Cell, Vol. 64, 875-878,
March
8, 1991, Copyright
0 1991 by Cell Press
T Cell Antigen Receptor Activation Pathways: The Tyrosine Kinase Connection Richard D. Klausner and Lawrence E. Samelson Cell Biology and Metabolism Branch National Institute of Child Health and Human Development National Institutes of Health Bethesda, Maryland 20892
The central requirement for the T cells of the immune system is the expression of cell surface receptors capable of distinguishing between self and foreign antigens. These recognition events must then be translated into approprilate biochemical signals that control cellular responses. Thlis two-step process of antigen recognition and signal transduction underlies the ability to regulate the maturation of T cells via thymic selection, wherein T cells are educated to distinguish self from nonself and to initiate, in mature T cells, either the immune response or, in some circumstances, anergy. These complex processes are subserved by the T cell antigen receptor (TCR). The TCR found on the surface of all T cells is a complex, hetero-oligomeric structure composed of six or seven different subunits (reviewed in Klausner et al., 1990). These can be divided into three distinct subgroups of proteins. The immunoglobulin-like, clonotypic chains, responsible for ligand binding, are present as heterodimers within the receptor complex. There are two major forms of receptor heterodimers: a8, found on most mature T cells, and ~8, found predominantlyon Tcells that are located in epithelia. The CD3 chains encompass three distinct, but closely related, subunits: two glycoproteins, y and 8, and one nonglycosylated protein, E, encoded by three homologous, clustered genes. The CD3 chains likely exist in the receptor as twlo subcomplexes of CD3 S-CD3 E and CD3 y-CD3 E (Koningetal., 1990; Blumbergetal., 1990).Thefinalgroup of subunits is the < family dimers. This family is distinct fro’rn CD3 genetically, structurally, and in its range of cellular expression. Three proteins, encoded by two genes, comprise the c family as known to date. They are 6, its alternately spliced form, n, and they chain of multisubunit FCC:receptors. The segregation of the chains of the TCR complex into multiple subunits allows the mixing and matching of these subunits with consequent diversification of receptor types. CD3containing TCR complexes must contain a 5 dimer, and three such dimers have been documented to date: c-c, G-n, and I;-FCE y (Orloff et al., 1990, and references therein). The functions of the individual chains of the TCR cornplex other than the clonotypic subunits are not known. It is reasonably assumed that the remaining subunits are essential for signal transduction. Currently information exists only for the ; chain, whose cytoplasmic tail appears to be essential for antigen-stimulated signaling (Frank et al., 1990). The recent work of Irving and Weiss (1991) elegantly addresses the role of this TCR subunit. They created a chimera comprised of the extracellular and transmembrane domains of the CD8 molecule and the
Minireview
intracellular domain of the < chain. This construct was transfected into the Jurkat T cell line and was expressed as a dimer on the cell surface. Antibody-mediated crosslinking of this chimeric structure resulted in activation of the T cells as assessed by increases in intracellular tyrosine phosphorylation, inositol phosphate production, intracellular calcium release, and IL-2 production. This work strongly suggests that the TCR < chain is involved in coupling the receptor to critical biochemical pathways. Biochemical Pathways of TCR Signaling A great deal of effort has been expended over the past decade aimed at documenting the early biochemical changes seen in response to TCR engagement. For these studies a variety of activating stimuli have been utilized including peptide or protein antigens, anti-receptor antibodies, and mitogenic lectins. Each of these stimuli has advantages and disadvantages. Specific antigen presented by appropriate MHC molecules is presumably the most physiologic ligand, although its use is limited to T cell clones or antigen-specific T cell hybridornas-cells whose signaling pathways may not be identical to those of either normal developing thyrnocytes or mature, peripheral T cells. Anti-receptor antibodies, which appear to mimic most, if not all, of the changes induced in response to physiologic ligand, have the advantage of inducing polyclonal activation and thus can be used on normal populations of T cells. They have the additional benefit of stimulating the TCR in the absence of non-T cells, which can introduce other receptor-ligand interactions. However, recent evidence suggests that there are some distinctions between the coupling mechanisms observed with physiologic ligands and anti-receptor antibodies. These differences include the sensitivity to intracellular levels of cyclic AMP and the requirement for an intact 6 chain (Frank et al., 1990). Mitogenic lectins have largely fallen out of favor because of the lack of definition of what they are binding to on the T cell surface. One of the events first recognized in studies on the biochemistry of T cell activation was a rapid change in cytoplasmic calcium levels. This observation was followed by the recognition that TCR engagement led to the hydrolysis of phosphatidylinositides (PI). The breakdown of PI occurs rapidly (within 30 s) of the engagement of the TCR complex. Thus it was clear that PI turnover is a very early event in TCR-mediated activation. These important findings allowed the TCR to be placed among the large group of diverse receptors that appear to function via the stirnulation of PI turnover, presumably through the activation of a PI-specific phospholipase C (PLC). It is well established that the breakdown of PI leads to the generation of at least two active biochemical messengers: diacylglycerol, capable of activating the serine/threonine kinase, protein kinase C; and inositol phosphates such as IPB, implicated in the release of intracellular calcium stores. The alterations in intracellular calcium levels observed upon TCR engagement include both release from intracellular stores and calcium influxes from the environ-
Cell 878
ment. Although the generation of IPs is clearly important in producing the changes in cytosolic calcium in T ceils, the exact molecular details underlying all of the changes in calcium fluxes still remain to be elucidated. The role of PI breakdown in T cell activation was made more compelling by numerous studies demonstrating that treatment of T cells with both phorbol esters (to activate protein kinase C) and calcium ionophores (to increase cytosolic calcium levels) could result in avariety of phenotypic changes similar to those observed with T cell activation via the antigen receptor, including the most commonly utilized activation readout: the production of IL-2 (discussed in Irving and Weiss, 1991). By the mid-1980s the question remained as to how the TCR induced PI turnover and whether other, independent biochemical events took place upon TCR engagement. It was proposed that the TCR activated PI turnover via coupling to a guanine nucleotide-binding protein; to date, however, no direct evidence has been found to implicate such a protein in this process. The picture of the early events of T cell signaling became more complex with a series of studies that examined early intracellular phosphorylations that followed receptor engagement by either physiologic ligand or anti-receptor antibody. Beginning with studies of the TCR complex itself, it was clear that at least two kinase pathways were activated and several subunits were phosphorylated in response to receptor engagement. One of these chains, CD3 y, was phosphorylated on serine residues. This phosphorylation could be mimicked by the addition of phorbol ester, or diacylglycerol, to cells and thus could be ascribed to the activation of protein kinase C. However, the iI,chain phosphorylation occurred on tyrosine residues and could not be mimicked by the addition of phorbol esters or diacylglycerol, with or without calcium ionophore. Moreover, depletion of intracellular protein kinase C by prolonged, high dose phorbol ester treatment had no effect on 1;chain tyrosine phosphorylation, but abrogated CD3 y phosphorylation. Thus an additional (tyrosine kinase) pathway was activated that could not be explained by the stimulation of PI turnover. Tyrosine Kinase Activation and PI Turnover It is clear that a number of receptors whose cytoplasmic domains contain a tyrosine kinase activate the turnover of PI. These include the EGF receptor and the PDGF receptor. To stimulate PI turnover, these receptors must activate their tyrosine kinase. A possible mechanism for this cascade has been found with the observation that the Pl-specific PLC-~1 is tyrosine phosphorylated directly or indirectly by the EGF or PDGF receptor after stimulation of the receptor with the appropriate ligand. Recent studies indicate that this tyrosine phosphorylation activates PLC-T (reviewed in Cantley et al., 1991). Thus there is now an excellent precedent that would allow one to place the activation of a tyrosine kinase as a proximal event to PI turnover in T cells. Such a model is currently supported indirectly by several pieces of information. First, following TCR engagement the time course for the activation of the tyrosine kinase, as assessed by the phosphorylation of a number of as yet
unidentified intracellular substrates, precedes the activation of PI breakdown, as assessed by the production of soluble inositol phosphates (June et al., 1990a). Tyrosine phosphorylation of several intracellular substrates is seen as early as 5 s after stimulation, while inositol phosphates are detected only after a lag of approximately 30 to 40 s. Second, the utilization of at least two inhibitors of tyrosine kinase function, genestein and herbimycin A, has demonstrated that the inhibition of tyrosine kinase activation results in failure of the TCR to activate PI turnover (Mustelin et al., 1990; June et al., 1990b). It is likely that a scenario similar to that observed for the EGF and PDGF receptors will directly explain the causal relationship between the TCR-activated tyrosine kinase and PI metabolism. The TCR-Coupled Tyrosine Kinase Pathway Any understanding of the many critical questions that remain to be answered about the tyrosine kinase pathway activated upon TCR stimulation absolutely requires the identification of the responsible kinase or kinases. The primary sequences of the identified components of the TCR complex demonstrate no recognizable kinase, or other enzyme, domains. On this basis it was concluded that the