, 29) , were grown in RPMI 1640 supplemented with 10% fetal calf serum, glutamine, penicillin, and streptomycin. Transfectants of JCaM1 were obtained by electroporation of 10^7 cells with 20-40 µg of plasmid DNA using 0.4-cm cuvettes at 250 V, 960 microfarads in Hepes-buffered saline (20 mM Hepes, pH 7.05, 137 mM NaCl, 5 mM KCl, 0.7 mM Na(2)HPO(4), 6 mM glucose), or RPMI 1640, 20% fetal calf serum. Stable transfectants were selected following electroporation by growth in the presence of G418, and clones were obtained by limiting dilution. The wild-type murine Lck cDNA or the LckR154K mutant described previously (24) were subcloned into the pBJ1-neo vector(30) , which uses the SRalpha promoter to drive expression of the inserted cDNA. The GST-Lck(SH2) plasmids were made by cloning StyI fragments from wild-type or LckR154K cDNAs into the EcoRI site of pGEX-KG(31) . The GST-Lck(SH2) fusion protein was purified out of bacterial sonicates using glutathione-Sepharose beads and eluted with free glutathione.

Transient Transfections

As has been described for Jurkat (32) , a derivative of JCaM1 expressing the SV40 large T antigen (JCaM1/TAg) was used to obtain higher levels of Lck expression as a result of the replication of plasmid DNA containing the SV40 origin of replication. JCaM1/TAg was electroporated with pBJ-neo-lck or pBJ-neo-LckR154K and an IL-2-luciferase reporter plasmid, pCLN15DeltaCX (kindly supplied by Gerry Crabtree). After 48 h the transfectants were stimulated with 50 ng/ml phorbol 12-myristate 13-acetate alone or with anti-TCR monoclonal antibody C305(33) . After 6 h at 37 °C, cells were harvested and lysed in 0.2 M phosphate buffer, pH 7.8, 1 mM dithiothreitol, and 1% Triton X-100. Luciferase activity was measured in a luminometer by incubating extracts in 3.3 mM ATP, 6.7 mM MgCl(2), and 0.33 mM luciferin.

Calcium Measurement

Cells were loaded with the fluorescent calcium-binding dye indo-1 (Molecular Probes) at 1 µM and then washed extensively. Cells were placed in a water-jacketed cuvette at 37 °C, and fluorescence levels were measured in a spectrofluorimeter before and after stimulation with saturating concentrations of antibody. The TCR was stimulated with the anti-TCR monoclonal antibody C305(33) . Calcium concentrations were determined using a K(d) of 250 nM for calcium binding to indo-1(34) .

Immunoprecipitations and Immunoblotting

Cells were stimulated at 37 °C for 2 min with the C305 monoclonal antibody, which is specific for the Jurkat TCR Vbeta chain. Cells were harvested and lysed in 1% Nonidet P-40, 10 mM Tris, pH 7.8, 150 mM NaCl, 1 mM phenylmethylsulfonyl fluoride, 0.4 mM orthovanadate, 10 mM NaF, 0.4 mM EDTA, 1 µg/ml leupeptin, 2 µg/ml pepstatin A. Lysates were cleared by centrifugation at 100,000 times g for 10 min at 4 °C. Prior to immunoprecipitation, lysates were further cleared by incubation with fixed Staphylococcus aureus cells. Lck, Fyn, and ZAP-70 were immunoprecipitated with rabbit anti-peptide antisera (Upstate Biotechnology), and the subunit of the TCR was immunoprecipitated using anti- peptide antisera kindly provided by Jeffrey Ravetch. Immunoprecipitates were collected on Protein A-Sepharose beads and washed in lysis buffer containing 0.5 M NaCl. For affinity precipitations with GST-Lck(SH2) fusion proteins, lysates prepared as described above were incubated with 1 µg of purified fusion protein bound to glutathione-agarose beads for 4-12 h and then washed extensively in lysis buffer. For immunoblotting, precipitates or Nonidet P-40 lysates were analyzed on SDS-polyacrylamide gels and transferred onto nitrocellulose or polyvinylidene difluoride membrane. Phosphotyrosine-containing proteins were detected using the 4G10 antibody, (Upstate Biotechnology), and Lck was detected using a monoclonal antibody (kindly provided by Anne Burkhardt and Joe Bolen) followed by goat anti-mouse antibody conjugated to either alkaline phosphatase or horseradish peroxidase. Bound antibodies were visualized using either nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate or by enhanced chemiluminescence. For detection of proteins transferred to nitrocellulose with GST-Lck(SH2), blots were incubated with the fusion protein at a concentration of 1 µg/ml in Tris-buffered saline containing 0.05% Tween 20. Bound fusion protein was detected with a monoclonal anti-GST antibody kindly supplied by Nicolai van Oers, followed by alkaline phosphatase-conjugated goat anti-mouse antibody.


