Vol. 12, No. 12

MOLECULAR AND CELLULAR BIOLOGY, Dec. 1992, p. 5438-5446 0270-7306/92/125438-09$02.00/0 Copyright ©) 1992, American Society for Microbiology

p598fv Tyrosine Kinase Associates with Multiple T-Cell Receptor Subunits through Its Unique Amino-Terminal Domain LISA K. TIMSON

GAUEN,1 A.-N. TONY KONG,2 LAWRENCE E. SAMELSON,2

AND ANDREY S. SHAW1* Department of Pathology, Box 8118, Washington University School of Medicine, 660 South Euclid, St. Louis, Missouri 63110,1 and Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development, Bethesda, Maryland 208922

Received 20 August 1992/Returned for modification 17 September 1992/Accepted 22 September 1992

Several lines of evidence link the protein tyrosine kinase p590' to the T-cell receptor. The molecular basis of this interaction has not been established. Here we show that the tyrosine kinase p59"' can associate with chimeric proteins that contain the cytoplasmic domains of CD3 epsilon, gamma, zeta (c), and eta. Mutational analysis of the C cytoplasmic domain demonstrated that the membrane-proximal 41 residues of ; are sufficient for p5905" binding and that at least two p59f"' binding domains are present. The association of p59ry' with the C chain was specific, as two closely related Src family protein tyrosine kinases, p60' and p56kk, did not associate with a chimeric protein that contained the cytoplasmic domain of C. Mutational analysis of p596"' revealed that a 10-amino-acid sequence in the unique amino-terminal domain of p596'n was responsible for the association with C. These findings support evidence that p5905" is fimctionally and structurally linked to the T-cell receptor. More importantly, these studies support a critical role for the unique amino-terminal domains of Src family kinases in the coupling of tyrosine kinases to the signalling pathways of cell surface receptors.

mice that lack p59l5& expression. Consistent with models implicating p596"' in T-cell activation, thymocytes from these mice are refractory to activation stimuli (1, 38). The association of specific Src family tyrosine kinases with cell surface receptors has implicated them in signal transduction pathways. For example, p561ck may be involved in T-cell activation via association with the T-cell membrane proteins CD4, CD8, and the interleukin-2 receptor (7, 31, 40). However, the specific interactions between p596"' and the T-cell receptor have not been elucidated. To determine exactly which component(s) of the receptor can associate with p596"' and to define the molecular basis for such an interaction, we developed a system to assess the ability of individual T-cell receptor subunits to bind to p596"'. Here we demonstrate that p596"' can associate specifically with cytoplasmic sequences present in the CD3 (, q, y, and £ chains. Mutational analysis of the t chain demonstrates that p596"' binds redundantly to sequences present in the cytoplasmic domain of t. Furthermore, the association of p5965' with t was specific, as two closely related kinases, p6Q" and p56 kk, did not associate with t. A 10-amino-acid sequence at the amino terminus of p596"' was found to confer specific binding to t. Our data demonstrate that p596"' can associate with multiple components of the T-cell receptor complex and identifies critical binding domains. This study extends the evidence supporting a role for p59fi" in early T-cell receptor signalling events and suggests strategies to directly test this hypothesis.

Antigen-induced activation of T lymphocytes is regulated by a multichain protein complex known as the T-cell receptor. This complex consists of at least six different proteins. Two of these chains, alpha and beta, are capable of recognizing a vast number of different antigens. The four remaining chains are invariant and are known as CD3 delta (8), epsilon (e), gamma (-y), and zeta (c). An alternatively spliced form of t, called eta (n), can be found as a component of some murine T-cell receptors. Because none of these proteins contains any intrinsic enzymatic activity, the mechanisms utilized by this complex to generate the signals that regulate the behavior of T lymphocytes are not known. Because tyrosine phosphorylation can be detected within seconds of T-cell receptor engagement and because tyrosine kinase inhibitors can block early signalling events in T cells (12, 20), protein tyrosine kinases are thought to be essential components of the signalling cascade. These data suggest that the T-cell receptor is directly coupled to a tyrosine protein kinase. Attention has focused on a member of the Src family of tyrosine kinases, p596"', because it can be coimmunoprecipitated with the T-cell receptor (32, 34) and because it colocalizes with the T-cell receptor in vivo (6). Functional evidence that p596"' participates in T-cell activation has been obtained from experiments using transgenic mice. T lymphocytes from transgenic mice that overexpress p596"' demonstrate enhanced biochemical responses during T-cell activation (3). Very early responses such as calcium mobilization and phosphotyrosine accumulation are affected in the T cells from these mice, supporting a role for p596y' in the earliest activation events. In a complementary series of experiments, T cells from transgenic mice expressing an enzymatically inactive form of p596"' (which acts in a dominant-negative manner) have reduced activation parameters, suggesting that p596"' is required for early activation events (3). More recently, homologous recombination has been used to generate *

