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T cell antigen receptor signal transduction
205
T cell antigen receptor signal transduction Dapeng Qian* and Arthur Weisst The T cell antigen receptor (TCR) initiates signal transduction by activating multiple cytoplasmic protein tyrosine kinases (PTKs). Considerable progress in the field of TCR signal transduction has been made in three areas recently: first, in understanding the structure and function of the PTK ZAP-70; second, in the elucidation of the function of the substrates and pathways downstream of the PTKs; and third, in the identification of molecules that negatively regulate TCR signalling.
Addresses Howard Hughes Medical Institute, Departments of Medicine and of Microbiology and Immunology, U426, University of California, San Francisco, CA 94143, USA *e-maih [email protected] te-maih [email protected] Current Opinion in Cell Biology 1997, 9:205-212
Electronic identifier: 0955-0674-009-00205 © Current Biology Ltd ISSN 0955-0674 Abbreviations CTLA-4 cytotoxic T lymphocyte antigen-4 ITAM immunoreceptor tyrosine-based activation motif KIR killer inhibitory receptor MAPK mitogen-activated protein kinase MAPKK MAPK kinase MHC major histocompatibility complex NFAT nuclear factor of activated T cells PH pleckstrin homology PLC phospholipase C PTK protein tyrosine kinase PTPese protein tyrosine phosphatase SH Src homology SHP SH2-containing tyrosine phosphatase SLP-76 SH2 domain leukocyte protein of 76 kDa TCR T cell antigen receptor ZAP-70 t-associated protein-70
[SH] 2 domain leukocyte protein of 76kDa), have been shown recently to function as important mediators in T C R signalling pathways (see below). Successful T C R signal transduction also requires control of the threshold, extent, and termination of T C R activation by molecules that function as negative regulators. Recent studies indicate that these negative regulators include other surface receptors expressed on T cells and certain protein tyrosine phosphatases (PTPases). This review will focus on these important advances in the study of T C R signal transduction. The T cell antigen
receptor
T h e T C R is an oligomeric complex that can be separated into two functional subunits, the ligand-binding and signal-transducing subunits (Fig. 1) [1]. The recently determined crystal structure of an ~13 dimer, that is, the ligand-binding subunit of the receptor, bound to a peptide and a class I major histocompatibility complex (MHC) molecule provides a solution to the puzzle of how the T C R simultaneously recognizes both peptide and MHC determinants [5°']. This is likely to represent a major step forward in understanding how agonist and antagonist peptides lead to distinct signal transduction events [6,7,8°].
Figure t
Ligandbinding Signaltransducing
Membrane Introduction
Engagement of the T cell antigen receptor (TCR) evokes a series of signal transduction events that are critical to the activation of T-cell functions during an immune response [1]. TCR-mediated signal transduction is also critical for T-cell development in the thymus [1]. The T C R lacks intrinsic enzymatic activity and initiates signal transduction by activating multiple cytoplasmic protein tyrosine kinases (PTKs) [1,2°]. Over the past year, significant progress has been made towards understanding the functions of these PTKs. T h e activation of these TCR-regulated PTKs results in the tyrosine phosphorylation of downstream substrates and the activation of downstream pathways [3°,4e1. T h e identity and function of many of these substrates and pathways have begun to be elucidated. Two such substrates, Vav and SLP-76 (Src homology
Ig-like ITAM 1997 Current Opinion in Cell Biology
Schematic illustration of the TCR. The majority of T cells express a clonotypic a~ heterodimer as their ligand-binding subunits. A small proportion of T cells use 7~5heterodimers as ligand-binding subunits (not shown). Complexed with either the o~1~dimer or the ~,~dimer are a group of at least six nonpolymorphic signal-transducing subunits, including a CD37-CD3E pair, a CD3E-CD38 pair, and a TCR~-~ homodimer. The ITAM is present as a single copy in each of the CD3 chains (7, E, and 5) and is triplicated in the TCR~ chain.
206
Cell regulation
T h e signal-transducing T C R subunits, that is, the CD3 and ~-family chains, contain conserved immunoreceptor tyrosine-based activation sequence motifs (ITAMs) within their cytoplasmic domains [1]. ITAMs are also found in other receptors of the hematopoietic cell lineage that are involved in antigen recognition. The consensus sequence for an ITAM is YXXL(X)6_8YXXL (single-letter code for amino acids). The major function of ITAMs is to interact with intracellular signalling molecules. Following T C R stimulation, the tyrosine residues within ITAMs become phosphorylated, creating binding sites for SH2 domain containing proteins. The binding of these proteins to ITAMs is a critical step in subsequent T C R signalling. Agents that interfere with this step completely abrogate downstream signalling events [9,10°].
Protein tyrosine kinases in TCR signalling Four families of PTKs are involved in TCR signalling Activation of PTKs represents one of the earliest signalling events following T C R stimulation. Src, Csk, Tec, and Syk are four families of cytoplasmic PTKs that are involved in T C R signalling (Fig. 2). T h e Src family PTKs implicated in T C R signalling are Lck and Fyn. Genetic studies demonstrated that Lck and Fyn are critical
for initiating T C R signalling and also play important roles in T-cell development [1,2",11°']. Studies with Lck- or Fyn-deficient mice suggest that Lck plays a more important role in mediating ITAM phosphorylation than does Fyn [12°]. Csk negatively regulates Src family PTKs and thus T C R signalling [1]. Csk phosphorylates the carboxy-terminal tyrosine in Lck and Fyn, and, by doing so, maintains these proteins in an inactive state. Dephosphorylation of the carboxy-terminal tyrosine is mediated by the transmembrane PTPase CD45, allowing Lck and Fyn to be activated by T C R stimulation [1]. The Tec family P T K preferentially expressed in T cells is Itk. Evidence for the involvement of Itk in TCR signalling comes from a recent study of mice deficient in Itk expression [13°]. The Itk-/- mice have fewer thymocytes and CD4+ T cells as well as defective T C R signalling [13°]. T h e mechanism by which Itk regulates T C R signalling has not been determined, however. Syk family PTKs include Syk and ZAP-70 (~-associated protein-70). T h e essential role of ZAP-70 in T C R
Figure 2
PTK
Family
Size (kDa)
Lck
Src
56
Myr
SH3
SH2
Kinase
Y
Csk
Csk
50 n
PH Itk
Tec
72
ZAP-70
Syk
70
TH
- -
-
_
_L~ ~T'~
y*
I
*y y* © 1997 Current Opinion in Cell Biology
Structures of some of the members of the four families of cytoplasmic PTKs that are involved in TCR signalling. The carboxy-terminal negative regulatory residue tyrosine 505 (Y) and the myristylated (Myr) amino terminus of Lck are indicated. The three major tyrosine residues ('ryrs 292, 492, and 493) of ZAP-70 that are phosphorylated in vivo are also indicated (Y*). TH, Tec homology domain.
