A Clue to Antigen Receptor Tails Bryan Irving and Arthur Weiss This information is current as of September 12, 2015.
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This article cites 22 articles, 9 of which you can access for free at: http://www.jimmunol.org/content/192/9/4013.full#ref-list-1 Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscriptions Submit copyright permission requests at: http://www.aai.org/ji/copyright.html Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/cgi/alerts/etoc
The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 9650 Rockville Pike, Bethesda, MD 20814-3994. Copyright © 2014 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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References
J Immunol 2014; 192:4013-4014; ; doi: 10.4049/jimmunol.1400660 http://www.jimmunol.org/content/192/9/4013
The
Pillars of Immunology
Journal of
Immunology
A Clue to Antigen Receptor Tails Bryan Irving* and Arthur Weiss†,‡
I
*CytomX Therapeutics, Inc., South San Francisco, CA 94080; †Rosalind Russell and Ephraim P. Engleman Arthritis Research Center, Division of Rheumatology, Department of Medicine, University of California, San Francisco, San Francisco, CA 94143; and ‡Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, CA 94143 Address correspondence and reprint requests to Dr. Arthur Weiss, University of California, San Francisco, 533 Parnassus Avenue, Room U-330, Box 0795, San Francisco, CA 94143. E-mail address: [email protected] Abbreviations used in this article: ITAM, immune tyrosine-based activation motif; SH2, Src homology 2. Copyright Ó 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00 www.jimmunol.org/cgi/doi/10.4049/jimmunol.1400660
So why did this letter not receive more attention and immediately expedite the field’s discovery of the ITAM? Reth pointed out that the motif is present in the CD3g and CD3d subunits, but absent from CD3e, the subunit that was then most implicated in signaling, as it was the target of most known mitogenic Abs directed against the TCR, for example, 2C11, OKT3, UCHT1, and Leu4. This unfortunate conclusion likely resulted from the publication of an erroneous CD3e sequence in 1984 that was later corrected via a published erratum (2). At the time, the prevailing data in TCR signaling supported a role for activation of the phosphatidylinositol pathway with its associated increase in calcium and protein kinase C activity (3), rather than the involvement of tyrosine phosphorylation. Although a requisite role for the tyrosine phosphatase CD45 had recently been demonstrated (4), it was the following year (1990) that tyrosine kinase inhibitors were shown to block initiation of TCR signaling (5, 6). Moreover, the requirement for CD45 confused the issue, because absence of a tyrosine phosphatase would be predicted to promote tyrosine phosphorylation. Finally, the discovery of the SH2 domain reactivity with PO4-Tyr, the key feature of the ITAM YxxL(I) motif, was still a year away (7). Work from several laboratories led to an appreciation of the signaling function of the ITAM motif. Unlike the CD3 chains, z had virtually no extracellular domain but became phosphorylated on multiple tyrosine residues upon receptor ligation (8, 9). This was reminiscent of the tyrosine kinase growth factor receptors whose cytoplasmic tails similarly contained multiple tyrosine residues that became trans autophosphorylated upon ligand binding. Ligand-induced phosphorylation of these receptors led to their association with signaling molecules to initiate the signaling cascade. Despite the structural complexity of the TCR and its absence of a catalytic domain, we hypothesized it might ultimately initiate signaling in a similar manner through its phosphorylated z dimer, with the other chains perhaps serving some sort of regulatory function. We tested this hypothesis by replacing the transmembrane and short extracellular domain of z with that of another dimeric protein, CD8, thereby