TCR must be coupled either directly or indirectly to a nonreceptor tyrosine kinase. An early candidate for such a nonreceptor tyrosine kinase arose with a discovery of a T cell-specific member of the Src family of tyrosine kinases, lck. The discovery that Ickwas tightly, noncovalently associated with the cytoplasmic domain of either the CD4 or CD8 molecule provided an intriguing mechanism by which the TCR and this tyrosine kinase could be indirectly coupled. The extracellular domains of CD4 and CD8 bind to MHC class II and class I molecules, respectively. Thus, upon binding of the TCR clonotypic chains to an antigen-MHC complex on a presenting cell, the TCR would be brought into close proximity with either a CD4 or CD8 molecule that could independently bind to the appropriate MHC molecule. Support for this model comes from the recent study of Glaichenhaus et al. (1991), wherein CD4-lck interaction was required for activation of a T cell clone. Several observations, however, raised problems with Ick being the sole TCR-coupled tyrosine kinase. First, whereas Ick could be demonstrated to be activated by the cross-linking of CD4 molecules, the cross-linking of the TCR in this study did not activate Ick (Veillette et al., 1989a). Second, the activation of Ick via CD4 cross-linking resulted in a pattern of intracellular substrate tyrosine phosphorylation different from that seen with the cross-linking of TCR (Veillette et al., 1989b; Luoand Sefton, 1990). Third, inTcellsexpressing y6 receptors, and hybridomas that lack either CD4 or CD8, the TCR remains coupled to a tyrosine kinase pathway. Such reservations do not, of course, rule out a central role for Ick in TCR-mediated activation events, especially where CD4 or CD8 are engaged. More recently, a new candidate for the TCR-coupled tyrosine kinase has been proposed. This is the fyn kinase, another member of the Src family. Unlike Ick, fyn is found in avariety of tissues. However, it has been demonstrated that the fyn present in T cells arises from a uniquely spliced form of the gene (Cooke and Perlmutter, 1990). When the
Mhireview 877
TCR complex from a murine hybridoma is immunoprecipitated with any one of a number of monoclonal antibodies under gentle detergent conditions, an additional complex of proteins can be observed (Samelson et al., 1990a). Under these solubilization conditions the specific TCR precipitates possess tyrosine kinase activity, and it can be demonstrated that the tyrosine kinase responsible for this activity is fyn; no Ick is observed in these precipitates. Under the same solubilization conditions, anti-fyn antibodies will coprecipitate the same complex of proteins, including the TCR. Although these findings may provide the structural basis for TCR coupling to a tyrosine kinase, it must still be demortstrated that fyn is actually activated upon TCR engagement. The observation that TCR and fyn interact does not exclude the possibility that the CD4-lck pair is brought into a multimolecular complex with the TCR upon antigenMHC engagement (Glaichenhaus et al., 1991). Either or both kinases could be activated under these circumstances. The two kinases could also phosphorylate each other in such a complex, resulting in either decreased or increased activity depending on the site(s) of phosphorylation. The mechanism by which a tyrosine kinase such as fyn or Ick might be activated via TCR engagement remains a central question in TCR signal transduction. Recent work suggests that an additional enzymatic component may be involved in this process. A number of tyrosine kinases of the Src family have been demonstrated to be tyrosine phosphorylated themselves. Dephosphorylation of a carboxyterminal tyrosine residue in pp6pm is associated with enhanced tyrosine kinase activity. Activation of Ick has been proposed tooccurviathis mechanism (reviewed in Cantley et al., 1991). The discovery that the CD45 molecule, present on the surface of the T cell, contains tyrosine phosphatase activity in its cytoplasmic domain may prove to be a critical finding in understanding the activation of Tcells(Charbonneau et al., 1988). Antibody-induced coaggregation of the TCR, or the stimulatory CD2 molecule, with the CD45 molecule has a marked inhibitory effect on T cell activation (Samelson et al., 1990b, and references therein). In Tcells that lack cell surface CD45, TCR occupancy fails to lead to proliferation, although the response to IL-2 is unaffected (Pingel and Thomas, 1989). TCR-mediated signaling in variants of human T cell tumor lines that lack CD45 is also abnormal. These cells fail to demonstrate elevations in inositol phosphates or intracellular calcium after TCR engagement, although these responses can be elicited via a G protein-coupled receptor. Signaling through the TCR in CD45negative T cell tumors such as HPB-ALL or Jurkat is also impaired. The ability of the TCR to function in these cells is reconstituted by the reintroduction of CD45. More direct evidence for the role of CD45 in the activation of the TCR-coupled tyrosine kinase pathway has been provided with the observation that this pathway Cannot be activated in CD45-negative T cells (Koretzky et al., 1991, and references therein). Studies using the drug phenylarsine oxide provide pharmacological support for the proximal role of tyrosine phosphatase activity in TCR signaling
