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Molecular and genetic insights into the role of protein tyrosine kinases in T cell receptor signaling
CLINICAL IMMUNOLOGYAND IMMUNOPATHOLOGY Vol. 76, No. 3, September, pp. S158-S162, 1995
Molecular and Genetic Insights into the Role of Protein Tyrosine Kinases in T Cell Receptor Signaling 1 ARTHUR WEISS
Departments of Medicine and of Microbiology and Immunology, Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, California 94143 INTRODUCTION
Recently, we have witnessed a convergence of progress in understanding the fundamental mechanisms involved in T cell biology and the molecular basis for a number of immune deficiency syndromes. Knowledge regarding signal transduction mechanisms of several receptors on T cells, such as the interleukin-2 (IL-2) receptor and T cell antigen receptor (TCR), has led to the identification of the molecular basis of T cell immune deficiency syndromes. These immune deficiency disorders have also provided unexpected insights into the biology of these receptors. This manuscript will focus on studies of the protein tyrosine kinases (PTKs) involved in TCR signal transduction. As a result of these studies, a rare form of the severe combined immune deficiency (SCID) syndrome was shown to result from mutations that inactivate the function of a protein PTK, ZAP-70, that participates in TCR signal transduction. The earliest event noted following TCR stimulation is the induced tyrosine phosphorylation of a variety of cellular proteins, including subunits of the TCR (CD3 and ~ chains), phospholipase C T1 (PLC T1), the product of the protooncogene v a v , and mitogen-activated protein kinase (MAP kinase) (1, 2). These phosphorylation events lead to a cascade of biochemical events which culminate in a cellular response. Phosphorylation of PLC 71 results in its activation (3). The activation of PLC vl is responsible for an increase in cytoplasmic-free calcium and activation of protein kinase C, events that are required for the induction of interleukin-2 gene transcription (2). Thus, the induction of protein tyrosine phosphorylation is critical for T cell responses. Considerable effort has focused on understanding how the TCR induces protein tyrosine phosphorylation. Unlike many PTK growth factor receptors, the TCR does not have PTK or protein tyrosine phosPresented as part of the fifth Jeffrey Modell Immunodeficiency Symposium titled "Advances in Primary Immunodeficiency Disease," October 10-11, 1994, Paris, France.
phatase (PTPases) domains. Instead, the TCR uses recently recognized structural domains contained in the cytoplasmic tails of the CD3 and ~ chains to interact with cytoplasmic PTKs (2). This interaction modifies a preexisting dynamic equilibrium b e t w e e n cellular PTKs and PTPases, leading to an increase in net protein tyrosine phosphorylation.
T H E TCR CD3 AND ~ C H A I N S I N T E R A C T W I T H CYTOPLASMIC PTKS
The TCR is an oligomeric complex consisting of an antigen-binding subunit and subunits involved in signaling. The Ti a~ heterodimer recognizes peptides bound to major histocompatibility complex molecules. The signal transduction function of the CD3 and chains has been most clearly established by studies employing chimeric receptors. In these chimeras, the cytoplasmic domains of the CD3 • or ~ chains have been linked to the extracellular and t r a n s m e m b r a n e domains of other transmembrane molecules (4-6). These chimeric receptors acquired the signal transduction function characteristic of the intact TCR complex. Stimulation of such chimeras with monoclonal antibodies reactive with their extracellular domains induced all of the events characteristic of the intact TCR. Thus, the cytoplasmic domains of both the CD3 • and ~ chains contain the sequence information necessary to couple the TCR to intracellular signal transduction machinery. This paradoxical redundancy of function of the CD3 and ~ chains was explained by mapping the functional domains in the cytoplasmic sequences in chimeric receptors (6-8). These studies revealed the presence of a functional sequence motif, termed ITAM (for immunoreceptor activation motif), which is triplicated within the cytoplasmic domain of ~ and contained as a single copy in each of the CD3 chains. The ITAM is sufficient and necessary to confer signal transduction function upon chimeric receptors. The ITAM motif, first noted by Reth (9), contains the consensus sequence of YXXL(X)6_sYXXL. Func-
S158 0090-1229/95 $12.00 Copyright © 1995 by Academic Press, Inc. All rights of reproduction in any form reserved.
PTKs IN TCR SIGNALTRANSDUCTION tional ITAMs are found in the non-ligand-binding subunits of other antigen receptors, including the B cell antigen receptor and the mast cell IgE Fc receptor. The ITAMs appear to have evolved from a common precursor, as their exon-intron organization is similar (10). E a c h of t h e a n t i g e n r e c e p t o r s c o n t a i n s m u l t i p l e ITAMs. The functional significance of the presence of multiple ITAMs within an oligomeric antigen receptor with a single ligand binding subunit is of considerable interest. Individual ITAMs may contain sufficient sequence disparity to interact with distinct intracellular proteins involved in signal transduction. Some studies suggest that this may be the case (6, 11). Alternatively, the presence of multiple ITAMs within a receptor with a single binding subunit may represent a means of signal amplification. Evidence to support this latter hypothesis was provided by linking a single copy or three copies of a ~ ITAM to the transmembrane and extracellular domains of CD8 (8). The chimera with three copies of the ~ ITAM induced greater increases in tyrosine phosphoproteins, cytoplasmic-free calcium, and interleukin-2 gene transcriptional activity than a chimeric receptor incorporating one copy of the motif. Indeed, the response of the chimeric receptor with three ITAMs was comparable to that of the wild-type ~ sequence, which contains three distinct ITAMs. Thus, multiple ITAMs within a single receptor are likely to represent a means of signal amplification, thereby increasing the sensitivity of the TCR to antigen.