enabling its separation from the TCR and independent expression of z in a chimeric form. We then assessed the signaling capacity of the z-chain independent of the TCR and even in a T cell line devoid of endogenous TCR expression. Surprisingly, oligomerization of the CD8/z chimera was sufficient to induce both early and late events associated with stimulation of the multichain TCR (10). At the same time, an independent study from the Seed laboratory utilizing a similar chimeric strategy demonstrated the capacity of z to induce mobilization of intracellular calcium and mediate cytolytic activity (11). These data demonstrated
Downloaded from http://www.jimmunol.org/ by guest on September 12, 2015
n science, context is essential for interpreting and integrating new observations into current paradigms. Without sufficient context, the significance of an important or insightful discovery may go unrealized until additional information is gleaned that brings its value to light. One such example was an observation made by Michael Reth during the early days of exploration into mechanisms of Ag receptor signaling in a correspondence to Nature in 1989 (1). In his very short note titled “Antigen receptor tail clue,” Reth drew attention to a consensus motif present in the cytoplasmic tails of several components of Ag receptors including invariant subunits of the TCR and BCR complexes, in addition to subunits of the FceRI receptor and the envelope protein of the bovine leukemia virus. The tyrosine and leucine (isoleucine)-based motif consists of only six consensus residues that occur in remarkably conserved spacing; it includes two acidic residues (Asp, Glu) with defined spacing upstream of two YxxL(I) groups separated by 7 aa (see figure 1 of Ref. 1). Because this motif occurs in components of multisubunit receptors with similar capacities to initiate signaling when cross-linked, Reth speculated presciently that “on cross-linking of receptor, the conserved amino acids from several chains may come together to form a binding site for the putative proteins that generate the signals resulting in proliferation and differentiation of the B and T cells . . . .” At the time of publication, this letter, despite its title, inspired curiosity but provided little in the way of clues or mechanistic insight into Ag receptor signaling. Today the letter is viewed as prophetic and the “Reth motif” is widely known as the immune tyrosine-based activation motif (ITAM) that serves as the basis for initiating signal transduction upon ligand engagement of several hematopoietic Ag receptors and a means by which viruses exploit Ag receptor signaling pathways to their benefit. The conserved YxxL pairs represent sites for phosphorylation by Src family kinases that, once phosphorylated, become stable docking sites for the tandem Src homology 2 (SH2) domains present in the Syk and ZAP70 cytoplasmic tyrosine kinases. These kinases cooperate to facilitate further signaling cascades that culminate in cellular activation.
4014
becomes phosphorylated by Lck to become fully activated to initiate the downstream signaling cascade that culminates in T cell activation. In retrospect, and armed today with a mechanistic understanding of early TCR signaling, one might question why Reth’s observation was not more obvious. The conserved spacing of the consensus residues was striking and their presence in multiple Ag receptor subunits certainly compelling. However, the letter’s significance could not be realized until new experimental work was conducted that provided the necessary context for our current, albeit still incomplete, understanding of ITAM function.
Disclosures The authors have no financial conflicts of interest.