(Garcia-Morales et al., 1990). This agent can be shown to potently inhibit tyrosine phosphatases, including CD45. At doses of phenylarsine oxide that completely inhibit CD45 in vitro, all receptor-activated tyrosine phosphorylations are shut off. Additional structural studies support the conclusion that the TCR and CD45 can interact. Under certain experimental conditions CD45 can be shown to associate with the TCR and CD4 molecules. Cocapping experiments suggest that the TCR, CD4, and Ick can form a stable complex in primed T cells (Dianzani et al., 1990). In addition, chemical cross-linking studies on a murine hybridoma suggest an interaction between the TCR and CD45 (Volarevic et al., 1990). A speculative model for the proximal events of T cell activation can be proposed to incorporate the recent biochemical observations (see Figure). At the surface of the T cell the TCR is associated directly or indirectly with fyn, and CD4 or CD8 is associated with Ick. Engagement of the TCR with antigen bound to MHC facilitates the interaction of these molecules with CD45 The appropriate apposition of the CD45 tyrosine phosphatase with fyn and Ick may result in the dephosphorylation and activation of the kinases. The relative importance of fyn and Ick remains to be shown, but activation of both may be synergistic. These kinases, once activated, phosphorylate a number of intracellular substrates including PLC-?I, which would result in PI breakdown, protein kinase C activation, and serine phosphorylation of a number of substrates in T cells such as the c-Raf kinase and on a protein (perhaps the GTPase activating protein GAP) whose phosphorylation leads to an increase in p2lras activity(Siegel et al., 1990; reviewed in Cantleyet al., 1991). Phosphorylation of the MAP kinase on tyrosine and threonine residues by distinct kinases results in activation of this enzyme, which in turn leads to phosphorylation and activation of the ribosomal S6 kinase (Anderson et al., 1990; Nel et al., 1990). Late events such as lymphokine production may also be regulated by tyrosine phosphorylation (O’Shea et al., 1990).
Cascade of TCR Activation
Cell 878
Conclusions and Open Issues After ten years of intense research into the biochemical basis of TCR signaling, a consensus is beginning to emerge about the shape of the cascade of events that underlie T cell activation. As with many growth factor receptors, a profusion of biochemical events take place upon engagement of the TCR. Despite obvious structural differences, as with growth factor receptors it appears that a critical and proximal event in signal transduction is the activation of a tyrosine kinase. This scheme can probably be extended to consideration of signal transduction in B cells. Recently it has been shown that ligation of the B cell antigen receptor (surface immunoglobulin) also leads to activation of tyrosine phosphorylation (Gold et al., 1990; Campbell and Sefton, 1990). A model for the TCR-mediated cascade of biochemical events is shown in the figure. Much remains to be proven about this scheme, and numerous mechanistic details need to be defined before we fully understand this critical problem. As more attention is focused on the proximal role of tyrosine kinase activation in the signaling pathways of the TCR, several issues remain to be clarified. What are the respective rolesof fyn, Ick, and othertyrosine kinases? How are they activated, and what is the exact role in this process of CD45 and possibly other tyrosine phosphatases? How does the TCR interact with fyn, and how do other activating cell surface molecules such as Thy-l, Ly-6, CD2, and others utilize the TCR for signaling? What are the identity and function of all the other tyrosinephosphorylated substrates? What has been, and remains, clear is that the activation of the Tcell is a complex process utilizing an ever-growing cast of molecules. References Anderson, N. G., Maker, Nature 343, 651-653.
J. L., Tonks,
N. K., and Sturgill,
T. W. (1990).
Blumberg, R. S., Ley, S., Sancho, J., Lonberg, N., Lacy, E., McDermott, F., Schad, V., Greenstein, J. L., and Terhorst, C. (1990). Proc. Natl. Acad. Sci USA 87,7220-7224. Campbell,
M., and Sefton,
B. M. (1990).
Cantley, L. C., Auger, K. FL, Carpenter, A., Kapeller, FL, and Soltoff, S. (1991).
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Charbonneau, H.,Tonks, N. K., Walsh, K. A., and Fischer, Proc. Natl. Acad. Sci. USA 85, 7182-7186. Cooke,
M P., and Perlmutter,
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U., Luqman, M., Roho, J., Yagi, J., Baron, J. L., Woods, A., C. A., and Bottomly, K. (1990). Eur. J. Immunol. 20,2249-
Frank, S. J., Niklinska, B. B., Orloff, D. G., Mercep, M., Ashwell, and Klausner, R. D. (1990). Science 249, 173-177.
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Garcia-Morales, P., Luong, E. T., Minami, Y., Klausner, R. D., and Samelson, L. E. (1990). Proc. Natl. Acad. Sci. USA 87, 9255-9259. Glaichenhaus, N., Shastri, Cell 64, 51 l-520. Gold, M. R.. Law, D.A., 813. Irving,
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June, C. H., Fletcher, M. C., Ledbetter, (1990a). J. Immunol. 144, 1591-1599.
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June, C. H., Fletcher, M. C., Ledbetter, J. A., Schieven, G. L., Siegel, J. N., Phillips, A. F., and Samelson, L. E. (1990b). Proc. Natl. Acad. Sci. USA 87, 7722-7726.