ITAMS I N T E R A C T WITH TWO D I S T I N C T FAMILIES OF P T K S
Biochemical and genetic studies have implicated at least two families of PTKs in TCR signal transduction. Two Src family members, Fyn and Lck, appear to play a role in TCR signaling. Under mild solubilization conditions, small amounts of Fyn coimmunoprecipitate with the TCR (12, 13). In mice, disruption of the fyn gene does not alter T cell development (14, 15). However, mature single positive (CD4 ÷ or CD8 ÷) thymocytes exhibit markedly diminished responses to TCR stimuli. Peripheral T cells have a much milder signaling defect and can respond to antigen. The other src family PTK, Lck, is associated with the cytoplasmic tails of the coreceptors CD4 and CD8 through an interaction involving cysteine residues in the cytoplasmic domains of these coreceptors and the unique N-terminal domain of Lck (16). Cell lines deficient in Lck kinase function have a marked impairment in TCR signal transduction (17, 18). Moreover, disruption of the lck gene results in a profound arrest in thymocyte development at an early double positive (CD4÷/CD8 ÷) stage (19). Thus, both of these PTKs appear to play important roles in TCR signal transduction. The Syk and ZAP-70 cytoplasmic PTKs are the only
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known members of another family of PTKs (20, 21). Unlike the Src PTKs, Syk and ZAP-70 lack an N-terminal myristilation site, an SH3 domain, and a C-terminal negative regulatory tyrosine phosphorylation site. Instead, Syk and ZAP-70 contain two tandem N-terminal SH2 domains and a more C-terminal kinase domain. ZAP-70 is expressed only in T cells and natural killer cells (21). However, Syk is expressed more broadly within the hematopoietic lineage (20). Syk is expressed at low levels in thymocytes and at even lower levels in peripheral T cells, compared to the high levels in B cells and myeloid cells (20, 22). In the J u r k a t T cell line, both ZAP-70 and Syk are recruited to the tyrosine phosphorylated ~ and CD3 ITAMs of the stimulated TCR (23). ZAP-70 has been shown to associate exclusively with the tyrosine-phosphorylated CD3 and ~ chains (21, 22). Both ZAP-70 and Syk are also inducibly phosphorylated on tyrosines following TCR stimulation (21, 22). These two families of PTKs appear to interact with an ITAM-containing receptor in a sequential manner (24). Stimulation of the TCR initially induces the tyrosine phosphorylation of ITAMs by a Src family member. This is likely to involve Lck or possibly Fyn in some cells. Genetic and biochemical studies support this model. In the J.CaM1 T cell line, which fails to express a functional Lck PTK, stimulation of the TCR fails to induce the phosphorylation of ~ or the CD3 chains. Likewise, in J.CaM1, ZAP-70 is not recruited to the TCR, nor is it tyrosine phosphorylated (24). These observations place Lck upstream of ZAP-70. These interactions have been studied in a heterologous cell system in further detail. In Cos 18, a Cos cell line stably expressing a CD8/~ chimera, transfection of either Lck or Fyn alone can induce low levels of phosphorylation of the CD8/~ chimera. The association of ZAP-70 or Syk with the CD8/~ chimera, as well as the optimal level of phosphorylation of ZAP-70 and Syk, requires Lck or Fyn. This is also associated with an increase in CD8/~ phosphorylation. Using mutants of Lck, we have shown that the kinase activity of Lck is required for the phosphorylation of CD8/~ as well as the recruitment and phosphorylation of ZAP-70. In contrast, the catalytic activity of ZAP-70 is not required for these events. These studies, using J.CaM1 and Cos 18 cells, provide strong evidence that Lck (or in some T cells Fyn) plays a role which is upstream of ZAP-70 in initiating signal transduction by the TCR. Both tyrosines within an ITAM are required for signal transduction and in the recruitment of ZAP-70. Mutation of either tyrosine eliminates the capability of the ITAM to confer signal transduction function upon chimeric receptors (6, 7). Thus, it is likely that phosphorylation of both tyrosines is required for the function of the ITAM and likely reflects the interaction of the ITAM with the SH2 domains of ZAP-70 or Syk. This notion has been confirmed with synthetic phos-
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phopeptides whose sequences are based on a ~ chain ITAM. Only the doubly phosphorylated ITAM could form a stable complex with ZAP-70 (24). Moreover, the phosphotyrosine binding activity of both SH2 domains of ZAP-70 is required for this interaction. This argues that only the doubly phosphorylated ITAM is able to interact with ZAP-70 and is mediated by the interaction of each SH2 domain with each phosphotyrosine residue in an ITAM. These observations offer an explanation for the requirement of two tyrosines within an ITAM for signal transduction function. The sequential interaction of the TCR ITAMs with these two families of PTKs is an attractive model since it incorporates into it the function of the coreceptors, CD4 and CD8. By binding to nonpolymorphic residues of MHC molecules, these coreceptors serve to enhance the sensitivity of the T cell to antigen (25). The cytoplasmic domains of CD4 and CD8 bind to the unique domain of Lck.'Thus, during antigen recognition, these coreceptors not only function to bind to MHC molecules and contribute to the binding energetics of the TCR/ major histocompatibility complex molecule/peptide, but also deliver and concentrate Lck to the ITAMs. This can augment signal transduction events (26). Once recruited to the TCR complex, the function of ZAP-70 is not so clear. In the Cos 18 cell system, a very large increase in cellular tyrosine phosphorylation is observed only if catalytically active ZAP-70 is cotransfected with either Lck or Fyn (21, 24). In further support of an important function of ZAP-70 is the correlation between the sequence motifs responsible for chain signal transduction, the ITAMs, and the sequence requirements for TCR and ZAP-70 interactions (8). However, the most compelling evidence that ZAP70 plays a critical role in TCR signal transduction comes from observations of a relatively rare form of the SCID syndrome.