MATERIALS AND METHODS DNA constructs and mutagenesis. pGZeta encodes the 461 residues of the extracellular domain of vesicular stomatitis virus glycoprotein (VSV G) and the transmembrane and cytoplasmic domains of the murine e chain (residues 10 to 143) (42). The GZeta chimera was made by using DNA fragments generated by polymerase chain reaction (PCR) (19). Two overlapping primers which spanned the VSV G/I

Corresponding author. 5438

VOL. 12, 1992

ASSOCIATION OF

junction were used to generate the hybrid DNA product, using the site-overlap extension method. The hybrid DNA product was ligated into the EcoRI site of the plasmid vector pBluescript SK+ (Stratagene). The truncation mutants of GZeta were generated by using PCR to insert a stop codon and anXbaI restriction site at the designated residue (denoted by an asterisk). After digestion, the PCR-generated DNA fragment was ligated into the EcoRI and XbaI sites of pGem3Z+. Plasmids encoding GZeta mutants with internal deletions were created by inverse PCR (8), using pGZeta as the template. G eta was generated by removing a downstream fragment of GZeta and replacing it with an equivalent fragment (SacI-SacI) of pBS--l (11) (provided by E. Reinherz). To construct G gamma, PCR was used to generate a DNA fragment that contained residues encoding the complete cytoplasmic domain of CD3 ry (residues 117 to 160) flanked by a 5' BamHI site and a 3' EcoRI site. This fragment was ligated with the EcoRI-BamHI fragment of pTMB (24) into the EcoRI site of pGem3Z+ oriented so it could be expressed by using the T7 promoter. This DNA fragment of pTMB encodes the extracellular and transmembrane domains of VSV G and four amino-terminal residues from the cytoplasmic domain. DNA sequences encoding the last three residues of CD3 y were removed by PCR and substituted with a stop codon and an EcoRI site. G epsilon was generated similarly to G gamma. PCR was used to generate a DNA fragment encoding the cytoplasmic domain of CD3 E (residues 133 to 185) flanked by BamHI and EcoRI sites. This fragment was ligated with the EcoRI-BamHI fragment of G gamma into the EcoRI site of pGem3Z+. The cDNA encoding the 5' sequences of murine p59?5f was obtained from S. Desiderio. The full-length p59fY" cDNA was obtained by using PCR technology (19). The Fyn/Src chimera was generated by substituting the XhoIHpaI fragment of pLS (36) with an equivalent PCR-generated fragment from p59'5", resulting in a chimeric cDNA encoding the first 125 amino acids of p59'5" and residues 125 to 531 of p60. A HpaI restriction site introduced at this position does not change the amino acid sequence in p59'5', p60, or p56'k. Similarly, the Src/Fyn chimera was generated by substituting the HpaI-XbaI fragment of pSL (36) with an equivalent fragment of p59'5" which was generated by PCR. The Fyn/Myc chimera was generated by PCR, using an oligonucleotide to append sequences encoding the epitope SMEQKLISEEDLN to the carboxy terminus of p59'5". Deletions of residues 11 to 25 (A11-25 Fyn/Myc), 26 to 50 (A26-50 Fyn/Myc), 51 to 75 (A51-75 Fyn/Myc), and 76 to 100 (A76-100 Fyn/Myc) of Fyn/Myc were created by inverse PCR, using pBS-Fyn/Myc as a template. The 1-10 Fyn/Src and 1-10 Src/Fyn/Myc chimeras were generated by inverse PCR, using pBS-Src (36) and pBS-Fyn/Myc, respectively, as templates. DNA sequence analysis (33) was performed to confirm the accuracy of all of the PCR-generated products for all of the DNA constructs. DNA transfections and in vitro kinase assays. HeLa cells (5 x 105) were infected with a recombinant vaccinia virus that expresses T7 RNA polymerase (4). Five micrograms of total DNA was then transfected into HeLa cells by using the liposome reagent Transfectase (15 p1; Bethesda Research Laboratories). The proportions of cotransfected DNAs were adjusted empirically to obtain comparable expression levels of the coexpressed proteins. Eleven hours after transfection, cells were lysed in a buffer containing 1% digitonin (Wako), 25 mM Tris (pH 8.0), 150 mM NaCl, 300 mM KCI, 25 mM