RESULTS

To study the function of the Lck SH2 domain in TCR signal transduction we took advantage of the Lck kinase-deficient cell line JCaM1. This mutant, which was derived from Jurkat, is defective in its response to stimulation of the TCR unless wild-type Lck is re-expressed by transfection(22) . To determine the importance of the SH2 domain in Lck function, we transfected JCaM1 with an IL-2-luciferase reporter plasmid and either wild-type Lck or Lck containing an arginine to lysine substitution at position 154 (LckR154K). The arginine at this position is crucial for SH2 domain function since it is responsible for making direct contact with the substrate phosphotyrosine residue(35, 36) . As shown in Fig. 1, stimulation of the TCR on JCaM1 leads to an induction of luciferase expression following transfection with wild-type Lck but not with the LckR154K mutant. This finding indicates that the SH2 domain function of Lck is essential at some point in the TCR signaling pathway that leads to IL-2 gene expression.


Figure 1: Induction of IL-2-luciferase activity in JCaM1 transfected with Lck+ or LckR154K. JCaM1/TAg cells were transfected with an IL-2-luciferase reporter plasmid and pBJ-neo-Lck+ or pBJ-neo-lckR154K, or without an Lck plasmid (mock). 48 h after transfection cells were left unstimulated or were stimulated with phorbol 12-myristate 13-acetate alone (solid bars), or with phorbol 12-myristate 13-acetate and anti-TCR antibody C305 (open bars). 6 h after stimulation, cells were lysed and luciferase activity was measured. The data are presented as the ratio of luciferase activity in cell extracts from stimulated and unstimulated cultures. In parallel experiments, immunoblotting cell lysates from transfectants demonstrated that equivalent levels of Lck+ and LckR154K were expressed. The data are representative of three experiments.



To understand how the SH2 domain of Lck contributes to TCR signaling, we undertook a biochemical analysis of JCaM1 transfectants expressing LckR154K to determine at what step the signaling pathway was blocked. Cell lines stably expressing LckR154K were obtained by electroporation of JCaM1. However, the level of Lck expression achieved was only 10-20% of levels normally found in Jurkat. Thus, a JCaM1 transfectant expressing similar levels of wild-type Lck was obtained as a matched control for the LckR154K transfectant (Fig. 2). One of the early events that occurs following TCR stimulation is an increase in intracellular calcium levels due to the induction of the phosphatidylinositol pathway. Analysis of calcium levels using the fluorescent calcium-binding dye indo-1 indicated that receptor stimulation was unable to elicit a significant rise in intracellular calcium in JCaM1 transfected with LckR154K (Fig. 3B). This result is in contrast with the rise in intracellular calcium that is observed following stimulation of the TCR on JCaM1 transfected with wild-type Lck (Fig. 3A and (22) ). Thus, the Lck SH2 domain is required for function of the TCR signaling pathway prior to the release of calcium from intracellular compartments.


Figure 2: Lck levels in lysates of Jurkat, JCaM1/Lck+, and JCaM1/LckR154K. Nonidet P-40 lysates of equivalent cell numbers were prepared from the indicated cell lines and analyzed on an 8% SDS-polyacrylamide gel, blotted onto nitrocellulose, and probed with anti-Lck antibody. The lower molecular weight band present in the JCaM1 transfectants is also present in parental JCaM1 lysates and represents the protein product of mis-spliced Lck mRNA expressed in JCaM1(22) .




Figure 3: Intracellular calcium levels in JCaM1/Lck+ (A), JCaM/LckR154K (B), and JCaM1 (C) following TCR stimulation. Calcium levels were measured at 37 °C in a spectrofluorimeter using the calcium-binding dye indo-1 described under ``Materials and Methods.'' Cells were stimulated with saturating amounts of anti-TCR antibody C305 as indicated. All three cell lines demonstrated a large increase in intracellular calcium in response to treatment with ionomycin.