ZAP-70 M U T A T I O N S A R E R E S P O N S I B L E F O R A F O R M OF T H E SCID S Y N D R O M E
Although most forms of SCID result from an absence or paucity of T and sometimes B cells, a minority of patients with SCID have normal or increased numbers of T cells that are functionally impaired. One such group of patients has been described which is uniquely characterized by the presence of CD4 ÷ T cells in peripheral blood but is deficient in CD8 ÷ T cells (27-29). The CD4 T cells are unusual in their failure to proliferate to typical TCR-dependent stimuli such as phytohemagglutinin and anti-CD3 monoclonal antibodies. These cells do respond to the combination of calcium ionophore and phorbol ester, reagents which mimic the downstream events induced by the TCR. They also respond to exogenous interleukin-2. Natural killer cell cytolytic activity and B cell responses to membrane
immunoglobulin stimulation are preserved. These observations suggest that these patients have a selective defect in which their CD4 T cells have a defect in TCR signal transduction. Indeed, analysis of freshly isolated cells or short-term T cell lines revealed that stimulation of the TCR on these CD4 ÷ T cells failed to induce increases in tyrosine phosphoproteins or in cytoplasmic-free calcium (28, 29). In order to identify the molecular basis for this selective defect in TCR signal transduction, the expression of PTKs and PTPases implicated in TCR signal transduction was assessed. Normal levels of the CD45 PTPase were detected in these cells. Subsequent analysis of the PTKs revealed that although normal levels of Lck, Fyn, and Syk protein could be detected by Western blot analysis, no ZAP-70 protein could be detected (27-29). To date, five affected families have been studied in detail. Three of these families are Canadian Mennonites and two m u t a n t alleles have been identified in these patients. One is a missense mutation in the kinase domain, and the other represents a point mutation in an intron which is responsible for a new splice acceptor site. ZAP-70 is an autosomal gene encoded on chromosome 2q12 (27, 29). Therefore, it is not surprising that the affected family members are homozygotes or compound heterozygotes inheriting mutant alleles from both parents who are heterozygous carriers. The patients in the other two families inherited m u t a n t alleles homozygously; they were the result of consanguinity. One mutation is a 13-bp deletion in the kinase domain (28). The transcripts that are expressed give rise to unstable proteins that lack kinase activity when analyzed in the Cos cell system. These data provide strong evidence that the mutations in ZAP-70 account for this rare form of the SCID syndrome. They also definitively establish the critical role that ZAP-70 plays in signal transduction by the TCR in CD4 ÷ peripheral T cells. Still unexplained, but perhaps providing unique and unexpected insights into T cell biology is the relative paucity of CD8 ÷ T cells in peripheral blood and the explanation for how CD4 ÷ T cells can develop in the absence of signal transduction by their TCRs. At least one study has shown that CD4 and CD8 antigens can be detected on cortical thymocytes (27). However, only CD4 antigens were detected on medullary thymocytes from such patients. This suggests a block in positive selection of CD8 ÷ but not CD4 ÷ thymocytes. Both CD4and CD8-positive cells express ZAP-70 (22). Thus, the differential expression of ZAP-70 does not explain the failure of CD8-positive cells to be selected. It is possible that the diminished association of Lck with CD8 compared to CD4 plays a role (30). Perhaps the CD4 lineage is favored under conditions of impaired signal transduction, i.e., in the absence of ZAP-70. If peripheral CD4 ÷ cells cannot signal, how then are these cells positively selected in the thymus? One possible expla-
PTKs IN TCR SIGNAL TRANSDUCTION