p59f" WITH T-CELL RECEPTOR SUBUNITS

5439

NaF, 100 pM sodium orthovanadate, and the protease inhibitor aprotinin (500 Kallikrein inhibitor units [KIU]/ml; Boehringer Mannheim). After incubation on ice for 5 min, lysates were spun in a microcentrifuge for 10 min at 4°C. Centrifugation of our cell lysates at 100,000 x g (which should have pelleted membrane fragments and insoluble material) had no effect on our results (data not shown). Supernatants, precleared with an irrelevant rabbit antiserum and PanSorbin (CalBiochem), were divided and then immunoprecipitated with a monoclonal antibody against VSV G (Il) (14), or a control monoclonal antibody (OKT4; American Type Culture Collection), and protein-A immobilized on Sepharose (Sigma). Tyrosine kinases were immunoprecipitated with rabbit polyclonal antibodies to p59'5" and p56kkc or with a monoclonal antibody to Src (EC10 [23]). Sodium deoxycholate (to a final concentration of 1%) was added to cell lysates used to immunoprecipitate tyrosine kinases. After immunoprecipitates were washed three times with phosphate-buffered saline, kinase assays were performed in a total volume of 15 pl containing 20 ,Ci of [y-32P]ATP, 25 mM N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES; pH 7.4), 150 mM NaCl, 5 mM MnCl2, 5 mM MgCl2, and 100 p,M sodium orthovanadate. Phosphorylated proteins were separated by 8% sodium dodecyl sulfate

(SDS)-polyacrylamide gel electrophoresis (PAGE). Partial proteolysis of phosphoproteins. Partial proteolysis of phosphoproteins was performed as previously described, using 10 ng of Staphylococcus aureus V8 protease (36). Digests were analyzed on 12% polyacrylamide gels. Immunoblotting. Immunoblotting was performed by using whole-cell lysates prepared from duplicate cultures of cells. (Analysis of cell lysates prepared with digitonin had very high backgrounds.) Proteins were separated by SDS-PAGE (8% polyacrylamide gel), transferred to nitrocellulose, and immunoblotted with polyclonal antibodies to VSV. The immunoblot in Fig. 1D was subsequently immunoblotted with polyclonal antibodies to p59y'5. Proteins were detected by using a horseradish peroxidase-conjugated goat antirabbit secondary antibody (Bio-Rad) and the enhanced chemiluminescence detection system (Amersham). RESULTS Association of p590" with the T-cell receptor C chain. To define the molecular basis for the interaction of p59'5" with the T-cell receptor, our strategy was to express individual T-cell receptor chains with p59y'5 in a nonlymphoid cell. Because some of the T-cell receptor chains are not stable or not properly transported to the cell surface when expressed alone, we used chimeric proteins that fused the extracellular domain of VSV G to the cytoplasmic domain of individual T-cell receptor subunits. This procedure allowed stable expression of individual subunits alone and allowed the use of the same antibody (against VSV G) for all of our coimmunoprecipitation and immunoblotting studies. We focused initially on the T-cell receptor ; chain because recent data demonstrate that sequences in the cytoplasmic domain of ; are sufficient to generate an activation signal in T cells (9, 15, 30, 41). A chimeric molecule consisting of the extracellular domain of VSV G fused to the transmembrane and cytoplasmic domains of CD3 t was generated (GZeta; Fig. 1A). This protein was recognized by antibodies to VSV G and was stably expressed and transported to the plasma membrane in HeLa cells (Fig. 1D, lane 3, and data not

shown).