References 1. Reth, M. 1989. Antigen receptor tail clue. Nature 338: 383–384. 2. van den Elsen, P., B. A. Shepley, J. Borst, J. E. Coligan, A. F. Markham, S. Orkin, and C. Terhorst. 1984. Isolation of cDNA clones encoding the 20K T3 glycoprotein of human T-cell receptor complex. Nature 312: 413–418. 3. Imboden, J. B., and J. D. Stobo. 1985. Transmembrane signalling by the T cell antigen receptor. Perturbation of the T3-antigen receptor complex generates inositol phosphates and releases calcium ions from intracellular stores. J. Exp. Med. 161: 446–456. 4. Pingel, J. T., and M. L. Thomas. 1989. Evidence that the leukocyte-common antigen is required for antigen-induced T lymphocyte proliferation. Cell 58: 1055– 1065. 5. June, C. H., M. C. Fletcher, J. A. Ledbetter, and L. E. Samelson. 1990. Increases in tyrosine phosphorylation are detectable before phospholipase C activation after T cell receptor stimulation. J. Immunol. 144: 1591–1599. 6. Mustelin, T., K. M. Coggeshall, N. Isakov, and A. Altman. 1990. T cell antigen receptor-mediated activation of phospholipase C requires tyrosine phosphorylation. Science 247: 1584–1587. 7. Moran, M. F., C. A. Koch, D. Anderson, C. Ellis, L. England, G. S. Martin, and T. Pawson. 1990. Src homology region 2 domains direct protein-protein interactions in signal transduction. Proc. Natl. Acad. Sci. USA 87: 8622–8626. 8. Samelson, L. E., M. D. Patel, A. M. Weissman, J. B. Harford, and R. D. Klausner. 1986. Antigen activation of murine T cells induces tyrosine phosphorylation of a polypeptide associated with the T cell antigen receptor. Cell 46: 1083–1090. 9. Weissman, A. M., L. E. Samelson, and R. D. Klausner. 1986. A new subunit of the human T-cell antigen receptor complex. Nature 324: 480–482. 10. Irving, B. A., and A. Weiss. 1991. The cytoplasmic domain of the T cell receptor z chain is sufficient to couple to receptor-associated signal transduction pathways. Cell 64: 891–901. 11. Romeo, C., and B. Seed. 1991. Cellular immunity to HIV activated by CD4 fused to T cell or Fc receptor polypeptides. Cell 64: 1037–1046. 12. Wegener, A. M., F. Letourneur, A. Hoeveler, T. Brocker, F. Luton, and B. Malissen. 1992. The T cell receptor/CD3 complex is composed of at least two autonomous transduction modules. Cell 68: 83–95. 13. Letourneur, F., and R. D. Klausner. 1992. Activation of T cells by a tyrosine kinase activation domain in the cytoplasmic tail of CD3e. Science 255: 79–82. 14. Romeo, C., M. Amiot, and B. Seed. 1992. Sequence requirements for induction of cytolysis by the T cell antigen/Fc receptor z chain. Cell 68: 889–897. 15. Irving, B. A., A. C. Chan, and A. Weiss. 1993. Functional characterization of a signal transducing motif present in the T cell antigen receptor z chain. J. Exp. Med. 177: 1093–1103. 16. Straus, D. B., and A. Weiss. 1992. Genetic evidence for the involvement of the lck tyrosine kinase in signal transduction through the T cell antigen receptor. Cell 70: 585–593. 17. Molina, T. J., K. Kishihara, D. P. Siderovski, W. van Ewijk, A. Narendran, E. Timms, A. Wakeham, C. J. Paige, K. U. Hartmann, A. Veillette, et al. 1992. Profound block in thymocyte development in mice lacking p56lck . Nature 357: 161–164. 18. Ostergaard, H. L., D. A. Shackelford, T. R. Hurley, P. Johnson, R. Hyman, B. M. Sefton, and I. S. Trowbridge. 1989. Expression of CD45 alters phosphorylation of the lck-encoded tyrosine protein kinase in murine lymphoma T-cell lines. Proc. Natl. Acad. Sci. USA 86: 8959–8963. 19. Chan, A. C., B. A. Irving, J. D. Fraser, and A. Weiss. 1991. The z chain is associated with a tyrosine kinase and upon T-cell antigen receptor stimulation associates with ZAP-70, a 70-kDa tyrosine phosphoprotein. Proc. Natl. Acad. Sci. USA 88: 9166–9170. 20. Chan, A. C., M. Iwashima, C. W. Turck, and A. Weiss. 1992. ZAP-70: a 70 kd protein-tyrosine kinase that associates with the TCR z chain. Cell 71: 649–662. 21. Iwashima, M., B. A. Irving, N. S. van Oers, A. C. Chan, and A. Weiss. 1994. Sequential interactions of the TCR with two distinct cytoplasmic tyrosine kinases. Science 263: 1136–1139. 22. Weiss, A. 1993. T cell antigen receptor signal transduction: a tale of tails and cytoplasmic protein-tyrosine kinases. Cell 73: 209–212.