nation is compensation by Syk for the loss of ZAP-70 in the thymus. Since Syk is expressed at higher levels in the thymus than in peripheral T cells (22), it may play a somewhat redundant role in thymus. However, Syk may not fully compensate for ZAP-70, explaining the failure to select the CD8 lineage. Thus, the preferential selection of the CD4 lineage may reflect the bias imposed by the preferential association of Lck with this coreceptor. Clearly, a more definitive explanation of the phenotype in these patients will await more detailed studies of the thymocytes from these patients as well as an available mouse model. CONCLUSION
Studies of the basic mechanisms involved in TCR signal transduction reveal the complex, yet highly coordinated, interaction of the receptor with distinct PTKs. Such studies have contributed to our understanding of the roles of the CD3 and ~ subunits of the TCR. In addition, insights into the roles of at least two distinct families of cytoplasmic PTKs have been provided. These studies have led to a molecular understanding of a rare form of a SCID syndrome. Conversely, the SCID syndrome provides strong genetic evidence for the critical role that the ZAP-70 PTK plays in TCR signal transduction. Finally, the unusual phenotype of the T cells in this SCID syndrome has led to new questions regarding the roles of the ZAP-70 and Syk PTKs in T cell development and function. REFERENCES
1. Samelson, L. E., and Klausner, R. D., Tyrosine kinases and tyrosine-based activation motifs: Current research on activation via the T cell antigen receptor. J. Biol. Chem. 267, 24913-24916, 1992. 2. Weiss, A., 'and Littman, D. R., Signal transduction by lymphocyte antigen receptors. Cell 76, 263-274, 1994. 3. Nishibe, S., Wahl, M. I., Hernandez-Sotomayor, S. M., Tonk, N. K., Rhee, S. G., and Carpenter, G., Increase of the catalytic activity of phospholipase C-T1 by tyrosine phosphorylation. Science 250, 1253-1256, 1990. 4. Irving, B., and Weiss, A., The cytoplasmic domain of the T cell receptor ~ chain is sufficient to couple to receptor-associated signal transduction pathways. Cell 64, 891-901, 1991. 5. Romeo, C., and Seed, B., Cellular immunity to HIV activated by CD4 fused to T cell or Fc receptor polypeptides. Cell 64, 10371046, 1991. 6. Letourneur, F., and Klausner, R. D., Activation of T cells by a tyrosine kinase activation domain in the cytoplasmic tail of CD3e. Science 255, 79-82, 1992. 7. Romeo, C., Amiot, M., and Seed, B., Sequence requirements for induction of cytolysis by the T cell antigen/Fc receptor ~ chain. Cell 88, 889-897, 1992. 8. Irving, B. A., Chan,. A. C., and Weiss, A., Functional characterization of a signal transducing motif present in the T cell receptor ~ chain. J. Exp. Med. 177, 1093-1103, 1993. 9. Reth, M., Antigen receptor tail clue. Nature 338, 383-384, 1989.
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10. Wegener, A.-M. K., Letourneur, F., Hoeveler, A., Brocker, T., Luton, F., and Malissen, B., The T cell receptor/CD3 complex is composed of at least two autonomous transduction molecules. Cell 68, 83-95, 1992. 11. Clark, M. R., Campbell, K. S., Kazlaukas, A., Johnson, S.A., Hertz, M., Potter, T. A., Pleiman, C., and Cambier, J. C., The B cell antigen receptor complex association of Ig-a and Ig-!8 with distinct cytoplasmic effectors. Science 258, 123-126, 1992. 12. Samelson, L.E., Phillips, A. F., Luong, E.T., and Klausner, R. D., Association of the fyn protein-tyrosine kinase with the T-cell antigen receptor. Proc. Natl. Acad. Sci. USA 87, 43584362, 1990. 13. Sarosi, G. P., Thomas, P. M., Egerton, M., Phillips, A. F., Kim, K. W., Bonvini, E., and Samelson, L. E., Characterization of the T cell antigen receptor-p60 ~y" protein tyrosine kinase association by chemical cross-linking. Int. Immunol. 4, 1211-1217, 1992. 