To determine the contribution of Lck SH2 domain function to the earliest signaling events, we examined induction of tyrosine phosphorylation of cellular proteins following receptor stimulation of the LckR154K transfectant. Lysates from unstimulated or stimulated transfectants were resolved on SDS-polyacrylamide gels and blotted with anti-phosphotyrosine antibody (Fig. 4). The LckR154K transfectant showed only a weak induction of tyrosine phosphoproteins compared with the matched wild-type control. This finding suggests that the Lck SH2 domain plays an important role in the initiation of the protein-tyrosine kinase pathway following TCR stimulation. To define this role in more detail we examined the tyrosine phosphorylation of the chain of the TCR and the ZAP-70 tyrosine kinase. Tyrosine phosphorylation of these proteins appears be required for their role in the initiation of receptor signaling(37, 38, 39) . Anti-phosphotyrosine immunoblots of or ZAP-70 immunoprecipitates from the transfectants showed that tyrosine phosphorylation of these signaling molecules induced by TCR stimulation was substantially reduced in the LckR154K transfectant (Fig. 5, A and B). These results show that the SH2 domain of Lck is required for the induction and/or maintenance of normal levels of and ZAP-70 tyrosine phosphorylation following TCR stimulation.


Figure 4: Induction of tyrosine phosphoproteins in JCaM1/Lck+ and JCaM1/LckR154K following anti-TCR stimulation. Nonidet P-40 lysates were prepared from the indicated cell lines; samples were either unstimulated or stimulated for 2 min at 37 °C with the anti-TCR antibody C305. After resolution on 10% SDS-polyacrylamide gels, proteins were transferred to nitrocellulose and probed with anti-phosphotyrosine antibody.




Figure 5: Induction of TCR and ZAP-70 tyrosine phosphorylation following TCR stimulation in JCaM1/Lck+ and JCaM1/LckR154K. A, was immunoprecipitated from Nonidet P-40 lysates prepared from unstimulated or anti-TCR-stimulated cells. Immunoprecipitates were analyzed on 11.5% SDS-polyacrylamide gels, transferred to nitrocellulose, and probed with anti-phosphotyrosine antibody. Stripping and reprobing the blot with anti- antibody demonstrated that equivalent levels of were immunoprecipitated from both cell lines. B, induction of ZAP-70 tyrosine phosphorylation following TCR stimulation in JCaM1/Lck+ and JCaM1/LckR154K. ZAP-70 was immunoprecipitated from Nonidet P-40 lysates prepared from unstimulated or anti-TCR-stimulated cells. Immunoprecipitates were analyzed on SDS-polyacrylamide gels, transferred to nitrocellulose, and probed with an anti-phosphotyrosine antibody. Parallel experiments demonstrated that the JCaM1/Lck+ and JCaM1/LckR154K cell lines express equivalent levels of ZAP-70 (data not shown).



Since SH2 domains can mediate protein interactions through the binding of phosphotyrosine residues it is likely that the essential role of the Lck SH2 domain in TCR signal transduction involves the association of Lck with a tyrosine phosphoprotein. To identify potential substrates of the Lck SH2 domain, we examined Lck immunoprecipitates from unstimulated and stimulated cell lysates of the Jurkat T cell line with anti-phosphotyrosine antibody. As shown in Fig. 6A, lane 4, several tyrosine phosphoproteins were observed in Lck immunoprecipitates from cells stimulated through the TCR. Most prominent among the tyrosine phosphoproteins was Lck itself, both the 56- and 60-kDa forms. We have confirmed the induced tyrosine phosphorylation of Lck by in vivo labeling studies (data not shown), and similar findings have been previously reported(40) . In addition to Lck we observed a 70-kDa tyrosine phosphoprotein. This phosphoprotein co-migrated with phospho-ZAP-70 present in immunoprecipitates from stimulated cells (Fig. 6A, lane 5). Although the Fyn tyrosine kinase was also tyrosine-phosphorylated following TCR stimulation, there was no detectable association of the 70-kDa tyrosine phosphoprotein (Fig. 6A, lane 2). We confirmed the identity of the 70-kDa tyrosine phosphoprotein by directly immunoblotting Lck immunoprecipitates with anti-ZAP-70 antibody (Fig. 6B). In addition to ZAP-70 we have also reproducibly observed a low level of a 20-23-kDa tyrosine phosphoprotein in Lck immunoprecipitates from stimulated cells. This phosphoprotein is at least in part the subunit of the TCR since it co-migrates with phospho- present in ZAP-70 immunoprecipitates and can be immunoprecipitated with anti- antiserum following solubilization of in vitro phosphorylated Lck immunoprecipitates (data not shown).