To determine whether p59'5"' could form a complex with

5440

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present in residues 84 to 143 of C, a GZeta mutant that lacked the membrane-proximal 51 cytoplasmic residues of C, GZetaA32-83, was generated. When GZetaA32-83 was coexpressed in HeLa cells with p596", in vitro kinase assays of VSV G immunoprecipitates indicated that p59' could still associate (Fig. 2). These results demonstrate that an additional p595'2 binding domain exists between residues 84 and 143 of C. Thus, at least two binding domains for p596' are contained within the C cytoplasmic domain. Consistent with this hypothesis was our observation that the amount of p596"' kinase activity coprecipitating with GZetaN84* and GZetaA32-83 was less than that coprecipitated by full-length GZeta (data not shown). Association of p596y' with CD3 i, CD3 fy, and CD3 e. To test whether other T-cell receptor subunits could bind p596', chimeric proteins that contained the cytoplasmic domains of the T-cell receptor q, y, and -chains were prepared. a is identical to C for the first 122 residues of the mature protein, 92 of which are cytoplasmic (11). The last 21 residues of ; and the last 63 residues of a are different. The 1 chain, therefore, contains the membrane-proximal p59"' binding domain of C identified above (between residues 32 and 83) and would be predicted to bind p596. As expected, VSV G immunoprecipitates of lysates from cells coexpress-

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FIG. 3. Association of p596' with chimeric proteins containing cytoplasmic sequences from CD3 C, , , andy. (A) Duplicate plates of HeLa cells were transfected with the indicated DNAs. After 11 h, cells from one set of plates were bsed in a digitonin-containing buffer, and in vitro kinase reactions were performed on immunoprecipitates prepared with antibodies against VSV G. Kinase reactions were separated on SDS-8% polyacrylamide gels. Positions (kilodaltons) of prestained molecular weight markers are indicated. (B) Expression of p59' in cotransfected cells as measured by in vitro kinase activity. Anti-p590 immunoprecipitates from one-third of the cell lysate described for panel A were prepared and tested for in vitro kinase activity. (C) Expression of VSV G chimeric proteins in cotransfected cells as measured by immunoblotting. Cells from the second set of plates (descnibed above) were lysed directly in 1 ml of sample buffer; 20 IlI of each lysate was analyzed on an SDS-8% polyacrylamide gel, transferred to nitrocellulose, and immunoblotted with antibodies to VSV.

ing G eta and p596" contained readily detectable p59'2" kinase activity (Fig. 3A, lane 3). Cytoplasmic sequences of " can, therefore, associate with p59'2". The ability of the CD3 -y chain to bind p596' was tested by generating a chimeric protein encoding the extracellular and transmembrane domains of VSV G and the cytoplasmic domain of CD3 -y (G gamma). This chimeric protein, however, was not transported to the cell membrane, as assessed by immunofluorescence or by the acquisition of resistance to endoglycosidase H, and appeared to be retained in the endoplasmic reticulum (ER). We noted that the last three C-terminal residues of CD3 -y are basic, containing two lysines and an arginine, which was reminiscent of the ER retention signal descnibed in the adenovirus protein E19 (10, 21). We therefore deleted the sequences encoding these residues from the G gamma cDNA to generate G gamma*. Consistent with reports by others (17, 18), G gamma* was not retained in the ER and was transported to the plasma membrane (data not shown), demonstrating that these C-terminal sequences constitute an ER retention signal. Both forms of G gamma were coexpressed with p596", and immunoprecipitates of both chimeric proteins contained p596' kinase activity (data not shown and Fig. 3A, lane 5). This result demonstrates that p59' can bind to sequences in the cytoplasmic domain of CI3 y. As with the assembly of CD4/p561k complexes (36), p596" can associate with CD3 y sequences even when retained in the ER. Cytoplasmic sequences of CD3 e were examined in a similar fashion. We generated a chimeric protein, G epsilon, which contains the extracellular and transmembrane domains of VSV G and the cytoplasmic domain of CD3 £. Like G gamma, G epsilon did not transport to the plasma membrane and was retained in the ER. Nevertheless, we tested