14. Appteby, M. W., Gross, J. A., Cooke, M. P., Levin, S. D., Qian, X., and Perlmutter, R. M., Defective T cell receptor signaling in mice lacking the thymic isoform of p59t~n. Cell 70, 751-763, 1992. 15. Stein, P. L., Lee, H.-M., Rich, S., and Soriano, P., pp5~~'" mutant mice display differential signaling in tbymocytes and peripheral T cells. Cell 70, 741-750, 1992. 16. Veillette, A., Abraham, N., Caron, L., and Davidson, D., The lymphocyte-specific tyrosine kinase p56 jck. Semin. Immunol. 3, 143-152, 1991. 17. Straus, D., and Weiss, A., Genetic evidence for the involvementof the Lck tyrosine kinase in signal transduction through the T cell antigen receptor. Cell 70, 585-593, 1992. 18. Karnitz, L., Sutor, S. L., Torigoe, T., Reed, J. C., Bell, M. P., McKean, D.J., Leibson, P.J., and Abraham, R.T., Effects of p56t'k on the growth and cytolytic effector function of an interleukin-2-dependent cytotoxic T-cell line. Mol. Cell. Biol. 12, 4521--4530, 1992. 19. Molina, T. J., Kishihara, K., Siderovski, D. P., van Ewijk, W., Narendran, A., Timms, E., Wakeham, A., Paige, C.J., Hartmann, K.-U., Veillette, A., Davidson, D., and Mak, T. W., Profound block in thymocyte development in mice lacking p561ck. Nature 357, 161-164, 1992. 20. Taniguchi, T., Kobayashi, T., Kondo, J., Takahashi, K., Nakamura, H., Suzuki, J., Nagai, K., Yamada, T., Nakamura, S., and Yamaamura, H., Molecular cloning of a porcine gene syk that encodes a 72-kDa protein-tyrosine kinase showing high susceptibility to proteolysis. J. Biol. Chem. 266, 15790-15796, 1991. 21. Chan, A. C., Iwashima, M., Turck, C. W., and Weiss, A., ZAP-70: A 70kD protein tyrosine kinase that associates with the TCR {: chain. Cell 71, 649-662, 1992. 22. Chan, A. C., van Oers, N. S. C., Tran, A., Turka, L., Law, C.-L., Ryan, J. C., Clark, E. A., and Weiss, A., Differential expression of ZAP-70 and Syk protein tyrosine kinases, and the role of this family of protein tyrosine kinases in T cell antigen receptor signaling. J. lmmunol. 152, 4758-4766, 1994. 23. Chan, A. C., Irving, B.A., Fraser, J. D., and Weiss, A., The {:-chain is associated with a tyrosine kinase and upon T cell antigen receptor stimulation associates with ZAP-70, a 70 kilodalton tyrosine phosphoprotein. Proc. Natl. Acad. Sci. USA 88, 9166-9170, 1991. 24. Iwashima, M., Irving, B. A., van Oers, N. S. C., Chan, A. C., and Weiss, A., Sequential interactions of the TCR with two distinct cytoplasmic tyrosine kinases. Science 263, 1136-1139, 1994. 25. Janeway, C.A., Jr., The T cell receptor as a multicomponent signalling machine: CD4/CD8 coreceptors and CD45 in T cell activation. Annu. Rev. Immunol. 10, 645-674, 1992. 26. Ledbetter, J. A., Gilliland, L. K., and Schieven, G. A., The interaction of CD4 with CD3/Ti regulates tyrosine phosphorylation of
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substrates during T cell activation. Semin. Immunol. 2, 99-106, 1990. 27. Arpaia, E., Shahar, M., Dadi, H., Cohen, A., and Roifman, C. M., Defective T cell receptor signaling and CD8÷ thymic selection in humans lacking ZAP-70 kinase. Cell 76, 947-958, 1994. 28. Elder, M. E., Lin, D., Clever, J., Cahn, A. C., Hope, T. J., Weiss, A., and Parslow, T. C-., Human severe combined immunodeficiency due to a defect in ZAP-70---A T-cell tyrosine receptorlinked tyrosine kinase. Science 264, 1596-1599, 1994. 29. Chan, A. C., Kadlecek, T. A., Elder, M. E., Filipovich, A. H., Kuo, Received March 16, 1995; accepted May 12, 1995
W.-L., Iwashima, M., Parslow, T. G., and Weiss, A., ZAP-70 deficiency in an autosomal recessive form of severe combined immunodeficiency. Science 264, 1599-1603, 1994. 30. Wiest, D. L., Yuan, L., Jefferson, J., Benveniste, P., Tsokos, M., K]ausner, R. D., Glimcher, L. H., Samelson, L. E., and Singer. A., Regulation of T cell receptor expression in immature CD4+CD8+ thymocytes by p56~c* tyrosine kinase: Basis for differential signaling by CD4 and CD8 in immature thymocytes expressing both coreceptor molecules. J. Exp. Med. 178, 17011712, 1993.