Klausner, R. D., Lippincott-Schwartz, Annu. Rev. Cell Biol. 6, 403-431. Koning, F., Malay, 20, 299-305.
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Koretzky, G. A., Picus, J., Schultz, Acad. Sci. USA, in press. Luo, K., and Sefton,
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Mol. Cell. Biol. 10, 5305-5313.
Mustelin, T., Coggeshall, K. M., Isakov, Science 247, 1584-l 587.
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Nel, A. E., Pollack, S., Landreth, G., Ledbetter, J. L., Hultin, L., Williams, K., Katz, R., and Ackerley, B. (1990). J. Immunol. 745971-979. Orloff, D. G., Ra, C., Frank, (1990). Nature 347, 189-191.
S. J., Klausner,
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O’Shea, J. J., Ashwell, J. D., Bailey,T. L.,Cross, L. E., and Klausner, R. D. (1990). Proc. Natl. Acad. Pingel, J. T., and Thomas, Samelson, L. E., Phillips, (199Oa). Proc. Natl. Acad.
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A. F., Luong, E. T., and Klausner, Sci. USA 87, 4358-4362.
Samelson, L. E., Fletcher, M. C., Ledbetter, (199Ob). J. Immunol. 745, 2448-2454.
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Siegel, J. N., Klausner, R. D., Rapp, U. R., and Samelson, J. Biol. Chem. 265, 18472-18480. Ullrich,
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Veillette, A., Bookman, M. A., Horak, E. M., Samelson, Bolen, J. B. (1989a). Nature 338, 257-259. Veillette, A., Bolen, J. B., and Bookman, 9, 4441-4446.
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Volarevic, S., Burns, C. M., Sussman, J. J., and Ashwell, Proc. Natl. Acad. Sci. USA 87, 7085-7089.
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March
8, 1991, Copyright
0 1991 by Cell Press
T Cell Antigen Receptor Activation Pathways: The Tyrosine Kinase Connection Richard D. Klausner and Lawrence E. Samelson Cell Biology and Metabolism Branch National Institute of Child Health and Human Development National Institutes of Health Bethesda, Maryland 20892
The central requirement for the T cells of the immune system is the expression of cell surface receptors capable of distinguishing between self and foreign antigens. These recognition events must then be translated into approprilate biochemical signals that control cellular responses. Thlis two-step process of antigen recognition and signal transduction underlies the ability to regulate the maturation of T cells via thymic selection, wherein T cells are educated to distinguish self from nonself and to initiate, in mature T cells, either the immune response or, in some circumstances, anergy. These complex processes are subserved by the T cell antigen receptor (TCR). The TCR found on the surface of all T cells is a complex, hetero-oligomeric structure composed of six or seven different subunits (reviewed in Klausner et al., 1990). These can be divided into three distinct subgroups of proteins. The immunoglobulin-like, clonotypic chains, responsible for ligand binding, are present as heterodimers within the receptor complex. There are two major forms of receptor heterodimers: a8, found on most mature T cells, and ~8, found predominantlyon Tcells that are located in epithelia. The CD3 chains encompass three distinct, but closely related, subunits: two glycoproteins, y and 8, and one nonglycosylated protein, E, encoded by three homologous, clustered genes. The CD3 chains likely exist in the receptor as twlo subcomplexes of CD3 S-CD3 E and CD3 y-CD3 E (Koningetal., 1990; Blumbergetal., 1990).Thefinalgroup of subunits is the < family dimers. This family is distinct fro’rn CD3 genetically, structurally, and in its range of cellular expression. Three proteins, encoded by two genes, comprise the c family as known to date. They are 6, its alternately spliced form, n, and they chain of multisubunit FCC:receptors. The segregation of the chains of the TCR complex into multiple subunits allows the mixing and matching of these subunits with consequent diversification of receptor types. CD3containing TCR complexes must contain a 5 dimer, and three such dimers have been documented to date: c-c, G-n, and I;-FCE y (Orloff et al., 1990, and references therein). The functions of the individual chains of the TCR cornplex other than the clonotypic subunits are not known. It is reasonably assumed that the remaining subunits are essential for signal transduction. Currently information exists only for the ; chain, whose cytoplasmic tail appears to be essential for antigen-stimulated signaling (Frank et al., 1990). The recent work of Irving and Weiss (1991) elegantly addresses the role of this TCR subunit. They created a chimera comprised of the extracellular and transmembrane domains of the CD8 molecule and the
Minireview