T cell antigen receptor signal transduction Oian and Weiss
signalling and T-cell development has been established by studies both of human patients with a rare immunodeficiency syndrome resulting from mutations in ZAP-70, and of mice deficient in ZAP-70 expression [14--17,18°°]. Although Syk is a critical P T K in B-cell antigen receptor signalling and B-cell development, it is not required for T C R signalling [19,20]. Syk-/- mice show largely normal T-cell development and T C R signalling, although the development of epithelial 78 T cells is impaired [19-21]. As ZAP-70 is a critical P T K in T C R signalling, the attempt to understand the mechanisms by which ZAP-70 regulates T C R signalling has been an area of intense investigation over the past year. The binding of ZAP-70 to the ITAMs
As has been described above, the initial step during T C R signalling is the tyrosine phosphorylation of ITAMs. This phosphorylation is mediated preferentially by Lck, though Fyn may play a role in some cells. Once phosphorylated, ITAMs recruit ZAP-70 to the activated receptor, facilitating subsequent tyrosine phosphorylation and activation of ZAP-70 by Lck or Fyn [22]. T h e binding of ZAP-70 to tyrosine-phosphorylated ITAMs is a high-affinity interaction [23-25]. Moreover, mutagenesis studies have demonstrated that the interaction of ZAP-70 with ITAMs requires both of the tandem SH2 domains of ZAP-70 and a doubly phosphorylated ITAM [22,26,27]. This requirement is explained by the recent solution of the crystal structure of the ZAP-70 SH2 domains bound to a doubly phosphorylated ITAM [28°°]. In this structure, the binding pocket for the amino-terminal phosphorylated tyrosine in the ITAM is formed by the carboxy-terminal SH2 domain alone of ZAP-70. T h e formation of the binding pocket for the carboxy-terminal phosphorylated tyrosine in the ITAM, however, requires residues provided by juxtaposition of both ZAP-70 SH2 domains. T h e conserved spacing between the two tyrosine residues within an ITAM is also important as it permits simultaneous binding of two ZAP-70 SH2 domains to the two phosphorylated tyrosines in the ITAM. Thus, the unique structure of ZAP-70 SH2 domains and the conserved features of ITAMs form the molecular basis for the high-affinity binding of ZAP-70 to ITAMs. Interestingly, in murine thymocytcs and ex v i v o T cells, Z A P - 7 0 is constitutively associated with the basally phosphorylated T C R ~ chain, and is not tyrosine-phosphorylated or activated until the T C R is stimulated [29,30]. These findings imply that the receptor signalling complex in these cells is already primed, but that an initiating event is still required to induce ZAP-70 phosphorylation and activation. Regulation of ZAP-70 activation by tyrosine phosphorylation
As for many other PTKs, TCR-induced tyrosine phosphorylation also plays important roles in regulating the catalytic activity and function of ZAP-70. T h e tyrosine
207
residues Y292, Y492, and Y493 are three major ZAP-70 sites that are phosphorylated following T C R stimulation in intact T cells [31,32°]. Y292 lies between the carboxyterminal SH2 domain and the kinase domain of ZAP-70, while Y492 and Y493 are located in the activation loop of the kinase domain. These sites are either positive or negative regulatory sites for ZAP-70 function [32°-35°]. Y493 is phosphorylated by Lck. T h e major function of phosphorylation of Y493 is to enable the activation of the kinase activity of ZAP-70 and to allow subsequent phosphorylation of Y292 and Y492 [32",33°]. T h e kinase(s) responsible for phosphorylating Y292 and Y492 is still not defined, but the kinase activity is likely to be mediated by the activated ZAP-70 itself. T h e phosphorylation of Y292 and Y492 results in negative regulatory functions. Phosphorylation of Y492 results in the downregulation of the kinase activity of ZAP-70 [32°,33°]. T h e mechanism by which Y292 mediates its negative regulatory function remains unclear, however. Mutation of Y292 does not affect the kinase activity of ZAP-70, change the ability of ZAP-70 to bind to tyrosine-phosphorylated ITAMs, or alter the ability of ZAP-70 to be tyrosine-phosphorylated [34",35°]. It is likely that Y292, once phosphorylated, serves as a binding site for an inhibitory protein. T h e phosphorylation sites in the ZAP-70-related P T K Syk have not been identified in T cells. Interestingly, recent evidence indicates that Syk has a different activation requirement to that of ZAP-70 [36°]. Mutant cell lines deficient in the expression of either Lck or CD45 are defective in T C R signalling. Overexpression of Syk, but not ZAP-70, restores T C R signalling in these mutant cell lines. Thus, the ability of Syk to function in T C R signalling does not require Lck and CD45, which are necessary upstream activators of ZAP-70. This is consistent with the fact that Syk has 100-fold higher intrinsic kinase activity than that of ZAP-70 and may be able to autophosphorylate tyrosine residues in its activation loop [37",38]. ZAP-70-binding proteins
In addition to binding to tyrosine-phosphorylated ITAMs, ZAP-70 has also been shown to bind Lck, Abl, rasGAP (GTPase-activating protein), Vav, the PTPase SHP (SH2-containing tyrosine phosphatase)-l, and Cbl [39-41,42"',43-45]. These interactions are mediated by tyrosine-phosphorylated ZAP-70 and either the SH2 domains of Lck, Abl, ras-GAP, Vav, and SHP-1 or a novel phosphotyrosine-binding domain in the amino terminus of Cbl. T h e interaction of ZAP-70 with such signalling proteins may have important functional consequences. First, these proteins may regulate the function of ZAP-70. Binding of SHP-1 to ZAP-70 results in a decrease in ZAP-70's kinase activity [42°°]. Binding of Cbl to ZAP-70 may sequester ZAP-70 molecules to prevent their access to the activated receptor. This notion is suggested by the observation that ZAP-70 that is complexed with Cbl is
208
Cell regulation
not i n the same pool of ZAP-70 as that which is bound to tyrosine-phosphorylated ITAMs [43]. Second, some of these proteins may serve as substrates for ZAP-70 and may mediate downstream signalling events.
Downstream
signalling
pathways
Several familiar pathways employed by many other receptor signalling systems are also activated following T C R stimulation [1,3"]. These include hydrolysis of inositol-containing phospholipids, Ca e÷ mobilization, and activation of the Ras/MAPK (mitogen-activated protein kinase) pathway. Many key enzymes of these pathways are directly regulated by PTKs. The enzymatic activity of phospholipase C (PLC) T1, a key enzyme in phosphatidylinositol metabolism, becomes activated when it is phosphorylated by PTKs following T C R stimulation [1]. T h e activation of the Ca z÷ channel inositol 1,4,5-trisphosphate (IP 3) receptor is also regulated by Fyn-mediated tyrosine phosphorylation [46"]. How PTKs couple T C R stimulation to Ras activation is not clear. T C R stimulation induces the association of several tyrosine-phosphorylated proteins with the Grb2-SOS complex [3"], which is involved in Ras regulation. These tyrosine-phosphorylated proteins include Shc, a prominent tyrosine-phosphorylated protein of 36kDa, and SLP-76.
In addition, Cbl, another tyrosine-phosphorylated protein, constitutively associates with Grb2 in T cells [3"]. Thus, Ras activation may be regulated by these Grb2-binding tyrosine-phosphorylated proteins. T h e Ras effector pathways include the Raf/MAPK kinase/extracellular signal regulated kinase cascade and the Ras-related GTPase Rac-1 [47"]. The importance of the Ras/MAPK pathway in T C R signalling is underscored by studies of this pathway in antigen-unresponsive anergic T cells [48",49"]. These cells have a block in the activation of the Ras/MAPK pathway and fail to produce IL-2. T h e critical role of Ras/MAPK pathway in T-cell development has also been demonstrated [50",51",52",53"]. Recent studies have also provided evidence that Vav and SLP-76, two proteins preferentially expressed in hematopoietic cells, are important mediators of T C R signalling. Vav-/- mice have arrested T-cell development and defective T C R signalling [54"'-56"']. Overexpression of Vav in T cells leads to basal activation of the nuclear factor of activated T cells (NFAT) and IL-2-driven transcriptional activity, which are further enhanced by T C R stimulation [57"']. Overexpression of SLP-76 also augments TCR-mediated activation of NFAT and the IL-2 promoter [58"]. Intriguingly, overexpression of both Vav and SLP-76 synergistically induces both basal and TCR-stimulated NFAT activation [59"]. Thus, Vav and
Table 1 Negative regulators of TCR signalling. Class
Molecule
Comments and references
Receptors
CD5
CD5 is constitutively expressed on T cells. Thymocytes from CD5 -/- mice exhibit increased TCR-mediated Ca 2+ mobilization, tyrosine phosphorylation of the TCR~ chain, PLC71, and Vav, and proliferative responses [66°]. The mechanism by which CD5 regulates TCR signalling has not been defined. KIRs constitute a family of receptors that are expressed on NK cells and a subset of CD4 + and CD8 + cells. Stimulation of KIRs inhibits TCR-stimulated activity and lymphokine production [67"°,68°]. The ligands for KIRs are class I MHC molecules. The cytoplasmic domains of KIRs contain an ITAM-like consensus amino acid sequence motif, YXXL(X)2sYXXL. KIRs become tyrosinephosphorylated upon stimulation and subsequently associate with the SH2 domains of SHP-1 [70",71°]. Mutation of both tyrosines in the ITAM-like motif of NKBI*, a member of the KIR family, abrogates the association of SHP-1 with NKB1 and abolishes the inhibitory functions of NKB1 [71"]. CTLA-4 is expressed late on T cells after TCR stimulation. Its ligands are members of the B7 family. CTLA-4 associates with SHP-2 [69"]. This association is probably mediated by the SH2 domains of SHP-2 and the tyrosine-phosphorylated amino acid sequence YVKM motif in the cytoplasmic tail of CTLA-4 [69°°]. Both the PTK pathway and the Ras/MAPK pathway are markedly hyperactivated in T cells from CTLA-4-/- mice [69"].