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whether G epsilon could associate with p59fi5n when coexpressed in HeLa cells. Anti-VSV G immunoprecipitates from G epsilon- and p59f5'-expressing cell lysates contained readily detectable p59fi5f kinase activity (Fig. 3A, lane 4). Thus, p595f can associate with cytoplasmic sequences of CD3 £. It is interesting to note that the levels of p595fl kinase activity coprecipitating with the G epsilon and G gamma chimeric proteins appeared reduced compared with the amount associated with GZeta or G eta. As the expression of p591Yf and all of the chimeric proteins appeared equivalent, as assessed by in vitro kinase activity and immunoblotting, respectively (Fig. 3B and C), these differences in associated p5O96 suggest that cytoplasmic sequences in CD3 y and E have a lower affinity for p596fl than does the comparable sequence in either ; or r. Specificity of p590' association with GZeta. The specificity of the GZeta/p59'5' interaction was examined by coexpressing two other Src family tyrosine kinases, p56kc and p60w", with GZeta. Coexpression of p56kck with GZeta resulted in only minimal levels of kinase activity in VSV G immunoprecipitates relative to controls (Fig. 4A). This finding was not due to lower levels of p561ck or GZeta expression. Immunoprecipitation with antibodies to p59'5" and p561ck demonstrated comparable kinase activities (Fig. 4B, lanes 1, 3, 4, and 5). Immunoblotting of cell lysates with anti-VSV antibodies demonstrated comparable GZeta expression when p59'5" and pS6lck were coexEressed with GZeta (Fig. 4C, lanes 3 and 5). Thus, p56k did not associate with the cytoplasmic domain of t. The differences in phosphorylation of GZeta seen in lanes 2 and 5 of Fig. 4A were most likely due to the differences in expression levels of GZeta (Fig. 4C, lanes 1 and 5).

233

FIG. 5. Inability of p60S7c to associate with cytoplasmic sequences of ;. (A) Cells from one set of duplicate plates of HeLa cells expressing the indicated DNAs were lysed, and immunoprecipitates prepared by using an antibody against VSV G. Kinase reactions were performed and analyzed as described for Fig. 3A. (B) Expression of p596'i and p60"' tyrosine kinases as measured by in vitro kinase activity. One-third of the cell lysates described in A was immunoprecipitated with an antibody to p59O' (lanes 1 and 2) or p6O0 (lane 3), tested for kinase activity, and analyzed by SDSPAGE (8% polyacrylamide gel) and autoradiography. (C) Expression of GZeta as measured by immunoblotting. The second plate of cells described above was analyzed for GZeta expression as described for Fig. 3C. Positions (kilodaltons) of size markers are indicated.

Similar results were obtained with p60. Coexpression of p60 with GZeta resulted in only minimal levels of kinase activity in VSV G immunoprecipitates relative to controls (Fig. 5A, lanes 1 to 3). Immunoprecipitation with antibodies to p59P5' and p60 demonstrated comparable kinase activity in the lysates (Fig. 5A, lanes 5 and 6). Immunoblotting of cell lysates with anti-VSV antibodies demonstrated comparable GZeta expression in all of the transfections (Fig. SB). Binding to the cytoplasmic domain of ; was, therefore, specific for p59'5". Sequences of p590' required for GZeta association. Because the Src family kinases differ primarily within the first 70 amino acids, the association of p59'5" with GZeta was probably due to sequences within the unique amino-terminal domain of p59f'i. To test this hypothesis, we constructed two chimeric proteins between p60 and p59y'5: a chimeric protein consisting of the amino-terminal 125 amino acids of p59y'5 joined to residues 125 to 533 of p60 (Fyn/Src; Fig. 6A), and a chimeric protein consisting of the amino-terminal 124 amino acids of p6O0 joined to residues 126 to 534 of p59fy'5 (Src/Fyn; Fig. 6A). As described previously, the junction of the hybrid cDNA is in a conserved region of p59'5" and p60. When Fyn/Src was coexpressed with GZeta, in vitro kinase assays of VSV G immunoprecipitates resulted in phosphorylation of both GZeta and Fyn/Src (Fig. 6B, lane 2). In contrast, no significant kinase activity was detected in GZeta immunoprecipitates when Src/Fyn was expressed with GZeta (Fig. 6B, lane 3). Kinase assays of immunoprecipitates prepared by using antibodies against p59y'5 and p60' demonstrated equivalent kinase activities in lysates containing Fyn/Src and Src/Fyn (Fig. 6B, lanes 4