intracellular domain of the < chain. This construct was transfected into the Jurkat T cell line and was expressed as a dimer on the cell surface. Antibody-mediated crosslinking of this chimeric structure resulted in activation of the T cells as assessed by increases in intracellular tyrosine phosphorylation, inositol phosphate production, intracellular calcium release, and IL-2 production. This work strongly suggests that the TCR < chain is involved in coupling the receptor to critical biochemical pathways. Biochemical Pathways of TCR Signaling A great deal of effort has been expended over the past decade aimed at documenting the early biochemical changes seen in response to TCR engagement. For these studies a variety of activating stimuli have been utilized including peptide or protein antigens, anti-receptor antibodies, and mitogenic lectins. Each of these stimuli has advantages and disadvantages. Specific antigen presented by appropriate MHC molecules is presumably the most physiologic ligand, although its use is limited to T cell clones or antigen-specific T cell hybridornas-cells whose signaling pathways may not be identical to those of either normal developing thyrnocytes or mature, peripheral T cells. Anti-receptor antibodies, which appear to mimic most, if not all, of the changes induced in response to physiologic ligand, have the advantage of inducing polyclonal activation and thus can be used on normal populations of T cells. They have the additional benefit of stimulating the TCR in the absence of non-T cells, which can introduce other receptor-ligand interactions. However, recent evidence suggests that there are some distinctions between the coupling mechanisms observed with physiologic ligands and anti-receptor antibodies. These differences include the sensitivity to intracellular levels of cyclic AMP and the requirement for an intact 6 chain (Frank et al., 1990). Mitogenic lectins have largely fallen out of favor because of the lack of definition of what they are binding to on the T cell surface. One of the events first recognized in studies on the biochemistry of T cell activation was a rapid change in cytoplasmic calcium levels. This observation was followed by the recognition that TCR engagement led to the hydrolysis of phosphatidylinositides (PI). The breakdown of PI occurs rapidly (within 30 s) of the engagement of the TCR complex. Thus it was clear that PI turnover is a very early event in TCR-mediated activation. These important findings allowed the TCR to be placed among the large group of diverse receptors that appear to function via the stirnulation of PI turnover, presumably through the activation of a PI-specific phospholipase C (PLC). It is well established that the breakdown of PI leads to the generation of at least two active biochemical messengers: diacylglycerol, capable of activating the serine/threonine kinase, protein kinase C; and inositol phosphates such as IPB, implicated in the release of intracellular calcium stores. The alterations in intracellular calcium levels observed upon TCR engagement include both release from intracellular stores and calcium influxes from the environ-
Cell 878
ment. Although the generation of IPs is clearly important in producing the changes in cytosolic calcium in T ceils, the exact molecular details underlying all of the changes in calcium fluxes still remain to be elucidated. The role of PI breakdown in T cell activation was made more compelling by numerous studies demonstrating that treatment of T cells with both phorbol esters (to activate protein kinase C) and calcium ionophores (to increase cytosolic calcium levels) could result in avariety of phenotypic changes similar to those observed with T cell activation via the antigen receptor, including the most commonly utilized activation readout: the production of IL-2 (discussed in Irving and Weiss, 1991). By the mid-1980s the question remained as to how the TCR induced PI turnover and whether other, independent biochemical events took place upon TCR engagement. It was proposed that the TCR activated PI turnover via coupling to a guanine nucleotide-binding protein; to date, however, no direct evidence has been found to implicate such a protein in this process. The picture of the early events of T cell signaling became more complex with a series of studies that examined early intracellular phosphorylations that followed receptor engagement by either physiologic ligand or anti-receptor antibody. Beginning with studies of the TCR complex itself, it was clear that at least two kinase pathways were activated and several subunits were phosphorylated in response to receptor engagement. One of these chains, CD3 y, was phosphorylated on serine residues. This phosphorylation could be mimicked by the addition of phorbol ester, or diacylglycerol, to cells and thus could be ascribed to the activation of protein kinase C. However, the iI,chain phosphorylation occurred on tyrosine residues and could not be mimicked by the addition of phorbol esters or diacylglycerol, with or without calcium ionophore. Moreover, depletion of intracellular protein kinase C by prolonged, high dose phorbol ester treatment had no effect on 1;chain tyrosine phosphorylation, but abrogated CD3 y phosphorylation. Thus an additional (tyrosine kinase) pathway was activated that could not be explained by the stimulation of PI turnover. Tyrosine Kinase Activation and PI Turnover It is clear that a number of receptors whose cytoplasmic domains contain a tyrosine kinase activate the turnover of PI. These include the EGF receptor and the PDGF receptor. To stimulate PI turnover, these receptors must activate their tyrosine kinase. A possible mechanism for this cascade has been found with the observation that the Pl-specific PLC-~1 is tyrosine phosphorylated directly or indirectly by the EGF or PDGF receptor after stimulation of the receptor with the appropriate ligand. Recent studies indicate that this tyrosine phosphorylation activates PLC-T (reviewed in Cantley et al., 1991). Thus there is now an excellent precedent that would allow one to place the activation of a tyrosine kinase as a proximal event to PI turnover in T cells. Such a model is currently supported indirectly by several pieces of information. First, following TCR engagement the time course for the activation of the tyrosine kinase, as assessed by the phosphorylation of a number of as yet