Molecular and Genetic Insights into the Role of Protein Tyrosine Kinases in T Cell Receptor Signaling 1 ARTHUR WEISS
Departments of Medicine and of Microbiology and Immunology, Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, California 94143 INTRODUCTION
Recently, we have witnessed a convergence of progress in understanding the fundamental mechanisms involved in T cell biology and the molecular basis for a number of immune deficiency syndromes. Knowledge regarding signal transduction mechanisms of several receptors on T cells, such as the interleukin-2 (IL-2) receptor and T cell antigen receptor (TCR), has led to the identification of the molecular basis of T cell immune deficiency syndromes. These immune deficiency disorders have also provided unexpected insights into the biology of these receptors. This manuscript will focus on studies of the protein tyrosine kinases (PTKs) involved in TCR signal transduction. As a result of these studies, a rare form of the severe combined immune deficiency (SCID) syndrome was shown to result from mutations that inactivate the function of a protein PTK, ZAP-70, that participates in TCR signal transduction. The earliest event noted following TCR stimulation is the induced tyrosine phosphorylation of a variety of cellular proteins, including subunits of the TCR (CD3 and ~ chains), phospholipase C T1 (PLC T1), the product of the protooncogene v a v , and mitogen-activated protein kinase (MAP kinase) (1, 2). These phosphorylation events lead to a cascade of biochemical events which culminate in a cellular response. Phosphorylation of PLC 71 results in its activation (3). The activation of PLC vl is responsible for an increase in cytoplasmic-free calcium and activation of protein kinase C, events that are required for the induction of interleukin-2 gene transcription (2). Thus, the induction of protein tyrosine phosphorylation is critical for T cell responses. Considerable effort has focused on understanding how the TCR induces protein tyrosine phosphorylation. Unlike many PTK growth factor receptors, the TCR does not have PTK or protein tyrosine phosPresented as part of the fifth Jeffrey Modell Immunodeficiency Symposium titled "Advances in Primary Immunodeficiency Disease," October 10-11, 1994, Paris, France.
phatase (PTPases) domains. Instead, the TCR uses recently recognized structural domains contained in the cytoplasmic tails of the CD3 and ~ chains to interact with cytoplasmic PTKs (2). This interaction modifies a preexisting dynamic equilibrium b e t w e e n cellular PTKs and PTPases, leading to an increase in net protein tyrosine phosphorylation.
T H E TCR CD3 AND ~ C H A I N S I N T E R A C T W I T H CYTOPLASMIC PTKS
The TCR is an oligomeric complex consisting of an antigen-binding subunit and subunits involved in signaling. The Ti a~ heterodimer recognizes peptides bound to major histocompatibility complex molecules. The signal transduction function of the CD3 and chains has been most clearly established by studies employing chimeric receptors. In these chimeras, the cytoplasmic domains of the CD3 • or ~ chains have been linked to the extracellular and t r a n s m e m b r a n e domains of other transmembrane molecules (4-6). These chimeric receptors acquired the signal transduction function characteristic of the intact TCR complex. Stimulation of such chimeras with monoclonal antibodies reactive with their extracellular domains induced all of the events characteristic of the intact TCR. Thus, the cytoplasmic domains of both the CD3 • and ~ chains contain the sequence information necessary to couple the TCR to intracellular signal transduction machinery. This paradoxical redundancy of function of the CD3 and ~ chains was explained by mapping the functional domains in the cytoplasmic sequences in chimeric receptors (6-8). These studies revealed the presence of a functional sequence motif, termed ITAM (for immunoreceptor activation motif), which is triplicated within the cytoplasmic domain of ~ and contained as a single copy in each of the CD3 chains. The ITAM is sufficient and necessary to confer signal transduction function upon chimeric receptors. The ITAM motif, first noted by Reth (9), contains the consensus sequence of YXXL(X)6_sYXXL. Func-
S158 0090-1229/95 $12.00 Copyright © 1995 by Academic Press, Inc. All rights of reproduction in any form reserved.
PTKs IN TCR SIGNALTRANSDUCTION tional ITAMs are found in the non-ligand-binding subunits of other antigen receptors, including the B cell antigen receptor and the mast cell IgE Fc receptor. The ITAMs appear to have evolved from a common precursor, as their exon-intron organization is similar (10). E a c h of t h e a n t i g e n r e c e p t o r s c o n t a i n s m u l t i p l e ITAMs. The functional significance of the presence of multiple ITAMs within an oligomeric antigen receptor with a single ligand binding subunit is of considerable interest. Individual ITAMs may contain sufficient sequence disparity to interact with distinct intracellular proteins involved in signal transduction. Some studies suggest that this may be the case (6, 11). Alternatively, the presence of multiple ITAMs within a receptor with a single binding subunit may represent a means of signal amplification. Evidence to support this latter hypothesis was provided by linking a single copy or three copies of a ~ ITAM to the transmembrane and extracellular domains of CD8 (8). The chimera with three copies of the ~ ITAM induced greater increases in tyrosine phosphoproteins, cytoplasmic-free calcium, and interleukin-2 gene transcriptional activity than a chimeric receptor incorporating one copy of the motif. Indeed, the response of the chimeric receptor with three ITAMs was comparable to that of the wild-type ~ sequence, which contains three distinct ITAMs. Thus, multiple ITAMs within a single receptor are likely to represent a means of signal amplification, thereby increasing the sensitivity of the TCR to antigen.