KIRs
CTLA-4
PTK
Csk
Csk negatively regulates Src family PTKs by phosphorylating the carboxy-terminal inhibitory tyrosine of Src family PTKs [1].
PTPases
SHP-1
The SH2 domains of SHP-1 bind to tyrosine-phosphorylated ZAP-70 following TCR stimulation [42°°]. This interaction results in an increase in the phosphatase activity of SHP-1 and a decrease in the kinase activity of ZAP-70 [42"]. Overexpression of wild-type SHP-1 enhances TCR-stimulated IL-2 production, while overexpression of a phosphatase-inactive SHP-1 decreases TCRstimulated IL-2 production [42"°]. SHP-1 also associates with KIRs [70",71"]. SHP-2 associates with CTLA-4 [69"].
SHP-2
*NKB1, inhibitory natural killer cell receptor 1.
T cell antigen receptor signal transduction Qian and Weiss
Figure 3
(a)
TCR
(b) C Lck )
ZAP-70
(¢)
(d)
(e) (f)
209
a cysteine-rich region, two SH3 domains, and an SH2 domain. SLP-76 has an amino-terminal acidic region containing tyrosine-phosphorylation sites, a central prolinerich region, and a carboxy-terminal SH2 domain. Thus, Vav and SLP-76 are likely to regulate T C R signalling, at least in part, through complex protein-protein interactions. Consistent with the functional cooperation between Vav and SLP-76, an activation-dependent physical interaction between Vav and SLP-76 has been detected [59°%60,61]. This interaction is mediated by tyrosine-phosphorylated SLP-76 and the SH2 domain of Vav. This interaction appears to be important for SLP-76 function, as mutation of putative Vav SH2 domain binding tyrosine residues in SLP-76 attenuates the ability of SLP-76 to augment TCR-stimulated N F A T or IL-2 gene activation [62",63"]. SLP-76 has also been shown to be a substrate of ZAP-70 [62"] and can associate with the two SH3 domains of Grb2 and an SH2 domain of PLCy1 [58",64]. Therefore, SLP-76 may function as an adaptor molecule linking ZAP-70 to the activation of Ras and Ca2÷ pathways. Vav contains a G E F domain and activates the JNK (Jun amino-terminal kinase) pathway in a Rac-dependent manner in fibroblasts [65]. However, whether Vav activates Rho/Rac/CDC42 pathways in T cells remains to be determined.
(g) Negative regulators of TCR signalling [~~?l~t,,~
Substratetyrosinephosphorylation and activationof downstreampathways
II ~
" @ Phosphorylation
© 1997c ..... t Opinionin CellBiology
IIq Iu
ZAP-70-bindingprotein ITAM
Model of proximal TCR signalling. (a) TCR stimulation leads to the activation of Lck which phosphorylates the tyrosines in an I'[AM of the TCR. (b) The tyrosine-phosphorylated ITAM provides high-affinity binding sites for the two SH2 domains of ZAP-70, resulting in the recruitment of ZAP-70 to the activated TCR. (c) The binding of ZAP-70 to the ITAM facilitates the phosphorylation of ZAP-70 at Tyr493 by Lck. (d) Phosphorylation of Tyr493 results in the activation of the kinase activity of ZAP-70. (e) Other tyrosine residues on ZAP-70 are subsequently phosphorylated. (f) Tyrosine-phosphorylated ZAP-70 binds to signalling molecules (ZAP-70-binding proteins) containing SH2 domains or a phosphotyrosine-binding domain. (g) ZAP-?O-binding proteins may serve as substrates for ZAP-70 and mediate the activation of downstream pathways.
SLP-76 function in T C R signalling pathways leading to IL-2 gene activation. T h e mechanism by which Vav and SLP-76 regulate T C R signalling is not fully understood. Both Vav and SLP-76 are tyrosine-phosphorylated following T C R stimulation and contain structural domains important in signal transduction. Vav has a putative G E F (guanine nucleotide exchange factor) domain for the Rho/Rac/CDC42 family of small GTPases, a PH (pleckstrin homology) domain,
T C R signalling requires a finely regulated balance between a positive signal that initiates the signalling response and a negative signal that controls the threshold, extent, and termination of T C R activation. In addition to the P T K Csk, two additional classes of molecules have recently been shown to function as negative regulators of T C R signalling, namely, negative regulatory receptors and PTPases (Table 1). Negative regulatory receptors include CD5 [66°], the killer inhibitory receptors (KIRs) [67"',68"], and cytotoxic T lymphocyte antigen-4 (CTLA-4) [69"']. CD5 is constitutively expressed on T cells, whereas CTLA-4 is only expressed late after T C R activation. T h e KIRs are expressed on natural killer cells and a subset of T cells. Thus, these receptors could provide negative signals at different stages of T C R activation and/or in a cell subset specific manner. How CD5 mediates its negative signal is not known, but both CTLA-4 and KIRs have been shown to mediate their effects by interacting with PTPases. CTLA-4 mediates its effect by interacting with SHP-2, consistent with the fact that T cells from CTLA-4--/- mice have hyperactivated P T K and Ras/MAPK pathways [69"]. T h e KIRs interact with SHP-1 through tyrosine-phosphorylated KIR residues and the SH2 domain of SHP-1 [70",71"]. Mutation of t-he tyrosines in KIRs abrogates both the association of KIR with SHP-1 and the ability of KIRs to inhibit T C R signalling [71"]. In addition to its role in KIR function, SHP-1 appears to have a more general negative regulatory function in T C R signalling by interacting with and downregulating ZAP-70 function, as has been mentioned above.
210
Cell regulation
Condusions Recent advances have provided a clearer picture of how proximal T C R signalling is initiated (Fig. 3). In addition, many downstream pathways and negative regulators of TCR signalling have been identified. It will be important in the near future to clearly establish the link between PTKs and the activation of downstream pathways, such as the Ras pathway. Other key questions for the future include: what are the effector pathways for Vav and SLP76? How are different downstream pathways coordinated to control T C R signalling? One unique feature of T-cell activation is the manifestation of many effector functions, including the expression of multiple lymphokine genes and the activation of cytolytic activity. Study of anergic T cells indicates that the Ras/MAPK pathway is critical for IL-2 gene expression. Expression of other lymphokine genes, such as the IFN-y gene, and the activation of cytolytic activity are largely unaffected in the absence of Ras/MAPK activation. Understanding the control of a given T cell effector function by a specific signalling pathway(s) will be an important challenge in the future.
Acknowledgements We wish to thank members of the Weiss laborators, for discussions and critical reading of the manuscript. D Qian is an associate and A Weiss is an investigator of the Howard Hughes Medical Institute.
References and recommended reading
receptor, blocks early T cell signaling. J Bio/Chem 1995, 270:944-948. 10. •
eian D, Mollenauer MN, Weiss A: Dominant-negative zetaassociated protein 70 inhibits T cell antigen receptor signaling. J Exp Med 1996, 183:611-620. Overexpression of a mutant ZAP-70 that contained only the two SH2 domains completely blocked both early and late TCR-mediated signalling events. This mutant was found to specifically target tyrosine-phosphorylated ITAMs. 11. •-
Van Oers NSC, Lowin-Kropf B, Finlay D, Connolly K, Weiss A: (x13T cell development is abolished in mice lacking both Lck and Fyn protein tyrosine kinases. Immunity 1996, 5:429-436. Combined disruption of the Lck and Fyn genes completely arrests T-cell development before the CD4+CD8 + stage, resulting in the absence of mature CD4 + and CD8 + T cells in peripheral lymphoid organs. This developmental block is more severe than that due to the disruption of Lck alone, which arrests T-cell development at the early CD4+CD8 + stage and still leaves small numbers of T cells in the peripheral lympoid organs, such as spleen and lymph nodes. These findings demonstrate critical roles of Lck and Fyn in T-cell development and also reveal the potential for functional redundancies of Src family PTKs. 12. •
Van Oers NSC, Killeen N, Weiss A: Lck regulates the tyrosine phosphorylation of the T cell receptor subunits and ZAP-70 in murine thymocytes. J Exp Med 1996, 183:1053-1062. Using thymocytes and lymph-node T cells from Lck- or Fyn-deficient mice, this study demonstrates that Lck is the primary Src family PTK required for tyrosine phosphorylation of the TCR~ chain and the ZAP-70 PTK. 13. •
Liao XC, Littman DR: Altered T cell receptor signaling and disrupted T cell development in mice lacking Itk. Immunity 1995, 3:757-769. This paper provides evidence that the Tec family PTK, Itk, is involved in TCR signalling and T-cell development. 14.