Downloaded from http://www.jimmunol.org/ by guest on September 12, 2015
for the first time a signaling capacity of one of the TCR invariant chains, and they pointed to the z dimer as the relevant signaling apparatus of the TCR. However, that hypothesis was short-lived and in need of modification, for shortly thereafter experiments from the Malissen laboratory suggested a comparable signaling function for the CD3 module in the context of a TCR essentially devoid of z cytoplasmic sequences and the Klausner group showed TCR-independent signaling by CD3e-containing chimeras (12, 13). Mutation of z and CD3 modules led to “re-identification” of the conserved motif originally described in the Reth letter and revealed the functional importance of the properly spaced YxxL pairs (13, 14). The motif is derived from an evolutionarily conserved two-exon structure that is used for signaling in several invariant chains associated with Ag receptors (12). Although these studies demonstrated sufficiency of the motif for T cell activation, they revealed little of the mechanistic basis for the consensus motif and spacing or the functional relevance of its multiple representations within the receptor. Were the motifs functionally redundant or did some possess unique functions? Triplication of a single motif in a chimeric context did confer enhanced function, supporting a role for amplification in the multiplicity (15). However, this result did not preclude the potential for unique properties within individual motifs that might synergize for optimal signaling in the native TCR context. Also, whereas the functional importance of the conserved tyrosine residues in the motif had been demonstrated, the role of their phosphorylation was still unclear. Both positive and negative roles had been proposed for z phosphorylation in regulating TCR function (12). Studies of the Src family kinase, Lck, and a 70-kDa z-associated protein helped reveal the true mechanistic function of the motif. A signaling defective mutant derived from Jurkat T cells (JCAM.1) was revealed to be deficient in Lck, and its signaling could be restored upon reconstitution with Lck (16). These data were consistent with the profound defect reported in T cell development in Lck-deficient mice (17), and they helped clarify the earlier results demonstrating potent inhibition of T cell activation by tyrosine kinase inhibitors (5). They also helped to resolve the apparent paradox of the positive regulatory requirement for the tyrosine phosphatase CD45 in initiation of TCR signaling. CD45 is critical for activating Lck through dephosphorylation of its C-terminal negative regulatory tyrosine (18). Despite the requisite presence of Lck for TCR function, a stable association between it and the TCR could not be shown. Instead, a 70-kDa tyrosine phosphorylated protein was reproducibly induced to associate with z shortly after TCR stimulation (19). Purification and cloning of this protein identified ZAP70 as a 70-kDa tyrosine kinase that contains two SH2 domains upstream of its kinase domain (20). Studies identifying a requisite sequential function for Lck and ZAP70 revealed the role of the conserved residues in the ITAM motif and refined our mechanistic understanding of early TCR signaling (21, 22). First, Lck phosphorylates tyrosine residues within the cytoplasmic ITAMs. These Lckmediated phosphorylations provide the basis for recruitment of ZAP70, whose interaction is stabilized via its two tandem SH2 domains interacting with the doubly phosphorylated YxxL pair that comprises the ITAM motif. The conserved spacing of 7–8 aa between the YxxL pairs is important to provide stable binding by ZAP70. Once recruited to an ITAM, ZAP70
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Subscriptions Permissions Email Alerts
This article cites 22 articles, 9 of which you can access for free at: http://www.jimmunol.org/content/192/9/4013.full#ref-list-1 Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscriptions Submit copyright permission requests at: http://www.aai.org/ji/copyright.html Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/cgi/alerts/etoc
The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 9650 Rockville Pike, Bethesda, MD 20814-3994. Copyright © 2014 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606.
Downloaded from http://www.jimmunol.org/ by guest on September 12, 2015
References
J Immunol 2014; 192:4013-4014; ; doi: 10.4049/jimmunol.1400660 http://www.jimmunol.org/content/192/9/4013
The
Pillars of Immunology
Journal of
Immunology
A Clue to Antigen Receptor Tails Bryan Irving* and Arthur Weiss†,‡
I
*CytomX Therapeutics, Inc., South San Francisco, CA 94080; †Rosalind Russell and Ephraim P. Engleman Arthritis Research Center, Division of Rheumatology, Department of Medicine, University of California, San Francisco, San Francisco, CA 94143; and ‡Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, CA 94143 Address correspondence and reprint requests to Dr. Arthur Weiss, University of California, San Francisco, 533 Parnassus Avenue, Room U-330, Box 0795, San Francisco, CA 94143. E-mail address: [email protected] Abbreviations used in this article: ITAM, immune tyrosine-based activation motif; SH2, Src homology 2. Copyright Ó 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00 www.jimmunol.org/cgi/doi/10.4049/jimmunol.1400660