ASSOCIATION OF

VOL. 12, 1992

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to 6), and VSV immunoblots demonstrated that GZeta was expressed at similar levels (Fig. 6C). Thus, the unique amino-terminal domain of p5905f was sufficient to direct

association with GZeta. To further define the residues in the unique amino-terminal domain of p5915f responsible for ; association, a set of p5915n mutants with deletions in the first 100 amino acids was prepared and tested for the ability to coprecipitate with GZeta. Because the p59fyt" antiserum that we used recognizes residues contained within the first 100 amino acids of p5915P, the p59fytn cDNA was extended with sequences that encode the epitope SMEQKLISEEDLN (Myc), which is recognized by monoclonal antibody 9E10. Four Myc-tagged p59l5f mutants containing internal deletions of the amino terminus were generated. Residues 11 to 25 (A11-25 Fyn/ Myc), 26 to 50 (A26-50 Fyn/Myc), 51 to 75 (A51-75 Fyn/ Myc), and 76 to 100 (A76-100 Fyn/Myc) were deleted from the amino terminus of p59'" (Fig. 7A). Each Fyn/Myc mutant was coexpressed with GZeta in HeLa cells and tested for association. Surprisingly, all four of these Fyn/ Myc mutants coimmunoprecipitated with GZeta with equal efficiency, as measured by in vitro kinase activity (data not shown). These data demonstrated that residues 11 to 100 of p59'5" were not required for association with GZeta and

p596

WITH T-CELL RECEPTOR SUBUNITS

5443

suggested that the critical residues were contained within the first 10 amino acids of p59fyn. Because the myristylation signal for all of the Src family of tyrosine kinases is thought to be contained within the first 10 residues, deletion of the first 10 residues of p59*fn would likely affect its membrane association. We therefore tested the importance of the first 10 amino acids of p59"Yn by generating two chimeric proteins. 1-10 Src/Fyn/Myc contains the first 10 amino acids of p60 joined to residue 11 of Fyn/Myc; 1-10 Fyn/Src contains the first 10 amino acids of p5961fn joined to residue 11 of p60 (Fig. 7A). Both chimeric proteins were coexpressed with GZeta in HeLa cells and tested for association by in vitro kinase assay. When the amino-terminal 10 residues of p595n were replaced with those of p60, no significant kinase activity was detected in the VSV G immunoprecipitation (Fig. 7B, lane 2). More importantly, transfer of the first 10 amino acids of p59&n to p60 conferred the ability of the chimeric protein to associate with GZeta (Fig. 7B, lane 3). Control immunoprecipitations demonstrated similar levels of kinase activity in the two lysates, and immunoblotting of duplicate lysates with the VSV antiserum confirmed equivalent levels of GZeta expression (Fig. 7C). The first 10 amino acids, therefore, direct the association of p59lYn with the cytoplasmic domain of t. DISCUSSION To study the capacity of individual T-cell receptor subunits to bind p591Yf, chimeric proteins that contained the cytoplasmic domains of various T-cell receptor subunits were generated and expressed in a nonlymphoid cell line (HeLa). This procedure allowed us to achieve stable expression of individual T-cell receptor subunits in an environment free of other T-cell receptor chains. Using this system, we have specifically defined the interaction between p59f5i and the T-cell receptor by demonstrating that multiple compocan bind to nents of the T-cell receptor, CD3 fy, s, t, and p5915f. To define the p596f5l binding domain, deletional analysis was used to demonstrate that at least two p595" binding domains are present in ; and that the 41 membraneproximal residues of; are sufficient for binding. The association of p595" and 4 was specific. Two closely related kinases, p6(Y'9 and ps6"k, did not associate with a fusion protein that contained the cytoplasmic domain of t. Furthermore, the unique amino-terminal domain of p59?5& was sufficient to direct a chimeric protein to associate with the ; chain. A 10-amino-acid sequence at the extreme amino terminus of p59lyf was found to confer specific binding of t. Although our detection of interactions between p59" and cytoplasmic sequences of the T-cell receptor t, 9, y, and £ chains depended on the use of the mild detergent digitonin, these are specific interactions because discrete sequences in both p5915l and ; are required for association. The instability of these complexes and their low affinities toward each other do suggest, however, that the approach that we chose was critical. The vaccinia virus T7 expression system generates very high levels of protein, and our VSV G chimeras form trimeric complexes in vivo (43). These factors probably resulted in very high local concentrations of expressed proteins in the membranes of the transfected cells. Even so, we estimate that only 1 to 5% of the p596141 molecules expressed was bound to ; and even less was bound to cytoplasmic sequences of q, -y, and £. These calculations compare favorably with those calculated for T-cell receptors in vivo (32, 34) and are consistent with the idea that this is a r,