Arpaia E, Shahar M, Dadi H, Cohen A, Roifman CM: Defective T cell receptor signaling and CD8 + thymic selection in humans lacking ZAP-70 kinase. Ceil 1994, 76:947-958.
15.
Chan AC, Kadlecek TA, Elder ME, Filipovich AH, Kuo WL, Iwashima M, Parslow TG, Weiss A: ZAP-70 deficiency in an autosomal recessive form of severe combined immunodeficiency. Science 1994, 264:1599-1601.
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Elder ME, Lin D, Clever J, Chan AC, Hope TJ, Weiss A, Parslow T: Human severe combined immunodeficiency due to a defect in ZAP-70, a T cell tyrosine kinase. Science 1994, 264:1596-1599.
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Gelfand E, Mazer B, Kadlecek T, Weinberg K, Weiss A: Absence of ZAP-70 prevents signaling through the antigen receptor on peripheral blood T cells but not thymocytes. J Exp Med 1995, 182:1057-1066.
Papers of particular interest, published within the annual period of review, have been highlighted as: • •• 1.
of special interest of outstanding interest Weiss A, Littman DR: Signal transduction by lymphocyte antigen receptors. Ceil 1994, 76:263-274.
2. his
Howe LR, Weiss A: Multiple kinases mediate T-cell-receptor signaling. Trends Biochem Sci 1995, 20:59-64. review discusses studies of the roles of Src and Syk family PTKs in TCR signalling. 3. Cantrell D: T cell antigen receptor signal transduction • pathways. Annu Rev Immuno11996, 14:259-274. An excellent review focusing on downstream TCR signalling pathways.
4. Wange RL, Samelson LE: Complex complexes: signaling at the • TCR. Immunity 1996, 5:197-205. A comprehensive recent review on TCR signalling. Garcia KC, Degano M, Stanfield RL, Brunmark A, Jackson MR, Peterson PA, Teyton L, Wilson IA: An a~ T cell receptor structure at 2.5A and its orientation in the TCR-MHC complex. Science 1996, 274:209-219. Describes the crystallographic structure of an cx~ TCR and its orientation when bound to a class I MHC peptide complex.
18. =•
Negishi I, Motoyama N, Nakayama K, Nakayama K, Senju S, Hatakeyama S, Zhang Q, Chan AC, Loh DY: Essential role for ZAP-70 in both positive and negative selection of thymocytes. Nature 1995, 376:435-438. The essential role of ZAP-70 in T-cell development was studied in ZAP-70knockout mice. This paper complements earlier studies of human patients with severe combined immunodeficiency resulting from mutations in ZAP70 [14-16]. 19.
TurnerM, Mee PJ, Costello PS, Williams O, Price AA, Duddy LP, Furlong MT, Geahlen RL, Tybulewicz VL: Perinatal lethality and blocked B-cell development in mice lacking the tyrosine kinase Syk. Nature 1995, 378:298-302.
20.
Cheng AM, Rowley B, Pao W, Hayday A, Bolen JB, Pawson T: Syk tyrosine kinase required for mouse viability and B-cell development. Nature 1995, 378:303-306.
21.
Mallick-Wood CA, Pao W, Cheng AM, Lewis JM, Kulkarni S, Bolen JB, Rowley B, Tigelaar RE, Pawson T, Hayday AC: Disruption of epithelial y8 T cell repertoires by mutation of the Syk tyrosine kinase. Proc Nat/Acad Sci USA 1996, 93:9704-9709.
22.
IwashimaM, Irving BA, Van Oers NSC, Chan AC, Weiss A: Sequential interactions of the TCR with two distinct cytoplasmic tyrosine kinases. Science 1994, 263:1136-1139.
23.
Isakov N, Wange RL, Burgess WH, Watts JD, Aebersold R, Samelson LE: ZAP-70 binding specificity to T cell receptor tyrosine-based activation motifs: the tandem SH2 domains of ZAP-70 bind distinct tyrosine-based activation motifs with varying affinity. J Exp Med 1995, 181:375-380.
24.
Bu JY, Shaw AS, Chan AC: Analysis of the interaction of ZAP70 and syk protein-tyrosine kinases with the T-cell antigen
5. •,
6.
Sloan-Lancaster J, Shaw AS, Rothbard JB, Allen PM: Partial T cell signaling: altered phospho-~ and lack of ZAP-70 recruitment in APL-induced T cell anergy. Ceil 1994, 79:913-922.
7.
Madrenas J, Wange RL, Wang JL, Isakov N, Samelson LE, Germain RN: ~ phosphorylation without ZAP-70 activation by TCR antagonists or partial agonists. Science 1995, 267:515-518.
8. •
Rabinowitz JD, Beeson C, Wulfing C, Tare K, Allen P, Davis MM, McConnell HM: Altered T cell receptor ligands trigger a subset of early T cell signals, Immunity1996, 5:125-135. This paper, together with [6,7), provides evidence that agonist and antagonist peptide ligands for the TCR induce distinct signalling events. 9.
Wange RL, Isakov N, Burke J, T.R., Otaka A, Roller PP, Watts JD, Aebersold R, Samelson LE: F2(PmP)2-TAM 3, a novel competitive inhibitor of the binding of ZAP-70 to the T cell antigen
T cell antigen receptor signal transduction Qian and Weiss
receptor by plasmon resonance. Proc Nat/Acad Sci USA 1995, 92:5106-5110. 25.
Osman N, Lucas SC, Turner H, Cantrell D: A comparison of the interaction of Shc and the tyrosine kinase ZAP-70 with the T cell antigen receptor zeta chain tyrosine-based activation motif. J Biol Chem 1995, 270:13981-13986.
26.
Koyasu S, Tse AGD, Moingeon P, Hussey RE, Mildonian A, Hannisian J, Clayton LK, Reinherz EL: Delineation of a T-cell activation motif required for binding of protein tyrosine kinases containing tandem SH2 domains. Proc Nat/Acad Sci USA 1994, 91:6693-6697.
27.
Kong GH, Bu JY, Kurosaki T, Shaw AS, Chan AC: Reconstitution of Syk function by the ZAP-70 protein tyrosine kinase. Immunity 1995, 2:485-492.
28. ••
Hatada MH, Lu X, Laird ER, Green J, Morgenstern JP, Lou M, Marr CS, Phillips TB, Ram MK, Theriault K et a/.: Molecular basis for interaction of the protein tyrosine kinase ZAP-70 with the T-cell receptor. Nature 1995, 376:32-38. I'he authors provide the first solution of the crystal structure of the tandem SH2 domains of ZAP-70 bound to a doubly phosphorylated ITAM. 29.
30.
31.
Van Oers NSC, Killeen N, Weiss A: ZAP-70 is constitutively associated with tyrosine phosphorylated TCR ~ in murine thymocytes and lymph node T cells. Immunity 1994, 1:675-685. Wiest A, Ashe JM, Abe R, Bolen JB, Singer A: TCR activation of ZAP-70 is impaired in CD4+CD8 + thymocytes as a consequence of intrathymic interactions that diminish available p561ck. Immunity 1996, 4:495-504. Watts JD, Affolter M, Krebs DL, Wange RL, Samelson LE, Aebersold R: Identification by electrospray ionization mass spectrometry of the sites of tyrosine phosphorylation induced in activated Jurkat T cells on the protein tyrosine kinase ZAP70. J Biol Chem 1994, 269:29520-29529.