So why did this letter not receive more attention and immediately expedite the field’s discovery of the ITAM? Reth pointed out that the motif is present in the CD3g and CD3d subunits, but absent from CD3e, the subunit that was then most implicated in signaling, as it was the target of most known mitogenic Abs directed against the TCR, for example, 2C11, OKT3, UCHT1, and Leu4. This unfortunate conclusion likely resulted from the publication of an erroneous CD3e sequence in 1984 that was later corrected via a published erratum (2). At the time, the prevailing data in TCR signaling supported a role for activation of the phosphatidylinositol pathway with its associated increase in calcium and protein kinase C activity (3), rather than the involvement of tyrosine phosphorylation. Although a requisite role for the tyrosine phosphatase CD45 had recently been demonstrated (4), it was the following year (1990) that tyrosine kinase inhibitors were shown to block initiation of TCR signaling (5, 6). Moreover, the requirement for CD45 confused the issue, because absence of a tyrosine phosphatase would be predicted to promote tyrosine phosphorylation. Finally, the discovery of the SH2 domain reactivity with PO4-Tyr, the key feature of the ITAM YxxL(I) motif, was still a year away (7). Work from several laboratories led to an appreciation of the signaling function of the ITAM motif. Unlike the CD3 chains, z had virtually no extracellular domain but became phosphorylated on multiple tyrosine residues upon receptor ligation (8, 9). This was reminiscent of the tyrosine kinase growth factor receptors whose cytoplasmic tails similarly contained multiple tyrosine residues that became trans autophosphorylated upon ligand binding. Ligand-induced phosphorylation of these receptors led to their association with signaling molecules to initiate the signaling cascade. Despite the structural complexity of the TCR and its absence of a catalytic domain, we hypothesized it might ultimately initiate signaling in a similar manner through its phosphorylated z dimer, with the other chains perhaps serving some sort of regulatory function. We tested this hypothesis by replacing the transmembrane and short extracellular domain of z with that of another dimeric protein, CD8, thereby enabling its separation from the TCR and independent expression of z in a chimeric form. We then assessed the signaling capacity of the z-chain independent of the TCR and even in a T cell line devoid of endogenous TCR expression. Surprisingly, oligomerization of the CD8/z chimera was sufficient to induce both early and late events associated with stimulation of the multichain TCR (10). At the same time, an independent study from the Seed laboratory utilizing a similar chimeric strategy demonstrated the capacity of z to induce mobilization of intracellular calcium and mediate cytolytic activity (11). These data demonstrated
Downloaded from http://www.jimmunol.org/ by guest on September 12, 2015
n science, context is essential for interpreting and integrating new observations into current paradigms. Without sufficient context, the significance of an important or insightful discovery may go unrealized until additional information is gleaned that brings its value to light. One such example was an observation made by Michael Reth during the early days of exploration into mechanisms of Ag receptor signaling in a correspondence to Nature in 1989 (1). In his very short note titled “Antigen receptor tail clue,” Reth drew attention to a consensus motif present in the cytoplasmic tails of several components of Ag receptors including invariant subunits of the TCR and BCR complexes, in addition to subunits of the FceRI receptor and the envelope protein of the bovine leukemia virus. The tyrosine and leucine (isoleucine)-based motif consists of only six consensus residues that occur in remarkably conserved spacing; it includes two acidic residues (Asp, Glu) with defined spacing upstream of two YxxL(I) groups separated by 7 aa (see figure 1 of Ref. 1). Because this motif occurs in components of multisubunit receptors with similar capacities to initiate signaling when cross-linked, Reth speculated presciently that “on cross-linking of receptor, the conserved amino acids from several chains may come together to form a binding site for the putative proteins that generate the signals resulting in proliferation and differentiation of the B and T cells . . . .” At the time of publication, this letter, despite its title, inspired curiosity but provided little in the way of clues or mechanistic insight into Ag receptor signaling. Today the letter is viewed as prophetic and the “Reth motif” is widely known as the immune tyrosine-based activation motif (ITAM) that serves as the basis for initiating signal transduction upon ligand engagement of several hematopoietic Ag receptors and a means by which viruses exploit Ag receptor signaling pathways to their benefit. The conserved YxxL pairs represent sites for phosphorylation by Src family kinases that, once phosphorylated, become stable docking sites for the tandem Src homology 2 (SH2) domains present in the Syk and ZAP70 cytoplasmic tyrosine kinases. These kinases cooperate to facilitate further signaling cascades that culminate in cellular activation.
4014
becomes phosphorylated by Lck to become fully activated to initiate the downstream signaling cascade that culminates in T cell activation. In retrospect, and armed today with a mechanistic understanding of early TCR signaling, one might question why Reth’s observation was not more obvious. The conserved spacing of the consensus residues was striking and their presence in multiple Ag receptor subunits certainly compelling. However, the letter’s significance could not be realized until new experimental work was conducted that provided the necessary context for our current, albeit still incomplete, understanding of ITAM function.