Figure 6: Association of ZAP-70 with Lck. A, anti-phosphotyrosine blot of Fyn, Lck, or ZAP-70 immunoprecipitates from unstimulated or stimulated Jurkat cells. The Fyn, Lck, or ZAP-70 kinases were immunoprecipitated from Nonidet P-40 lysates of unstimulated or anti-TCR-stimulated Jurkat cells with anti-peptide antisera, resolved on SDS-polyacrylamide gels, transferred to nitrocellulose, and probed with anti-phosphotyrosine antibody. B, ZAP-70 blot of Lck immunoprecipitates from unstimulated or stimulated Jurkat cells. Immunoprecipitates of Lck from Nonidet P-40 lysates of unstimulated or anti-TCR-stimulated cells were resolved on SDS-polyacrylamide gels, transferred to nitrocellulose, and blotted with anti-ZAP-70 antibody. The bands at 50-55 kDa are immunoglobulin heavy chain from the immunoprecipitating antisera.



The presence of ZAP-70 in Lck immunoprecipitates could be the result of an interaction between the Lck SH2 domain and tyrosine-phosphorylated ZAP-70. To investigate this we constructed glutathione S-transferase fusion proteins using either the wild-type or the R154K mutant form of the Lck SH2 domain. These fusion proteins were then incubated with lysates from unstimulated or stimulated Jurkat cells, and the presence of ZAP-70 in the precipitates was determined by immunoblotting (Fig. 7). These results show that the Lck SH2 domain can associate with ZAP-70 from stimulated cells. The R154K mutation disrupted the interaction between the Lck SH2 domain and ZAP-70 (Fig. 7, lanes 3 and 4) as well as the interaction between the Lck SH2 domain and other tyrosine phosphoproteins (data not shown). Since other proteins are present in the GST-Lck(SH2) precipitates the interaction between the fusion protein and ZAP-70 could be indirect. To address this possibility we probed blots of ZAP-70 and immunoprecipitates using a GST-Lck SH2 domain fusion protein (Fig. 8). These studies showed that the fusion protein was capable of directly interacting with both ZAP-70 and . This interaction was enhanced by TCR stimulation, suggesting that it was mediated by the increased tyrosine phosphorylation of ZAP-70 and following receptor stimulation. These results indicate that the interaction of Lck with ZAP-70 and can be mediated by a direct interaction between the Lck SH2 domain and phosphotyrosine residues on ZAP-70 and .


Figure 7: Association of ZAP-70 with wild-type or R154K GST-Lck(SH2) fusion protein. Lysates from unstimulated or anti-TCR-stimulated Jurkat cells were incubated with 1 µg of GST-Lck+(SH2) or GST-LckR154K(SH2) fusion protein. The associated proteins were then analyzed on SDS-polyacrylamide gels, immunoblotted with anti-ZAP-70 antibody, and developed using a chemiluminescence detection system. The immunoblot was exposed for 1 min. Extended exposure of the immunoblot revealed bands corresponding to ZAP-70 in the GST-LckR154K(SH2) fusion protein precipitates. However, this association may be nonspecific, as it was unaltered by TCR stimulation.




Figure 8: GST-Lck(SH2) fusion protein blot of ZAP-70 or TCR immunoprecipitates from unstimulated or anti-TCR-stimulated Jurkat cells. ZAP-70 and immunoprecipitates were prepared from Nonidet P-40 lysates of unstimulated or anti-TCR-stimulated cells. Immunoprecipitates were resolved on SDS-polyacrylamide gels, transferred to nitrocellulose, and probed first with purified GST-Lck(SH2) fusion protein, and then with anti-GST antibody as described under ``Materials and Methods.''