32. •
Chan AC, Dalton M, Johnson R, Kong GH, Wang T, Thoma R, KurosakiT: Activation of ZAP-7O kinase activity by phosphorylation of tyrosine 493 is required for lymphocyte antigen receptor function. EMBO J 1995, 14:2499-2508. See annotation [35"]. 33.
Wange RL, Guitian R, Isakov N, Watts JD, Aebersold R, Samelson LE: Activating and inhibitory mutations in adjacent tyrosines in the kinase domain of ZAP-70. J Biol Chem 1995, 270:18730-18733. See annotation [35"]. •
34.
Kong G, Dalton M, Wardenburg JB, Straus D, Kurosaki T, Chan AC: Distinct tyrosine phosphorylation sites in ZAP-70 mediate activation and negative regulation of antigen receptor function. Mol Cell Bio/1996, 16:5026-5035. See annotation [35"]. •
35.
Zhao Q, Weiss A: Enhancement of lymphocyte responsiveness by a gain-of-function mutation of ZAP-70. Mol Cell Bio11996, 16:6765-6774. This paper, together with [39"-34"], characterizes the functions of three major in vivo tyrosine phosphorylation sites (Y292, Y492, and Y493) in ZAP-70. The authors of [35"] also demonstrate that the interdomain between the carboxy-terminal SH2 domain and the kinase domain has an overall negative regulatory function, as deletion of this region results in a ZAP-70 mutant with enhanced function. •
36. •
Chu DH, Spits H, Peyron JF, Rowley RB, Bolen JB, Weiss A: The Syk protein tyrosine kinase can function independently of CD45 or Lck in T cell antigen receptor signaling. EMBO J 1996, 15:6251-6261. This study shows that Syk has different activation requirements to those of ZAP-70 in order to mediate TCR signalling. 37.
Latour S, Chow LML, Veillette A: Differential intrinsic enzymatic activity of Syk and Zap-70 protein-tyrosine kinases. J Bio/ Chem 1996, 271:22782-22790. Using chimeras of Syk and ZAP-70, the authors demonstrate that Syk has much higher intrinsic kinase activity than does ZAP-70. •
36.
39.
Couture C, Baler G, Oekten C, Williams S, Telford D, MarieCardine A, Baier-Bitterlioh G, Fischer S, Burn P, Altman A, Muste~in T: Activation of p561ck by p72syk through physical association and N-terminal tyrosine phosphorylation. Mol Cell Bio11994, 14:5249-5258. Thome M, Duplay P, Guttinger M, Acuto O: Syk and ZAP-7O mediate recruitment of p561ck/CD4 to the activated T cell receptor/CD3/zeta complex. J Exp Med 1995, 181:1997-2006.
211
40.
Neumeister EN, Zhu Y, Richard S, Terhorst C, Chan AS, Shaw AS: Binding of ZAP-70 to phosphorylated T-cell receptor ~ and q enhances its autophosphorylation and generates specific binding sites for SH2 domain-containing proteins. Mol Cell Biol 1995, 15:3171-3178.
41.
KatzavS, Sutherland M, Packham G, Yi T, Weiss A: The protein tyrosine kinase ZAP-70 can associate with the SH2 domain of proto-vav. J Bio/Chem 1994, 269:32579-32585.
42. ••
Plas DR, Johnson R, Pingel JT, Matthews RJ, Dalton M, Roy G, Chan AC, Thomas ML: Direct regulation of ZAP-70 by SHP-1 in T cell antigen receptor signaling. Science 1996, 272:1173-1176. The authors provide evidence that SHP-1 functions as a negative regulator of TCR signalling by interacting with ZAP-70 and downregulating the kinase activity of ZAP-70. 43.
Weil R, Cloutier JF, Fournel M, Veillette A: Regulation of Zap-70 by Src family tyrosine protein kinases in an antigen-specific T-celt line. J Biol Chem 1995, 270:2791-2799.
44.
FournelM, Davidson D, Wail R, Veillette A: Association of tyrosine protein kinase Zap-70 with the protooncogene product p120c-cbl in T lymphocytes. J Exp Med 1996, 183:301-306.
45.
Lupher MII, Reedquist KA, Miyake S, Langdon VVY, Band H: A novel phosphotyrosine-binding domain in the N-terminal transforming region of Cbl interacts directly and selectively with ZAP-70 in T cells. J Biol Chem 1996, 271:24063-24068.
46. •
JayaramanT, Ondrias K, Ondriasova E, Marks AR: Regulation of the inositol 1,4,5-trisphosphate receptor by tyrosine phosphorylation. Science 1996, 272:1492-1494. This paper shows that the Ca 2+ channel inositol 1,4,5-trisphosphate (IP3) receptor can be regulated by Fyn-mediated tyrosine phosphorylation. 47. •
Genot E, Cleverley S, Henning S, Cantrell D: Mutiple p21ras effector pathways regulate nuclear factor of activated T cells. EMBO J 1996, 15:3923-3933. The authors of this paper studied the effector pathways that mediate the activity of Ras in inducing NFAT activation and show that Raf/MAPKK/MAPK, Rao-1, and an unidentified effector pathway are required for Ras function. 48.
FieldsPE, Gajewski TF, Fitch FW: Blocked Ras activation in anergic CD4 + T cells. Science 1996, 271:1276-1278. ~ee annotation [49"']. 49. •-
Li W, Whaley CD, Mondino A, Mueller DL: Blocked signal transduction to the ERK and JNK protein kinases in anergic CD4 + T cells. Science 1996, 271:1272-1276. The antigen-unresponsive anergic T cells are characterized by their inability to produce IL-2. These two papers [48"',49"'] provide evidence that this unresponsiveness is due a block in the activation of the Ras/MAPK pathway. 50. ••
Alberola-Ila J, Forbush KA, Seger R, Krebs EG, Permulter RM: Selective requirement for MAP kinase activation in thymocyte differentiation. Nature 1995, 373:620-623. See annotation [51"]. 51. •
Swan KA, Alberola-Ila J, Gross JA, Appleby MW, Forbush KA, ThomasJF, Permulter RM: Involvement of p21ras distinguishes positive and negative selection in thymocytes. EMBO J 1995, 14:276-285. These papers [50",51"] provide evidence that the Ras/MAPK pathway is required for positive selection (maturation of T cells that react with foreign antigens), but not for negative selection (death of self-reactive T cells), during T-cell development in the thymus. 52.
Swat W, Shinkai Y, Cheng HL, Davidson L, AIt FW: Activated Ras signals differentiation and expansion of CD4+8 + thymocytes. Proc Nat/Acad Sci USA 1996, 93:4683-4687 See annotation [53"']. •
53. ••
Crompton T, Gilmour KC, Owen MJ: The MAP kinase pathway controls differentiation from double-negative to doublepositive thymocyte. Cell 1996, 86:243-251. This paper, together with [52"], provides evidence that the Ras/MAPK pathway is involved in early T-cell development from the CD4~DS- stage to the CD4+CD8 + stage. 54. •.
FischerKD, Zmuldzinas A, Gardner S, Barbacid M, Bernstein A, Guidos C: Defective T-cell receptor signalling and positive selection of Vav-deficient CD4 + CD8 + thymocytes. Nature 1995, 374:474-477. See annotation [57"°]. 55. ••
Tarakhovsky A, Turner M, Schaal S, Joseph Mee P, Duddy LP, Rajewsky K, Tybulewicz VU: Defective antigen receptor-
212
Cell regulation
mediated proliferation of B and T cells in the absence of Vav. Nature 1995, 374:467-470. See annotation [57°']. Zhang R, AIt FW, Davidson L, Orkin SH, Swat W: Defective signalling through the T- and B-cell antigen receptors in lymphoid cells lacking the vav proto-oncogene. Nature 1995, 374:470-473. See annotation [57"].
during TCR signalling. In addition, [62 °] shows that SLP-76 is a substrate for ZAP-70. 64.