Disclosures The authors have no financial conflicts of interest.
References 1. Reth, M. 1989. Antigen receptor tail clue. Nature 338: 383–384. 2. van den Elsen, P., B. A. Shepley, J. Borst, J. E. Coligan, A. F. Markham, S. Orkin, and C. Terhorst. 1984. Isolation of cDNA clones encoding the 20K T3 glycoprotein of human T-cell receptor complex. Nature 312: 413–418. 3. Imboden, J. B., and J. D. Stobo. 1985. Transmembrane signalling by the T cell antigen receptor. Perturbation of the T3-antigen receptor complex generates inositol phosphates and releases calcium ions from intracellular stores. J. Exp. Med. 161: 446–456. 4. Pingel, J. T., and M. L. Thomas. 1989. Evidence that the leukocyte-common antigen is required for antigen-induced T lymphocyte proliferation. Cell 58: 1055– 1065. 5. June, C. H., M. C. Fletcher, J. A. Ledbetter, and L. E. Samelson. 1990. Increases in tyrosine phosphorylation are detectable before phospholipase C activation after T cell receptor stimulation. J. Immunol. 144: 1591–1599. 6. Mustelin, T., K. M. Coggeshall, N. Isakov, and A. Altman. 1990. T cell antigen receptor-mediated activation of phospholipase C requires tyrosine phosphorylation. Science 247: 1584–1587. 7. Moran, M. F., C. A. Koch, D. Anderson, C. Ellis, L. England, G. S. Martin, and T. Pawson. 1990. Src homology region 2 domains direct protein-protein interactions in signal transduction. Proc. Natl. Acad. Sci. USA 87: 8622–8626. 8. Samelson, L. E., M. D. Patel, A. M. Weissman, J. B. Harford, and R. D. Klausner. 1986. Antigen activation of murine T cells induces tyrosine phosphorylation of a polypeptide associated with the T cell antigen receptor. Cell 46: 1083–1090. 9. Weissman, A. M., L. E. Samelson, and R. D. Klausner. 1986. A new subunit of the human T-cell antigen receptor complex. Nature 324: 480–482. 10. Irving, B. A., and A. Weiss. 1991. The cytoplasmic domain of the T cell receptor z chain is sufficient to couple to receptor-associated signal transduction pathways. Cell 64: 891–901. 11. Romeo, C., and B. Seed. 1991. Cellular immunity to HIV activated by CD4 fused to T cell or Fc receptor polypeptides. Cell 64: 1037–1046. 12. Wegener, A. M., F. Letourneur, A. Hoeveler, T. Brocker, F. Luton, and B. Malissen. 1992. The T cell receptor/CD3 complex is composed of at least two autonomous transduction modules. Cell 68: 83–95. 13. Letourneur, F., and R. D. Klausner. 1992. Activation of T cells by a tyrosine kinase activation domain in the cytoplasmic tail of CD3e. Science 255: 79–82. 14. Romeo, C., M. Amiot, and B. Seed. 1992. Sequence requirements for induction of cytolysis by the T cell antigen/Fc receptor z chain. Cell 68: 889–897. 15. Irving, B. A., A. C. Chan, and A. Weiss. 1993. Functional characterization of a signal transducing motif present in the T cell antigen receptor z chain. J. Exp. Med. 177: 1093–1103. 16. Straus, D. B., and A. Weiss. 1992. Genetic evidence for the involvement of the lck tyrosine kinase in signal transduction through the T cell antigen receptor. Cell 70: 585–593. 17. Molina, T. J., K. Kishihara, D. P. Siderovski, W. van Ewijk, A. Narendran, E. Timms, A. Wakeham, C. J. Paige, K. U. Hartmann, A. Veillette, et al. 1992. Profound block in thymocyte development in mice lacking p56lck . Nature 357: 161–164. 18. Ostergaard, H. L., D. A. Shackelford, T. R. Hurley, P. Johnson, R. Hyman, B. M. Sefton, and I. S. Trowbridge. 1989. Expression of CD45 alters phosphorylation of the lck-encoded tyrosine protein kinase in murine lymphoma T-cell lines. Proc. Natl. Acad. Sci. USA 86: 8959–8963. 19. Chan, A. C., B. A. Irving, J. D. Fraser, and A. Weiss. 1991. The z chain is associated with a tyrosine kinase and upon T-cell antigen receptor stimulation associates with ZAP-70, a 70-kDa tyrosine phosphoprotein. Proc. Natl. Acad. Sci. USA 88: 9166–9170. 20. Chan, A. C., M. Iwashima, C. W. Turck, and A. Weiss. 1992. ZAP-70: a 70 kd protein-tyrosine kinase that associates with the TCR z chain. Cell 71: 649–662. 21. Iwashima, M., B. A. Irving, N. S. van Oers, A. C. Chan, and A. Weiss. 1994. Sequential interactions of the TCR with two distinct cytoplasmic tyrosine kinases. Science 263: 1136–1139. 22. Weiss, A. 1993. T cell antigen receptor signal transduction: a tale of tails and cytoplasmic protein-tyrosine kinases. Cell 73: 209–212.
