CLINICAL IMMUNOLOGY AND 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 W E I S S
Departments of Medicine and of Microbiology and Immunology, Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, California 94143
I N T R O D U C T I O N
Recently, we have witnessed a convergence of prog- ress 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 im- mune deficiency syndromes. These immune deficiency disorders have also provided unexpected insights into the biology of these receptors. This manuscript will fo- cus 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 im- mune deficiency (SCID) syndrome was shown to result from mutations that inactivate the function of a pro- tein 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 prod- uct of the protooncogene vav , and mitogen-activated protein kinase (MAP kinase) (1, 2). These phosphory- lation events lead to a cascade of biochemical events which culminate in a cellular response. Phosphoryla- tion of PLC 71 results in its activation (3). The activa- tion of PLC vl is responsible for an increase in cyto- plasmic-free calcium and activation of protein kinase C, events that are required for the induction of inter- leukin-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 phosphoryla- tion. Unlike many PTK growth factor receptors, the TCR does not have PTK or protein tyrosine phos-
Presented as par t 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 re- cently recognized structural domains contained in the cytoplasmic tails of the CD3 and ~ chains to interact with cytoplasmic PTKs (2). This interaction modifies a preexis t ing dynamic equi l ibr ium be tween cel lular PTKs and PTPases, leading to an increase in net pro- tein tyrosine phosphorylation.
THE TCR CD3 AND ~ CHAINS INTERACT WITH CYTOPLASMIC PTKS
The TCR is an oligomeric complex consisting of an antigen-binding subunit and subunits involved in sig- naling. 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 ransmembrane do- mains of other t ransmembrane molecules (4-6). These chimeric receptors acquired the signal transduction function characterist ic of the intact TCR complex. Stimulation of such chimeras with monoclonal antibod- ies 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 machin- ery.
This paradoxical redundancy of function of the CD3 and ~ chains was explained by mapping the functional domains in the cytoplasmic sequences in chimeric re- ceptors (6-8). These studies revealed the presence of a functional sequence motif, termed ITAM (for immuno- receptor 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-
tional ITAMs are found in the non-ligand-binding sub- units 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 precur- sor, as their exon-intron organization is similar (10). Each of the an t igen recep tors conta ins mul t ip le 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 se- quence 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 sig- nal amplification. Evidence to support this lat ter hy- pothesis was provided by linking a single copy or three copies of a ~ ITAM to the t ransmembrane and extra- cellular domains of CD8 (8). The chimera with three copies of the ~ ITAM induced greater increases in ty- rosine phosphoproteins, cytoplasmic-free calcium, and interleukin-2 gene transcriptional activity than a chi- meric receptor incorporating one copy of the motif. In- deed, the response of the chimeric receptor with three ITAMs was comparable to that of the wild-type ~ se- quence, which contains three distinct ITAMs. Thus, multiple ITAMs within a single receptor are likely to represent a means of signal amplification, thereby in- creasing the sensitivity of the TCR to antigen.
ITAMS INTERACT WITH TWO DISTINCT FAMILIES OF PTKS
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 con- ditions, 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). How- ever, mature single positive (CD4 ÷ or CD8 ÷) thymo- cytes exhibit markedly diminished responses to TCR stimuli. Peripheral T cells have a much milder signal- ing 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 in- teraction involving cysteine residues in the cytoplas- mic domains of these coreceptors and the unique N-ter- minal domain of Lck (16). Cell lines deficient in Lck kinase function have a marked impairment in TCR sig- nal transduction (17, 18). Moreover, disruption of the lck gene results in a profound arrest in thymocyte de- velopment 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
known members of another family of PTKs (20, 21). Unlike the Src PTKs, Syk and ZAP-70 lack an N-ter- minal myristilation site, an SH3 domain, and a C-ter- minal negative regulatory tyrosine phosphorylation site. Instead, Syk and ZAP-70 contain two tandem N-terminal SH2 domains and a more C-terminal ki- nase 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 Ju rka 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 asso- ciate exclusively with the tyrosine-phosphoryla ted 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 tyro- sine phosphorylation of ITAMs by a Src family mem- ber. 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 heterolo- gous 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 phos- phorylation 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 recrui tment and phosphorylation of ZAP-70. In contrast, the catalytic activity of ZAP-70 is not re- quired 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 sig- nal 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 phos- phorylation of both tyrosines is required for the func- tion 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-
S160 ARTHUR WEISS
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 interac- tion of each SH2 domain with each phosphotyrosine residue in an ITAM. These observations offer an expla- nation 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 cyto- plasmic 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 histocompatibil i ty 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 cotrans- fected with either Lck or Fyn (21, 24). In further sup- port of an important function of ZAP-70 is the correla- tion between the sequence motifs responsible for chain signal transduction, the ITAMs, and the se- quence requirements for TCR and ZAP-70 interactions (8). However, the most compelling evidence that ZAP- 70 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 ARE R E S P O N S I B L E F O R A FORM OF THE 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 periph- eral 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 phytohemag- glutinin and anti-CD3 monoclonal antibodies. These cells do respond to the combination of calcium iono- phore and phorbol ester, reagents which mimic the downstream events induced by the TCR. They also re- spond to exogenous interleukin-2. Natural killer cell cytolytic activity and B cell responses to membrane
immunoglobulin stimulation are preserved. These ob- servations 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 iso- lated cells or short-term T cell lines revealed that stim- ulation of the TCR on these CD4 ÷ T cells failed to in- duce increases in tyrosine phosphoproteins or in cyto- plasmic-free calcium (28, 29).
In order to identify the molecular basis for this se- lective defect in TCR signal transduction, the expres- sion of PTKs and PTPases implicated in TCR signal transduction was assessed. Normal levels of the CD45 PTPase were detected in these cells. Subsequent anal- ysis of the PTKs revealed that although normal levels of Lck, Fyn, and Syk protein could be detected by West- ern blot analysis, no ZAP-70 protein could be detected (27-29). To date, five affected families have been stud- ied in detail. Three of these families are Canadian Mennonites and two mutant alleles have been identi- fied 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 en- coded on chromosome 2q12 (27, 29). Therefore, it is not surprising that the affected family members are ho- mozygotes or compound heterozygotes inheriting mu- tant alleles from both parents who are heterozygous carriers. The patients in the other two families inher- ited mutant alleles homozygously; they were the result of consanguinity. One mutat ion is a 13-bp deletion in the kinase domain (28). The transcripts that are ex- pressed give rise to unstable proteins that lack kinase activity when analyzed in the Cos cell system. These data provide strong evidence that the mutat ions in ZAP-70 account for this rare form of the SCID syn- drome. 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 CD4- and 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 com- pared to CD4 plays a role (30). Perhaps the CD4 lin- eage is favored under conditions of impaired signal transduction, i.e., in the absence of ZAP-70. If periph- eral CD4 ÷ cells cannot signal, how then are these cells positively selected in the thymus? One possible expla-
PTKs IN TCR SIGNAL TRANSDUCTION S161
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 im- posed by the preferential association of Lck with this coreceptor. Clearly, a more definitive explanation of the phenotype in these patients will await more de- tailed 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 co- ordinated, interaction of the receptor with distinct PTKs. Such studies have contributed to our under- standing 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 pro- vided. These studies have led to a molecular under- standing of a rare form of a SCID syndrome. Con- versely, the SCID syndrome provides strong genetic evidence for the critical role that the ZAP-70 PTK plays in TCR signal transduction. Finally, the unusual phe- notype 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.
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