unidentified intracellular substrates, precedes the activation of PI breakdown, as assessed by the production of soluble inositol phosphates (June et al., 1990a). Tyrosine phosphorylation of several intracellular substrates is seen as early as 5 s after stimulation, while inositol phosphates are detected only after a lag of approximately 30 to 40 s. Second, the utilization of at least two inhibitors of tyrosine kinase function, genestein and herbimycin A, has demonstrated that the inhibition of tyrosine kinase activation results in failure of the TCR to activate PI turnover (Mustelin et al., 1990; June et al., 1990b). It is likely that a scenario similar to that observed for the EGF and PDGF receptors will directly explain the causal relationship between the TCR-activated tyrosine kinase and PI metabolism. The TCR-Coupled Tyrosine Kinase Pathway Any understanding of the many critical questions that remain to be answered about the tyrosine kinase pathway activated upon TCR stimulation absolutely requires the identification of the responsible kinase or kinases. The primary sequences of the identified components of the TCR complex demonstrate no recognizable kinase, or other enzyme, domains. On this basis it was concluded that the TCR must be coupled either directly or indirectly to a nonreceptor tyrosine kinase. An early candidate for such a nonreceptor tyrosine kinase arose with a discovery of a T cell-specific member of the Src family of tyrosine kinases, lck. The discovery that Ickwas tightly, noncovalently associated with the cytoplasmic domain of either the CD4 or CD8 molecule provided an intriguing mechanism by which the TCR and this tyrosine kinase could be indirectly coupled. The extracellular domains of CD4 and CD8 bind to MHC class II and class I molecules, respectively. Thus, upon binding of the TCR clonotypic chains to an antigen-MHC complex on a presenting cell, the TCR would be brought into close proximity with either a CD4 or CD8 molecule that could independently bind to the appropriate MHC molecule. Support for this model comes from the recent study of Glaichenhaus et al. (1991), wherein CD4-lck interaction was required for activation of a T cell clone. Several observations, however, raised problems with Ick being the sole TCR-coupled tyrosine kinase. First, whereas Ick could be demonstrated to be activated by the cross-linking of CD4 molecules, the cross-linking of the TCR in this study did not activate Ick (Veillette et al., 1989a). Second, the activation of Ick via CD4 cross-linking resulted in a pattern of intracellular substrate tyrosine phosphorylation different from that seen with the cross-linking of TCR (Veillette et al., 1989b; Luoand Sefton, 1990). Third, inTcellsexpressing y6 receptors, and hybridomas that lack either CD4 or CD8, the TCR remains coupled to a tyrosine kinase pathway. Such reservations do not, of course, rule out a central role for Ick in TCR-mediated activation events, especially where CD4 or CD8 are engaged. More recently, a new candidate for the TCR-coupled tyrosine kinase has been proposed. This is the fyn kinase, another member of the Src family. Unlike Ick, fyn is found in avariety of tissues. However, it has been demonstrated that the fyn present in T cells arises from a uniquely spliced form of the gene (Cooke and Perlmutter, 1990). When the
Mhireview 877
TCR complex from a murine hybridoma is immunoprecipitated with any one of a number of monoclonal antibodies under gentle detergent conditions, an additional complex of proteins can be observed (Samelson et al., 1990a). Under these solubilization conditions the specific TCR precipitates possess tyrosine kinase activity, and it can be demonstrated that the tyrosine kinase responsible for this activity is fyn; no Ick is observed in these precipitates. Under the same solubilization conditions, anti-fyn antibodies will coprecipitate the same complex of proteins, including the TCR. Although these findings may provide the structural basis for TCR coupling to a tyrosine kinase, it must still be demortstrated that fyn is actually activated upon TCR engagement. The observation that TCR and fyn interact does not exclude the possibility that the CD4-lck pair is brought into a multimolecular complex with the TCR upon antigenMHC engagement (Glaichenhaus et al., 1991). Either or both kinases could be activated under these circumstances. The two kinases could also phosphorylate each other in such a complex, resulting in either decreased or increased activity depending on the site(s) of phosphorylation. The mechanism by which a tyrosine kinase such as fyn or Ick might be activated via TCR engagement remains a central question in TCR signal transduction. Recent work suggests that an additional enzymatic component may be involved in this process. A number of tyrosine kinases of the Src family have been demonstrated to be tyrosine phosphorylated themselves. Dephosphorylation of a carboxyterminal tyrosine residue in pp6pm is associated with enhanced tyrosine kinase activity. Activation of Ick has been proposed tooccurviathis mechanism (reviewed in Cantley et al., 1991). The discovery that the CD45 molecule, present on the surface of the T cell, contains