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Kinase Activity VSV Immunoblot FIG. 7. Identification of residues that direct association of p59f" with C. (A) Schematic diagram of p596, mutants. Open boxes represent p59' sequences; striped boxes represent the Myc epitope appended to the carboxy termini of the p59f" mutants; closed boxes represent sequences from p6O0. Binding to C was determined after coexpression of each mutant with GZeta in HeLa cells by measuring p59'5 autokinase activity in VSV G immunoprecipitations. The clear presence of phosphorylated p59f" was scored as +. The efficiencies of all transfections were tested by measuring kinase activities of immunoprecipitates prepared with antibody 9E10 and immunoblotting cell lysates with antibodies to VSV. (B) In vitro kinase reactions of VSV G immunoprecipitates from cells expressing the indicated proteins. Positions (kilodaltons) of molecular weight markers are indicated. (C) Expression of chimeric kinases and GZeta. A portion of the lysates described above was immunoprecipitated with an antibody to p59f" (lanes 1 and 2) or p60^ (lane 3) and tested for kinase activity. A duplicate plate of cells was lysed directly in 1 ml of sample buffer and analyzed for GZeta expression as described for Fig. 3C. myr

-

1-10 FYN/SRC

low-affinity interaction. As some complexes probably disassociate during cell lysis, the actual stoichiometry of binding is likely to be higher than 1 to 5%. Although CD3 y and apparently have affinities toward p59f)f that are even lower, these complexes are probably physiologically relevant because T-cell receptor complexes that lack ; in vivo can still bind p59y'5 and can still signal (13). Our demonstration that and y can bind p595' could account for these findings. Association of p59'5" with specific components of the T-cell receptor is consistent with models in which single T-cell receptor chains can initiate signals that result in T-cell activation. Recent studies show that chimeric proteins containing the cytoplasmic domains of CD3 t, q, and are capable by themselves of generating signals that result in the activation of T cells (9, 15, 16, 29, 30, 41). Most of the studies have focused on signalling by the CD3 e chain. When expressed in T cells, chimeric proteins that contain the extracellular domain of CD4, CD8, CD16, or the interleukin-2 receptor and the cytoplasmic domain of t can initiate multiple biochemical pathways characteristic of T-cell activation. Mapping studies have demonstrated that the amino acid sequences required for signalling are contained within an 18-residue motif that is repeated three times in the cytoplasmic domain of CD3 e (29). This motif is characterized by two tyrosines spaced 9 or 10 residues apart, with conserved acidic residues and leucines spaced at fixed lengths from the tyrosines. The mechanism used by this tyrosine-containing domain to generate signals is unknown. e

As first noted by Reth, the tyrosine-containing signalling motif present redundantly in ; is also present in the CD3 8, e, -y, and q chains (28). Unlike X, which contains three of these motifs, a contains two of these motifs, and CD3 -y, and 8 each contain only one motif. On the basis of our data, it is plausible that the tyrosine-containing signalling motif may function through its ability to bind p59fn. The minimal p593"' binding domain of t that we have defined between residues 32 and 73 contains the most membrane proximal of the three tyrosine-containing signalling motifs. We have also shown that the p59'5n binding domain in t is redundant. The presence of multiple p59'" binding domains in t and q is supported by their apparent increased affinities toward p59Pn in comparison with CD3 e and -y. In addition, we observed that GZeta mutants lacking one or two of the tyrosine-containing signalling motifs have a lower affinity for p59'" (data not shown). We are currently engaged in a comprehensive study to determine the exact requirements for association of ; cytoplasmic sequences with p59?Sf. Is the minimal binding domain that we have defined equivalent to the minimal signalling domain as defined by others? Romeo et al. generated a series of e cytoplasmic truncation mutants to define the minimal signalling domain (29). They found that the ; cytoplasmic domain truncated to position 65 (three residues beyond the second tyrosine) could still signal but that a truncation to position 59 could not signal. In contrast, we found that the cytoplasmic domain of t truncated to residue 73 could still bind p59'5" but that a e,