JackmanJK, Motto DG, Sun CI, Tanemoto M, Turck CW, Peltz GA, Koretzky GA, Findell PR: Molecular cloning of SLP-76, a 76-kDa tyrosine phosphoprotein associated with Grb2 in T cells. J Biol Chem 1995, 270:7029-7032.
65.
Crespo P, Bustelo XR, Aaronson DS, Coso OA, Lopez-Barahona M, Barbacid M, Gutkind JS: Rac-1 dependent stimulation of the JNK/SAPK signalling pathway by Vav. Oncogene 1996, 13:455-460.
56. o•
57. *•
Wu J, Katzav S, Weiss A: A functional T cell receptor signaling pathway is required for p95ray activity. Mol Cell Biol 1995, 15:4337-4346. The authorsof these papersused Vav-/- mice [54°'-56 °"] or Vawoverexpressing T cells [57 "°] to demonstrate that Vav is an important mediator of TCR signalling. 58. •
Motto DG, Ross SE, Wu J, Hendricks-Taylor LR, Koretzky GA: Implication of the GRB2-associated phosphoprotein SLP-76 in T cell receptor-mediated interleukin 2 production. J Exp Med 1996, 183:1937-1943. Overexpression of SLP-76 resulted in enhanced TCR-mediated IL-2 gene expression, suggesting a role of SLP-76 in TCR signalling. 59. •-
Wu J, Motto DG, Koretzky GA, Weiss A: Vav and SLP-76 interact and functionally cooperate in IL-2 gene activation, immunity 1996, 4:593-602. This study demonstrates a functional cooperation between Vav and SLP76 during TCR signalling, in addition to an activation-dependent physical interaction between these two molecules. 60.
61.
Onodera H, Motto DG, Koretzky GA, Rothstein DM: Differential regulation of activation-induced tyrosine phosphorylation and recruitment of SLP-76 to Vav by distinct isoforms of the CD45 protein-tyrosine phosphatase. J Biol Chem 1996, 271:22225-22230. TuostoL, Michel F, Acuto O: p95vav associates with tyrosine phosphorylated SLP-76 in antigen-stimulated T cells. J Exp Med 1996, 184:1161-1166.
62. •
Wardenburg JB, Fu C, Jackman JK, Flotow H, Wilkinson SE, Williams DH, Johnson R, Kong G, Chan AC, Findell PR: Phosphorylation of SLP-76 by the ZAP-70 protein-tyrosine kinase is required for T-cell receptor function. J Biol Chem 1996, 271:19641-19644. See annotation [63"]. 63. •
FangN, Motto DG, Ross SE, Koretzky GA: Tyrosine 113, 128, and 145 of SLP-76 are required for optimal augmentation of NFAT promoter activity. J Immunol 1996, 157:3769-3773. The authors of [62",63 °] show that the putative Vav SH2 domain binding tyrosine residues in SLP-76 are required for SLP-76 to exhibit its function
66. •
Tarakhovsky A, Kanner SB, Hombach J, Ledbetter JA, Muller W, Killeen N, Rajewsky K: A role for CD5 in TCR-mediated signal transduction and thymocyte selection. Science 1995, 269:535-537. This paper provides evidence that CD5 is a negative regulator of TCR signalling. 67. ••
Phillips JH, Gumperz JE, Parham P, Lanier LL: Superantigendependent, cell-mediated cytotoxicity inhibited by MHC class I receptors on T lymphocytes. Science 1995, 268:403-405. Describes the identification of KIRs as negative regulators of TCR-activated cytotoxicity. 68. •
D'Andrea A, Chang C, Phillips JH, Lanier LL: Regulation of T cell lymphokine production by killer cell inhibitory receptor recognition of self HLA class I alleles. J Exp Med 1996, 184:789-794. This paper shows that KIRs can inhibit TCR-stimulated lymphokine production. 69. •.
Marengere LE, Waterhouse P, Duncan GS, Mittrucker HW, Fang GS, Mak TW: Regulation of T cell receptor signaling by tyrosine phosphatase SYP association with CTLA-4. Science 1996, 272:1170-11 73. This paper demonstrates that CTLA-4 is a negative regulator of TCR signalling. CTLA-4 interacts with SHP-2, and both the PTK pathway and the Ras/MAPK pathway are all hyperactivated in CTLA-4-/- mice. 70. •
Burshtyn DN, Scharenberg AM, Wagtmann N, Rajagopalan S, Berrada K, Yi T, Kinet J-P, Long EO: Recruitment of tyrosine phosphatase HCP by the killer cell inhibitory receptor. Immunity 1996, 4:77-85. See annotation [71"]. 71. •
Fry AM, Lanier LL, Weiss A: Phosphotyrosine in the killer cell inhibitory receptor motif of NKB1 are required for negative signaling and for association with protein tyrosine phosphatase 1C. J Exp Med 1996, 184:295-300. The authors of [70",71"] show that KIRs mediate their negative regulatory functions via interations with SHP-1.
T cell antigen receptor signal transduction Dapeng Qian* and Arthur Weisst The T cell antigen receptor (TCR) initiates signal transduction by activating multiple cytoplasmic protein tyrosine kinases (PTKs). Considerable progress in the field of TCR signal transduction has been made in three areas recently: first, in understanding the structure and function of the PTK ZAP-70; second, in the elucidation of the function of the substrates and pathways downstream of the PTKs; and third, in the identification of molecules that negatively regulate TCR signalling.
Addresses Howard Hughes Medical Institute, Departments of Medicine and of Microbiology and Immunology, U426, University of California, San Francisco, CA 94143, USA *e-maih [email protected] te-maih [email protected] Current Opinion in Cell Biology 1997, 9:205-212
Electronic identifier: 0955-0674-009-00205 © Current Biology Ltd ISSN 0955-0674 Abbreviations CTLA-4 cytotoxic T lymphocyte antigen-4 ITAM immunoreceptor tyrosine-based activation motif KIR killer inhibitory receptor MAPK mitogen-activated protein kinase MAPKK MAPK kinase MHC major histocompatibility complex NFAT nuclear factor of activated T cells PH pleckstrin homology PLC phospholipase C PTK protein tyrosine kinase PTPese protein tyrosine phosphatase SH Src homology SHP SH2-containing tyrosine phosphatase SLP-76 SH2 domain leukocyte protein of 76 kDa TCR T cell antigen receptor ZAP-70 t-associated protein-70
[SH] 2 domain leukocyte protein of 76kDa), have been shown recently to function as important mediators in T C R signalling pathways (see below). Successful T C R signal transduction also requires control of the threshold, extent, and termination of T C R activation by molecules that function as negative regulators. Recent studies indicate that these negative regulators include other surface receptors expressed on T cells and certain protein tyrosine phosphatases (PTPases). This review will focus on these important advances in the study of T C R signal transduction. The T cell antigen
receptor
T h e T C R is an oligomeric complex that can be separated into two functional subunits, the ligand-binding and signal-transducing subunits (Fig. 1) [1]. The recently determined crystal structure of an ~13 dimer, that is, the ligand-binding subunit of the receptor, bound to a peptide and a class I major histocompatibility complex (MHC) molecule provides a solution to the puzzle of how the T C R simultaneously recognizes both peptide and MHC determinants [5°']. This is likely to represent a major step forward in understanding how agonist and antagonist peptides lead to distinct signal transduction events [6,7,8°].
Figure t
Ligandbinding Signaltransducing
Membrane Introduction
Engagement of the T cell antigen receptor (TCR) evokes a series of signal transduction events that are critical to the activation of T-cell functions during an immune response [1]. TCR-mediated signal transduction is also critical for T-cell development in the thymus [1]. The T C R lacks intrinsic enzymatic activity and initiates signal transduction by activating multiple cytoplasmic protein tyrosine kinases (PTKs) [1,2°]. Over the past year, significant progress has been made towards understanding the functions of these PTKs. T h e activation of these TCR-regulated PTKs results in the tyrosine phosphorylation of downstream substrates and the activation of downstream pathways [3°,4e1. T h e identity and function of many of these substrates and pathways have begun to be elucidated. Two such substrates, Vav and SLP-76 (Src homology
Ig-like ITAM 1997 Current Opinion in Cell Biology
Schematic illustration of the TCR. The majority of T cells express a clonotypic a~ heterodimer as their ligand-binding subunits. A small proportion of T cells use 7~5heterodimers as ligand-binding subunits (not shown). Complexed with either the o~1~dimer or the ~,~dimer are a group of at least six nonpolymorphic signal-transducing subunits, including a CD37-CD3E pair, a CD3E-CD38 pair, and a TCR~-~ homodimer. The ITAM is present as a single copy in each of the CD3 chains (7, E, and 5) and is triplicated in the TCR~ chain.