DISCUSSION

Our analysis indicates that the SH2 domain of Lck plays a crucial role in TCR signal transduction. Expression of an Lck cDNA containing an arginine to lysine substitution within the SH2 domain (LckR154K) was unable to restore signaling function to the Lck-deficient cell line JCaM1. The failure to restore signaling function to JCaM1 occurred at the earliest steps in the signal transduction pathway. Unlike the wild-type Lck transfectant, TCR stimulation of JCaM1 transfected with LckR154K did not increase the level of intracellular calcium or result in a significant induction of tyrosine phosphoproteins. More detailed analysis showed that two of the most proximal signaling events, tyrosine phosphorylation of the TCR chain as well as tyrosine phosphorylation and recruitment of the ZAP-70 tyrosine kinase, were substantially reduced in the LckR154K transfectant. These results indicate that the function of the Lck SH2 domain is crucial for initiating signal transduction events following TCR stimulation.

Crystallographic analysis of the Lck SH2 domain complexed with a phosphopeptide indicates that the arginine residue at position 154 makes direct contact with the phosphotyrosine residue of the peptide (35) . Substitution of the analogous arginine residue in c-Abl with lysine eliminates binding to phosphotyrosine(36) . Additionally, we have found that this mutation eliminates the ability of the Lck SH2 domain to associate with tyrosine phosphoproteins (data not shown). The fact that the LckR154K mutant is unable to function in the TCR signaling pathway suggests that the interaction of Lck with a tyrosine phosphoprotein is crucial for initiation of TCR-mediated signaling. Anti-phosphotyrosine blots of Lck immunoprecipitates revealed that a 70-kDa tyrosine phosphoprotein associated with Lck immediately following receptor stimulation. Direct blotting of Lck immunoprecipitates demonstrated that this protein was the ZAP-70 tyrosine kinase. To investigate whether the interaction of the two kinases was mediated by SH2-phosphotyrosine interaction we blotted ZAP-70 immunoprecipitates with a purified GST-LckSH2 domain fusion protein. These blots demonstrated that the association of Lck and ZAP-70 following TCR stimulation could be the result of a direct interaction between phosphotyrosine residues on ZAP-70 and the LckSH2 domain. We found that purified GST-LckSH2 domain fusion protein could associate with ZAP-70 in lysates from stimulated cells and confirmed that this interaction was disrupted by the R154K mutation. Similar biochemical results have been reported by Duplay et al.(41) .

Anti-phosphotyrosine blots of Lck immunoprecipitates also revealed the weak association of a tyrosine phosphoprotein that co-migrated with the TCR chain present in ZAP-70 immunoprecipitates. Lck immunoprecipitates labeled by in vitro phosphorylation with [P]ATP do contain , as demonstrated by reprecipitation (data not shown). Blotting of immunoprecipitates with purified GST-LckSH2 fusion protein further demonstrated a potential direct interaction between Lck and the chain. Lck has been found to associate with in natural killer cells in the context of the FcRIIIA(42) ; however, it has been difficult to demonstrate such an association in T cells. The presence of only a low level of in Lck immunoprecipitates may reflect a very unstable association of the two molecules, and it may also reflect an indirect association in vivo since has been shown to bind ZAP-70 tightly(24, 43) . It is likely that serves as a substrate for Lck in T cells, and the two molecules may remain associated following phosphorylation of via the SH2 domain of Lck, but this interaction may be displaced by the stronger binding of the dual SH2 domains of ZAP-70 to tyrosine-phosphorylated (44) .

It appears likely that the interaction between Lck and ZAP-70 mediated by the SH2 domain is crucial in the initiation of TCR signaling. Co-expression of Lck and ZAP-70 in Sf9 cells leads to the phosphorylation of ZAP-70 on the same sites as observed in activated T cells and also results in an increase in ZAP-70 kinase activity(39) . Cross-linking of the TCR leads to the Lck-dependent phosphorylation of the TCR and CD3 cytoplasmic domains as well as the recruitment of ZAP-70. Presumably, the co-localization of Lck and ZAP-70 could lead to a direct interaction of the two kinases, mediated by recognition of phosphotyrosine residues on ZAP-70 by the Lck SH2 domain. Once bound, Lck may further phosphorylate and activate ZAP-70, thereby leading to the phosphorylation and activation of downstream signaling molecules. It is unclear whether Lck binds ZAP-70 at a site of autophosphorylation or at a site previously phosphorylated by Lck (or another kinase). This question could be addressed in part by analyzing the ability of Lck to interact with a kinase-deficient mutant of ZAP-70.