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ASSOCIATION OF p596 WITH T-CELL RECEPTOR SUBUNITS

truncation to residue 69 could not. It is possible that the discrepancy between our results is due to differences in the constructs used (VSV G versus CD16), different sensitivities of the systems used, or the possibility that the p5915n binding motif is distinct from the tyrosine-containing signalling motif. Unfortunately, mapping studies reported by others used internal deletions rather than truncations to define the minimal signalling motif of t (16, 41). We plan to test the capacity of our constructs to signal so that a direct correlation can be made between p5915n binding and T-cell receptor signalling. Our finding that the domain of p59"' responsible for specific binding to t is contained within the first 10 residues of p59fi5f is surprising given that this domain is thought to also contain a signal for myristylation. It is, however, consistent with the hypothesis that membrane receptors recognize both specific amino-terminal sequences of myristylated proteins and the fatty acid moiety (35). Examination of the amino-terminal 10 residues of different Src family kinases shows sequence diversity as well as the presence of three positions of high conservation. Glycine at position 2 is required for myristylation and is, therefore, invariant for all myristylated proteins. Position 3 is cysteine for all Src family members except p6O'. Similarly, position 7 is lysine in all Src family members except p561ck. The other residues vary between the kinases and may determine the specificity of binding to putative myristyl-protein receptors. It is interesting to note that the first 10 amino acids of p58P' are very similar to those of p59fi, with amino acid differences at only three positions. We plan to test whether p58f' can associate with GZeta. Studies of p60 support the idea that myristyl-protein receptors recognize sequences within the first 10 residues of myristylated proteins. Because myristylation of p6(Y' is not sufficient for stable association with membranes (2, 5), and because binding of p60 to artificial liposomes is dependent on the presence of membrane proteins (25), stable membrane association of myristylated proteins is thought to require additional proteins. In support of this view, binding to cell membranes of a myristylated p60 peptide containing residues 1 to 11 is sequence specific and saturable and may interact with a 32-kDa membrane protein (26, 27). Because t appears to bind to a sequence within the first 10 residues of p5915f, and because it presumably facilitates membrane association, t may be a myristyl-protein receptor. It is, however, an interaction that is of significantly lower affinity than is the interaction of Src with its receptor. This interpretation, however, would support the idea that myristic acid facilitates specific protein-protein interactions in cellular membranes. Examples include the interactions between G-protein subunits (39), cytochrome b with cytochrome b5 reductase (22), and CD4 with p56k (37). We have implicated at least one mechanism for coupling tyrosine phosphorylation pathways to the multisubunit T-cell receptor complex. The demonstration that the protein tyrosine kinase p59?n can specifically associate with cytoplasmic sequences of the CD3 y, e, t, and rj chains suggest that signalling by these molecules may be mediated by p591Yf. Further studies will be important to determine whether regions of these proteins involved in binding the amino-terminal domain of p5915f are also critical for T-cell receptor signalling. More importantly, these studies support a critical role for the unique amino-terminal domains of Src family kinases in the coupling of tyrosine kinases to the signalling pathways of cell surface receptors.

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ACKNOWLEDGMENTS We thank M. Thomas, M. Olszowy, E. Cahir McFarland, J. Matthews, P. Conolly, and T. Woodford-Thomas for helpful comments. We thank J. Bolen, S. Parsons, S. Desiderio, and E. Reinherz for providing the antibodies to Fyn and Src and the cDNAs to Fyn and n, respectively. A. Schipper and W. Shaw provided excellent technical assistance. This project was initiated in the laboratory of Jack Rose, whose support and advice are warmly acknowledged. This work was supported by grants from the Edward Mallinkrodt Jr. Foundation, the Lucille Markey Charitable Trust, and the National Institutes of Health.

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