206
Cell regulation
T h e signal-transducing T C R subunits, that is, the CD3 and ~-family chains, contain conserved immunoreceptor tyrosine-based activation sequence motifs (ITAMs) within their cytoplasmic domains [1]. ITAMs are also found in other receptors of the hematopoietic cell lineage that are involved in antigen recognition. The consensus sequence for an ITAM is YXXL(X)6_8YXXL (single-letter code for amino acids). The major function of ITAMs is to interact with intracellular signalling molecules. Following T C R stimulation, the tyrosine residues within ITAMs become phosphorylated, creating binding sites for SH2 domain containing proteins. The binding of these proteins to ITAMs is a critical step in subsequent T C R signalling. Agents that interfere with this step completely abrogate downstream signalling events [9,10°].
Protein tyrosine kinases in TCR signalling Four families of PTKs are involved in TCR signalling Activation of PTKs represents one of the earliest signalling events following T C R stimulation. Src, Csk, Tec, and Syk are four families of cytoplasmic PTKs that are involved in T C R signalling (Fig. 2). T h e Src family PTKs implicated in T C R signalling are Lck and Fyn. Genetic studies demonstrated that Lck and Fyn are critical
for initiating T C R signalling and also play important roles in T-cell development [1,2",11°']. Studies with Lck- or Fyn-deficient mice suggest that Lck plays a more important role in mediating ITAM phosphorylation than does Fyn [12°]. Csk negatively regulates Src family PTKs and thus T C R signalling [1]. Csk phosphorylates the carboxy-terminal tyrosine in Lck and Fyn, and, by doing so, maintains these proteins in an inactive state. Dephosphorylation of the carboxy-terminal tyrosine is mediated by the transmembrane PTPase CD45, allowing Lck and Fyn to be activated by T C R stimulation [1]. The Tec family P T K preferentially expressed in T cells is Itk. Evidence for the involvement of Itk in TCR signalling comes from a recent study of mice deficient in Itk expression [13°]. The Itk-/- mice have fewer thymocytes and CD4+ T cells as well as defective T C R signalling [13°]. T h e mechanism by which Itk regulates T C R signalling has not been determined, however. Syk family PTKs include Syk and ZAP-70 (~-associated protein-70). T h e essential role of ZAP-70 in T C R
Figure 2
PTK
Family
Size (kDa)
Lck
Src
56
Myr
SH3
SH2
Kinase
Y
Csk
Csk
50 n
PH Itk
Tec
72
ZAP-70
Syk
70
TH
- -
-
_
_L~ ~T'~
y*
I
*y y* © 1997 Current Opinion in Cell Biology
Structures of some of the members of the four families of cytoplasmic PTKs that are involved in TCR signalling. The carboxy-terminal negative regulatory residue tyrosine 505 (Y) and the myristylated (Myr) amino terminus of Lck are indicated. The three major tyrosine residues ('ryrs 292, 492, and 493) of ZAP-70 that are phosphorylated in vivo are also indicated (Y*). TH, Tec homology domain.
T cell antigen receptor signal transduction Oian and Weiss
signalling and T-cell development has been established by studies both of human patients with a rare immunodeficiency syndrome resulting from mutations in ZAP-70, and of mice deficient in ZAP-70 expression [14--17,18°°]. Although Syk is a critical P T K in B-cell antigen receptor signalling and B-cell development, it is not required for T C R signalling [19,20]. Syk-/- mice show largely normal T-cell development and T C R signalling, although the development of epithelial 78 T cells is impaired [19-21]. As ZAP-70 is a critical P T K in T C R signalling, the attempt to understand the mechanisms by which ZAP-70 regulates T C R signalling has been an area of intense investigation over the past year. The binding of ZAP-70 to the ITAMs
As has been described above, the initial step during T C R signalling is the tyrosine phosphorylation of ITAMs. This phosphorylation is mediated preferentially by Lck, though Fyn may play a role in some cells. Once phosphorylated, ITAMs recruit ZAP-70 to the activated receptor, facilitating subsequent tyrosine phosphorylation and activation of ZAP-70 by Lck or Fyn [22]. T h e binding of ZAP-70 to tyrosine-phosphorylated ITAMs is a high-affinity interaction [23-25]. Moreover, mutagenesis studies have demonstrated that the interaction of ZAP-70 with ITAMs requires both of the tandem SH2 domains of ZAP-70 and a doubly phosphorylated ITAM [22,26,27]. This requirement is explained by the recent solution of the crystal structure of the ZAP-70 SH2 domains bound to a doubly phosphorylated ITAM [28°°]. In this structure, the binding pocket for the amino-terminal phosphorylated tyrosine in the ITAM is formed by the carboxy-terminal SH2 domain alone of ZAP-70. T h e formation of the binding pocket for the carboxy-terminal phosphorylated tyrosine in the ITAM, however, requires residues provided by juxtaposition of both ZAP-70 SH2 domains. T h e conserved spacing between the two tyrosine residues within an ITAM is also important as it permits simultaneous binding of two ZAP-70 SH2 domains to the two phosphorylated tyrosines in the ITAM. Thus, the unique structure of ZAP-70 SH2 domains and the conserved features of ITAMs form the molecular basis for the high-affinity binding of ZAP-70 to ITAMs. Interestingly, in murine thymocytcs and ex v i v o T cells, Z A P - 7 0 is constitutively associated with the basally phosphorylated T C R ~ chain, and is not tyrosine-phosphorylated or activated until the T C R is stimulated [29,30]. These findings imply that the receptor signalling complex in these cells is already primed, but that an initiating event is still required to induce ZAP-70 phosphorylation and activation. Regulation of ZAP-70 activation by tyrosine phosphorylation
As for many other PTKs, TCR-induced tyrosine phosphorylation also plays important roles in regulating the catalytic activity and function of ZAP-70. T h e tyrosine
207
residues Y292, Y492, and Y493 are three major ZAP-70 sites that are phosphorylated following T C R stimulation in intact T cells [31,32°]. Y292 lies between the carboxyterminal SH2 domain and the kinase domain of ZAP-70, while Y492 and Y493 are located in the activation loop of the kinase domain. These sites are either positive or negative regulatory sites for ZAP-70 function [32°-35°]. Y493 is phosphorylated by Lck. T h e major function of phosphorylation of Y493 is to enable the activation of the kinase activity of ZAP-70 and to allow subsequent phosphorylation of Y292 and Y492 [32",33°]. T h e kinase(s) responsible for phosphorylating Y292 and Y492 is still not defined, but the kinase activity is likely to be mediated by the activated ZAP-70 itself. T h e phosphorylation of Y292 and Y492 results in negative regulatory functions. Phosphorylation of Y492 results in the downregulation of the kinase activity of ZAP-70 [32°,33°]. T h e mechanism by which Y292 mediates its negative regulatory function remains unclear, however. Mutation of Y292 does not affect the kinase activity of ZAP-70, change the ability of ZAP-70 to bind to tyrosine-phosphorylated ITAMs, or alter the ability of ZAP-70 to be tyrosine-phosphorylated [34",35°]. It is likely that Y292, once phosphorylated, serves as a binding site for an inhibitory protein. T h e phosphorylation sites in the ZAP-70-related P T K Syk have not been identified in T cells. Interestingly, recent evidence indicates that Syk has a different activation requirement to that of ZAP-70 [36°]. Mutant cell lines deficient in the expression of either Lck or CD45 are defective in T C R signalling. Overexpression of Syk, but not ZAP-70, restores T C R signalling in these mutant cell lines. Thus, the ability of Syk to function in T C R signalling does not require Lck and CD45, which are necessary upstream activators of ZAP-70. This is consistent with the fact that Syk has 100-fold higher intrinsic kinase activity than that of ZAP-70 and may be able to autophosphorylate tyrosine residues in its activation loop [37",38]. ZAP-70-binding proteins
In addition to binding to tyrosine-phosphorylated ITAMs, ZAP-70 has also been shown to bind Lck, Abl, rasGAP (GTPase-activating protein), Vav, the PTPase SHP (SH2-containing tyrosine phosphatase)-l, and Cbl [39-41,42"',43-45]. These interactions are mediated by tyrosine-phosphorylated ZAP-70 and either the SH2 domains of Lck, Abl, ras-GAP, Vav, and SHP-1 or a novel phosphotyrosine-binding domain in the amino terminus of Cbl. T h e interaction of ZAP-70 with such signalling proteins may have important functional consequences. First, these proteins may regulate the function of ZAP-70. Binding of SHP-1 to ZAP-70 results in a decrease in ZAP-70's kinase activity [42°°]. Binding of Cbl to ZAP-70 may sequester ZAP-70 molecules to prevent their access to the activated receptor. This notion is suggested by the observation that ZAP-70 that is complexed with Cbl is
208
Cell regulation
not i n the same pool of ZAP-70 as that which is bound to tyrosine-phosphorylated ITAMs [43]. Second, some of these proteins may serve as substrates for ZAP-70 and may mediate downstream signalling events.