tyrosine phosphatase activity in its cytoplasmic domain may prove to be a critical finding in understanding the activation of Tcells(Charbonneau et al., 1988). Antibody-induced coaggregation of the TCR, or the stimulatory CD2 molecule, with the CD45 molecule has a marked inhibitory effect on T cell activation (Samelson et al., 1990b, and references therein). In Tcells that lack cell surface CD45, TCR occupancy fails to lead to proliferation, although the response to IL-2 is unaffected (Pingel and Thomas, 1989). TCR-mediated signaling in variants of human T cell tumor lines that lack CD45 is also abnormal. These cells fail to demonstrate elevations in inositol phosphates or intracellular calcium after TCR engagement, although these responses can be elicited via a G protein-coupled receptor. Signaling through the TCR in CD45negative T cell tumors such as HPB-ALL or Jurkat is also impaired. The ability of the TCR to function in these cells is reconstituted by the reintroduction of CD45. More direct evidence for the role of CD45 in the activation of the TCR-coupled tyrosine kinase pathway has been provided with the observation that this pathway Cannot be activated in CD45-negative T cells (Koretzky et al., 1991, and references therein). Studies using the drug phenylarsine oxide provide pharmacological support for the proximal role of tyrosine phosphatase activity in TCR signaling
(Garcia-Morales et al., 1990). This agent can be shown to potently inhibit tyrosine phosphatases, including CD45. At doses of phenylarsine oxide that completely inhibit CD45 in vitro, all receptor-activated tyrosine phosphorylations are shut off. Additional structural studies support the conclusion that the TCR and CD45 can interact. Under certain experimental conditions CD45 can be shown to associate with the TCR and CD4 molecules. Cocapping experiments suggest that the TCR, CD4, and Ick can form a stable complex in primed T cells (Dianzani et al., 1990). In addition, chemical cross-linking studies on a murine hybridoma suggest an interaction between the TCR and CD45 (Volarevic et al., 1990). A speculative model for the proximal events of T cell activation can be proposed to incorporate the recent biochemical observations (see Figure). At the surface of the T cell the TCR is associated directly or indirectly with fyn, and CD4 or CD8 is associated with Ick. Engagement of the TCR with antigen bound to MHC facilitates the interaction of these molecules with CD45 The appropriate apposition of the CD45 tyrosine phosphatase with fyn and Ick may result in the dephosphorylation and activation of the kinases. The relative importance of fyn and Ick remains to be shown, but activation of both may be synergistic. These kinases, once activated, phosphorylate a number of intracellular substrates including PLC-?I, which would result in PI breakdown, protein kinase C activation, and serine phosphorylation of a number of substrates in T cells such as the c-Raf kinase and on a protein (perhaps the GTPase activating protein GAP) whose phosphorylation leads to an increase in p2lras activity(Siegel et al., 1990; reviewed in Cantleyet al., 1991). Phosphorylation of the MAP kinase on tyrosine and threonine residues by distinct kinases results in activation of this enzyme, which in turn leads to phosphorylation and activation of the ribosomal S6 kinase (Anderson et al., 1990; Nel et al., 1990). Late events such as lymphokine production may also be regulated by tyrosine phosphorylation (O’Shea et al., 1990).
Cascade of TCR Activation
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Conclusions and Open Issues After ten years of intense research into the biochemical basis of TCR signaling, a consensus is beginning to emerge about the shape of the cascade of events that underlie T cell activation. As with many growth factor receptors, a profusion of biochemical events take place upon engagement of the TCR. Despite obvious structural differences, as with growth factor receptors it appears that a critical and proximal event in signal transduction is the activation of a tyrosine kinase. This scheme can probably be extended to consideration of signal transduction in B cells. Recently it has been shown that ligation of the B cell antigen receptor (surface immunoglobulin) also leads to activation of tyrosine phosphorylation (Gold et al., 1990; Campbell and Sefton, 1990). A model for the TCR-mediated cascade of biochemical events is shown in the figure. Much remains to be proven about this scheme, and numerous mechanistic details need to be defined before we fully understand this critical problem. As more attention is focused on the proximal role of tyrosine kinase activation in the signaling pathways of the TCR, several issues remain to be clarified. What are the respective rolesof fyn, Ick, and othertyrosine kinases? How are they activated, and what is the exact role in this process of CD45 and possibly other tyrosine phosphatases? How does the TCR interact with fyn, and how do other activating cell surface molecules such as Thy-l, Ly-6, CD2, and others utilize the TCR for signaling? What are the identity and function of all the other tyrosinephosphorylated substrates? What has been, and remains, clear is that the activation of the Tcell is a complex process utilizing an ever-growing cast of molecules. References Anderson, N. G., Maker, Nature 343, 651-653.
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