An alternative explanation for the role of the Lck SH2 domain in TCR signaling is that it is required not only for a direct interaction between Lck and ZAP-70, but also for the phosphorylation of the TCR cytoplasmic domains, which is required for the initial recruitment of ZAP-70 to the TCR. This is consistent with the observation that phosphorylation is reduced in JCaM1 transfected with the LckR154K mutant. Recent studies on the phosphorylation of the carboxyl-terminal tail of RNA polymerase II, or p130, by the Abl tyrosine kinase has indicated that SH2 domain function is required for complete phosphorylation of the substrate(45, 46) . Presumably the SH2 domain allows the kinase to remain in association with the substrate, thereby promoting phosphorylation of multiple tyrosine residues. This model is consistent with the observation that substrate specificity of tyrosine kinases correlates with the specificity of their SH2 domains(47) . However, unlike the Abl kinase, mutation of the Lck SH2 domain does not significantly reduce the ability of Lck to phosphorylate the cytoplasmic domain of in vitro (data not shown). This may not be surprising in light of the fact that only one of six tyrosines in the cytoplasmic domain is found in the preferred context for Lck SH2 binding, YEEI(48) . Alternatively, the reduction in phosphorylation observed with the SH2 domain mutant may be due to reduced protection of phospho- from dephosphorylation by tyrosine phosphatases. Phospho- could be protected through a direct interaction with Lck, as discussed above. In addition, ZAP-70 has been shown to protect phospho- from dephosphorylation in COS cells(24) . The protection of phospho- may be enhanced by the SH2 domain-mediated interaction between Lck and ZAP-70.

In previous work a functional interaction between Lck and ZAP-70 was observed when both kinases were transfected into COS cells(24) . Co-expression of the kinases led to enhanced tyrosine phosphorylation of cellular proteins and the association of ZAP-70 with the tyrosine-phosphorylated cytoplasmic domain of the TCR chain. In contrast to our findings with the JCaM1 transfectants, in COS cells Lck with the R154K mutation functioned as well as wild-type Lck. The different requirements for LckSH2 domain function probably reflect a difference in the level of expression between the two experimental systems. The high level expression of transfected cDNA clones in COS cells may have overcome the requirement for the LckSH2 domain to mediate a specific interaction between the two kinases or between Lck and the cytoplasmic domain.

In summary, our results indicate that the phosphotyrosine binding function of the Lck SH2 domain is crucial for the role of Lck in T cell receptor signal transduction. Lck carrying a mutation in the phosphotyrosine binding pocket of the SH2 domain is unable to restore even the earliest signaling events to an Lck-deficient cell line. The major tyrosine phosphoprotein that associates with Lck is the ZAP-70 tyrosine kinase. TCR chain also associates with Lck but apparently much more weakly than ZAP-70. Further experiments showed that the Lck SH2 domain could mediate a direct interaction between Lck and ZAP-70, as well as between Lck and . Since the Lck SH2 domain mutant is defective in signaling function, it is likely that the interaction between Lck and ZAP-70 and/or possibly , is essential for the initiation of TCR signal transduction.


FOOTNOTES

*
This work was supported in part by National Institutes of Health Grant GM39553 (to A. W.), a senior postdoctoral fellowship from the California Division of the American Cancer Society (to D. B. S.), and the Gastrointestinal Research Foundation. 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: University of Chicago, Department of Medicine/MC6084, 5841 S. Maryland Ave., Chicago, IL 60637. Tel.: 312-702-4708; Fax: 312-702-2281.

Current address: Howard Hughes Medical Institute, Washington University School of Medicine, St. Louis, MO 63110.

(^1)
The abbreviations used are: TCR, T cell receptor; SH2, Src homology domain 2; GST, glutathione S-transferase; IL-2, interleukin 2.


ACKNOWLEDGEMENTS

We thank Gerry Crabtree, Jeffrey Ravetch, Anne Burkhardt, Joe Bolen, and Nicolai van Oers for providing reagents.


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