ITAMS I N T E R A C T WITH TWO D I S T I N C T FAMILIES OF P T K S
Biochemical and genetic studies have implicated at least two families of PTKs in TCR signal transduction. Two Src family members, Fyn and Lck, appear to play a role in TCR signaling. Under mild solubilization conditions, small amounts of Fyn coimmunoprecipitate with the TCR (12, 13). In mice, disruption of the fyn gene does not alter T cell development (14, 15). However, mature single positive (CD4 ÷ or CD8 ÷) thymocytes exhibit markedly diminished responses to TCR stimuli. Peripheral T cells have a much milder signaling defect and can respond to antigen. The other src family PTK, Lck, is associated with the cytoplasmic tails of the coreceptors CD4 and CD8 through an interaction involving cysteine residues in the cytoplasmic domains of these coreceptors and the unique N-terminal domain of Lck (16). Cell lines deficient in Lck kinase function have a marked impairment in TCR signal transduction (17, 18). Moreover, disruption of the lck gene results in a profound arrest in thymocyte development at an early double positive (CD4÷/CD8 ÷) stage (19). Thus, both of these PTKs appear to play important roles in TCR signal transduction. The Syk and ZAP-70 cytoplasmic PTKs are the only
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known members of another family of PTKs (20, 21). Unlike the Src PTKs, Syk and ZAP-70 lack an N-terminal myristilation site, an SH3 domain, and a C-terminal negative regulatory tyrosine phosphorylation site. Instead, Syk and ZAP-70 contain two tandem N-terminal SH2 domains and a more C-terminal kinase domain. ZAP-70 is expressed only in T cells and natural killer cells (21). However, Syk is expressed more broadly within the hematopoietic lineage (20). Syk is expressed at low levels in thymocytes and at even lower levels in peripheral T cells, compared to the high levels in B cells and myeloid cells (20, 22). In the J u r k a t T cell line, both ZAP-70 and Syk are recruited to the tyrosine phosphorylated ~ and CD3 ITAMs of the stimulated TCR (23). ZAP-70 has been shown to associate exclusively with the tyrosine-phosphorylated CD3 and ~ chains (21, 22). Both ZAP-70 and Syk are also inducibly phosphorylated on tyrosines following TCR stimulation (21, 22). These two families of PTKs appear to interact with an ITAM-containing receptor in a sequential manner (24). Stimulation of the TCR initially induces the tyrosine phosphorylation of ITAMs by a Src family member. This is likely to involve Lck or possibly Fyn in some cells. Genetic and biochemical studies support this model. In the J.CaM1 T cell line, which fails to express a functional Lck PTK, stimulation of the TCR fails to induce the phosphorylation of ~ or the CD3 chains. Likewise, in J.CaM1, ZAP-70 is not recruited to the TCR, nor is it tyrosine phosphorylated (24). These observations place Lck upstream of ZAP-70. These interactions have been studied in a heterologous cell system in further detail. In Cos 18, a Cos cell line stably expressing a CD8/~ chimera, transfection of either Lck or Fyn alone can induce low levels of phosphorylation of the CD8/~ chimera. The association of ZAP-70 or Syk with the CD8/~ chimera, as well as the optimal level of phosphorylation of ZAP-70 and Syk, requires Lck or Fyn. This is also associated with an increase in CD8/~ phosphorylation. Using mutants of Lck, we have shown that the kinase activity of Lck is required for the phosphorylation of CD8/~ as well as the recruitment and phosphorylation of ZAP-70. In contrast, the catalytic activity of ZAP-70 is not required for these events. These studies, using J.CaM1 and Cos 18 cells, provide strong evidence that Lck (or in some T cells Fyn) plays a role which is upstream of ZAP-70 in initiating signal transduction by the TCR. Both tyrosines within an ITAM are required for signal transduction and in the recruitment of ZAP-70. Mutation of either tyrosine eliminates the capability of the ITAM to confer signal transduction function upon chimeric receptors (6, 7). Thus, it is likely that phosphorylation of both tyrosines is required for the function of the ITAM and likely reflects the interaction of the ITAM with the SH2 domains of ZAP-70 or Syk. This notion has been confirmed with synthetic phos-
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phopeptides whose sequences are based on a ~ chain ITAM. Only the doubly phosphorylated ITAM could form a stable complex with ZAP-70 (24). Moreover, the phosphotyrosine binding activity of both SH2 domains of ZAP-70 is required for this interaction. This argues that only the doubly phosphorylated ITAM is able to interact with ZAP-70 and is mediated by the interaction of each SH2 domain with each phosphotyrosine residue in an ITAM. These observations offer an explanation for the requirement of two tyrosines within an ITAM for signal transduction function. The sequential interaction of the TCR ITAMs with these two families of PTKs is an attractive model since it incorporates into it the function of the coreceptors, CD4 and CD8. By binding to nonpolymorphic residues of MHC molecules, these coreceptors serve to enhance the sensitivity of the T cell to antigen (25). The cytoplasmic domains of CD4 and CD8 bind to the unique domain of Lck.'Thus, during antigen recognition, these coreceptors not only function to bind to MHC molecules and contribute to the binding energetics of the TCR/ major histocompatibility complex molecule/peptide, but also deliver and concentrate Lck to the ITAMs. This can augment signal transduction events (26). Once recruited to the TCR complex, the function of ZAP-70 is not so clear. In the Cos 18 cell system, a very large increase in cellular tyrosine phosphorylation is observed only if catalytically active ZAP-70 is cotransfected with either Lck or Fyn (21, 24). In further support of an important function of ZAP-70 is the correlation between the sequence motifs responsible for chain signal transduction, the ITAMs, and the sequence requirements for TCR and ZAP-70 interactions (8). However, the most compelling evidence that ZAP70 plays a critical role in TCR signal transduction comes from observations of a relatively rare form of the SCID syndrome.