Downloaded from http://www.jimmunol.org/ by guest on September 12, 2015
for the first time a signaling capacity of one of the TCR invariant chains, and they pointed to the z dimer as the relevant signaling apparatus of the TCR. However, that hypothesis was short-lived and in need of modification, for shortly thereafter experiments from the Malissen laboratory suggested a comparable signaling function for the CD3 module in the context of a TCR essentially devoid of z cytoplasmic sequences and the Klausner group showed TCR-independent signaling by CD3e-containing chimeras (12, 13). Mutation of z and CD3 modules led to “re-identification” of the conserved motif originally described in the Reth letter and revealed the functional importance of the properly spaced YxxL pairs (13, 14). The motif is derived from an evolutionarily conserved two-exon structure that is used for signaling in several invariant chains associated with Ag receptors (12). Although these studies demonstrated sufficiency of the motif for T cell activation, they revealed little of the mechanistic basis for the consensus motif and spacing or the functional relevance of its multiple representations within the receptor. Were the motifs functionally redundant or did some possess unique functions? Triplication of a single motif in a chimeric context did confer enhanced function, supporting a role for amplification in the multiplicity (15). However, this result did not preclude the potential for unique properties within individual motifs that might synergize for optimal signaling in the native TCR context. Also, whereas the functional importance of the conserved tyrosine residues in the motif had been demonstrated, the role of their phosphorylation was still unclear. Both positive and negative roles had been proposed for z phosphorylation in regulating TCR function (12). Studies of the Src family kinase, Lck, and a 70-kDa z-associated protein helped reveal the true mechanistic function of the motif. A signaling defective mutant derived from Jurkat T cells (JCAM.1) was revealed to be deficient in Lck, and its signaling could be restored upon reconstitution with Lck (16). These data were consistent with the profound defect reported in T cell development in Lck-deficient mice (17), and they helped clarify the earlier results demonstrating potent inhibition of T cell activation by tyrosine kinase inhibitors (5). They also helped to resolve the apparent paradox of the positive regulatory requirement for the tyrosine phosphatase CD45 in initiation of TCR signaling. CD45 is critical for activating Lck through dephosphorylation of its C-terminal negative regulatory tyrosine (18). Despite the requisite presence of Lck for TCR function, a stable association between it and the TCR could not be shown. Instead, a 70-kDa tyrosine phosphorylated protein was reproducibly induced to associate with z shortly after TCR stimulation (19). Purification and cloning of this protein identified ZAP70 as a 70-kDa tyrosine kinase that contains two SH2 domains upstream of its kinase domain (20). Studies identifying a requisite sequential function for Lck and ZAP70 revealed the role of the conserved residues in the ITAM motif and refined our mechanistic understanding of early TCR signaling (21, 22). First, Lck phosphorylates tyrosine residues within the cytoplasmic ITAMs. These Lckmediated phosphorylations provide the basis for recruitment of ZAP70, whose interaction is stabilized via its two tandem SH2 domains interacting with the doubly phosphorylated YxxL pair that comprises the ITAM motif. The conserved spacing of 7–8 aa between the YxxL pairs is important to provide stable binding by ZAP70. Once recruited to an ITAM, ZAP70
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