ZAP-70 M U T A T I O N S A R E R E S P O N S I B L E F O R A F O R M OF T H E SCID S Y N D R O M E
Although most forms of SCID result from an absence or paucity of T and sometimes B cells, a minority of patients with SCID have normal or increased numbers of T cells that are functionally impaired. One such group of patients has been described which is uniquely characterized by the presence of CD4 ÷ T cells in peripheral blood but is deficient in CD8 ÷ T cells (27-29). The CD4 T cells are unusual in their failure to proliferate to typical TCR-dependent stimuli such as phytohemagglutinin and anti-CD3 monoclonal antibodies. These cells do respond to the combination of calcium ionophore and phorbol ester, reagents which mimic the downstream events induced by the TCR. They also respond to exogenous interleukin-2. Natural killer cell cytolytic activity and B cell responses to membrane
immunoglobulin stimulation are preserved. These observations suggest that these patients have a selective defect in which their CD4 T cells have a defect in TCR signal transduction. Indeed, analysis of freshly isolated cells or short-term T cell lines revealed that stimulation of the TCR on these CD4 ÷ T cells failed to induce increases in tyrosine phosphoproteins or in cytoplasmic-free calcium (28, 29). In order to identify the molecular basis for this selective defect in TCR signal transduction, the expression of PTKs and PTPases implicated in TCR signal transduction was assessed. Normal levels of the CD45 PTPase were detected in these cells. Subsequent analysis of the PTKs revealed that although normal levels of Lck, Fyn, and Syk protein could be detected by Western blot analysis, no ZAP-70 protein could be detected (27-29). To date, five affected families have been studied in detail. Three of these families are Canadian Mennonites and two m u t a n t alleles have been identified in these patients. One is a missense mutation in the kinase domain, and the other represents a point mutation in an intron which is responsible for a new splice acceptor site. ZAP-70 is an autosomal gene encoded on chromosome 2q12 (27, 29). Therefore, it is not surprising that the affected family members are homozygotes or compound heterozygotes inheriting mutant alleles from both parents who are heterozygous carriers. The patients in the other two families inherited m u t a n t alleles homozygously; they were the result of consanguinity. One mutation is a 13-bp deletion in the kinase domain (28). The transcripts that are expressed give rise to unstable proteins that lack kinase activity when analyzed in the Cos cell system. These data provide strong evidence that the mutations in ZAP-70 account for this rare form of the SCID syndrome. They also definitively establish the critical role that ZAP-70 plays in signal transduction by the TCR in CD4 ÷ peripheral T cells. Still unexplained, but perhaps providing unique and unexpected insights into T cell biology is the relative paucity of CD8 ÷ T cells in peripheral blood and the explanation for how CD4 ÷ T cells can develop in the absence of signal transduction by their TCRs. At least one study has shown that CD4 and CD8 antigens can be detected on cortical thymocytes (27). However, only CD4 antigens were detected on medullary thymocytes from such patients. This suggests a block in positive selection of CD8 ÷ but not CD4 ÷ thymocytes. Both CD4and CD8-positive cells express ZAP-70 (22). Thus, the differential expression of ZAP-70 does not explain the failure of CD8-positive cells to be selected. It is possible that the diminished association of Lck with CD8 compared to CD4 plays a role (30). Perhaps the CD4 lineage is favored under conditions of impaired signal transduction, i.e., in the absence of ZAP-70. If peripheral CD4 ÷ cells cannot signal, how then are these cells positively selected in the thymus? One possible expla-
PTKs IN TCR SIGNAL TRANSDUCTION
nation is compensation by Syk for the loss of ZAP-70 in the thymus. Since Syk is expressed at higher levels in the thymus than in peripheral T cells (22), it may play a somewhat redundant role in thymus. However, Syk may not fully compensate for ZAP-70, explaining the failure to select the CD8 lineage. Thus, the preferential selection of the CD4 lineage may reflect the bias imposed by the preferential association of Lck with this coreceptor. Clearly, a more definitive explanation of the phenotype in these patients will await more detailed studies of the thymocytes from these patients as well as an available mouse model. CONCLUSION
Studies of the basic mechanisms involved in TCR signal transduction reveal the complex, yet highly coordinated, interaction of the receptor with distinct PTKs. Such studies have contributed to our understanding of the roles of the CD3 and ~ subunits of the TCR. In addition, insights into the roles of at least two distinct families of cytoplasmic PTKs have been provided. These studies have led to a molecular understanding of a rare form of a SCID syndrome. Conversely, the SCID syndrome provides strong genetic evidence for the critical role that the ZAP-70 PTK plays in TCR signal transduction. Finally, the unusual phenotype of the T cells in this SCID syndrome has led to new questions regarding the roles of the ZAP-70 and Syk PTKs in T cell development and function. REFERENCES
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