Downstream
signalling
pathways
Several familiar pathways employed by many other receptor signalling systems are also activated following T C R stimulation [1,3"]. These include hydrolysis of inositol-containing phospholipids, Ca e÷ mobilization, and activation of the Ras/MAPK (mitogen-activated protein kinase) pathway. Many key enzymes of these pathways are directly regulated by PTKs. The enzymatic activity of phospholipase C (PLC) T1, a key enzyme in phosphatidylinositol metabolism, becomes activated when it is phosphorylated by PTKs following T C R stimulation [1]. T h e activation of the Ca z÷ channel inositol 1,4,5-trisphosphate (IP 3) receptor is also regulated by Fyn-mediated tyrosine phosphorylation [46"]. How PTKs couple T C R stimulation to Ras activation is not clear. T C R stimulation induces the association of several tyrosine-phosphorylated proteins with the Grb2-SOS complex [3"], which is involved in Ras regulation. These tyrosine-phosphorylated proteins include Shc, a prominent tyrosine-phosphorylated protein of 36kDa, and SLP-76.
In addition, Cbl, another tyrosine-phosphorylated protein, constitutively associates with Grb2 in T cells [3"]. Thus, Ras activation may be regulated by these Grb2-binding tyrosine-phosphorylated proteins. T h e Ras effector pathways include the Raf/MAPK kinase/extracellular signal regulated kinase cascade and the Ras-related GTPase Rac-1 [47"]. The importance of the Ras/MAPK pathway in T C R signalling is underscored by studies of this pathway in antigen-unresponsive anergic T cells [48",49"]. These cells have a block in the activation of the Ras/MAPK pathway and fail to produce IL-2. T h e critical role of Ras/MAPK pathway in T-cell development has also been demonstrated [50",51",52",53"]. Recent studies have also provided evidence that Vav and SLP-76, two proteins preferentially expressed in hematopoietic cells, are important mediators of T C R signalling. Vav-/- mice have arrested T-cell development and defective T C R signalling [54"'-56"']. Overexpression of Vav in T cells leads to basal activation of the nuclear factor of activated T cells (NFAT) and IL-2-driven transcriptional activity, which are further enhanced by T C R stimulation [57"']. Overexpression of SLP-76 also augments TCR-mediated activation of NFAT and the IL-2 promoter [58"]. Intriguingly, overexpression of both Vav and SLP-76 synergistically induces both basal and TCR-stimulated NFAT activation [59"]. Thus, Vav and
Table 1 Negative regulators of TCR signalling. Class
Molecule
Comments and references
Receptors
CD5
CD5 is constitutively expressed on T cells. Thymocytes from CD5 -/- mice exhibit increased TCR-mediated Ca 2+ mobilization, tyrosine phosphorylation of the TCR~ chain, PLC71, and Vav, and proliferative responses [66°]. The mechanism by which CD5 regulates TCR signalling has not been defined. KIRs constitute a family of receptors that are expressed on NK cells and a subset of CD4 + and CD8 + cells. Stimulation of KIRs inhibits TCR-stimulated activity and lymphokine production [67"°,68°]. The ligands for KIRs are class I MHC molecules. The cytoplasmic domains of KIRs contain an ITAM-like consensus amino acid sequence motif, YXXL(X)2sYXXL. KIRs become tyrosinephosphorylated upon stimulation and subsequently associate with the SH2 domains of SHP-1 [70",71°]. Mutation of both tyrosines in the ITAM-like motif of NKBI*, a member of the KIR family, abrogates the association of SHP-1 with NKB1 and abolishes the inhibitory functions of NKB1 [71"]. CTLA-4 is expressed late on T cells after TCR stimulation. Its ligands are members of the B7 family. CTLA-4 associates with SHP-2 [69"]. This association is probably mediated by the SH2 domains of SHP-2 and the tyrosine-phosphorylated amino acid sequence YVKM motif in the cytoplasmic tail of CTLA-4 [69°°]. Both the PTK pathway and the Ras/MAPK pathway are markedly hyperactivated in T cells from CTLA-4-/- mice [69"].
KIRs
CTLA-4
PTK
Csk
Csk negatively regulates Src family PTKs by phosphorylating the carboxy-terminal inhibitory tyrosine of Src family PTKs [1].
PTPases
SHP-1
The SH2 domains of SHP-1 bind to tyrosine-phosphorylated ZAP-70 following TCR stimulation [42°°]. This interaction results in an increase in the phosphatase activity of SHP-1 and a decrease in the kinase activity of ZAP-70 [42"]. Overexpression of wild-type SHP-1 enhances TCR-stimulated IL-2 production, while overexpression of a phosphatase-inactive SHP-1 decreases TCRstimulated IL-2 production [42"°]. SHP-1 also associates with KIRs [70",71"]. SHP-2 associates with CTLA-4 [69"].
SHP-2
*NKB1, inhibitory natural killer cell receptor 1.
T cell antigen receptor signal transduction Qian and Weiss
Figure 3
(a)
TCR
(b) C Lck )
ZAP-70
(¢)
(d)
(e) (f)
209
a cysteine-rich region, two SH3 domains, and an SH2 domain. SLP-76 has an amino-terminal acidic region containing tyrosine-phosphorylation sites, a central prolinerich region, and a carboxy-terminal SH2 domain. Thus, Vav and SLP-76 are likely to regulate T C R signalling, at least in part, through complex protein-protein interactions. Consistent with the functional cooperation between Vav and SLP-76, an activation-dependent physical interaction between Vav and SLP-76 has been detected [59°%60,61]. This interaction is mediated by tyrosine-phosphorylated SLP-76 and the SH2 domain of Vav. This interaction appears to be important for SLP-76 function, as mutation of putative Vav SH2 domain binding tyrosine residues in SLP-76 attenuates the ability of SLP-76 to augment TCR-stimulated N F A T or IL-2 gene activation [62",63"]. SLP-76 has also been shown to be a substrate of ZAP-70 [62"] and can associate with the two SH3 domains of Grb2 and an SH2 domain of PLCy1 [58",64]. Therefore, SLP-76 may function as an adaptor molecule linking ZAP-70 to the activation of Ras and Ca2÷ pathways. Vav contains a G E F domain and activates the JNK (Jun amino-terminal kinase) pathway in a Rac-dependent manner in fibroblasts [65]. However, whether Vav activates Rho/Rac/CDC42 pathways in T cells remains to be determined.
(g) Negative regulators of TCR signalling [~~?l~t,,~
Substratetyrosinephosphorylation and activationof downstreampathways
II ~
" @ Phosphorylation
© 1997c ..... t Opinionin CellBiology
IIq Iu
ZAP-70-bindingprotein ITAM
Model of proximal TCR signalling. (a) TCR stimulation leads to the activation of Lck which phosphorylates the tyrosines in an I'[AM of the TCR. (b) The tyrosine-phosphorylated ITAM provides high-affinity binding sites for the two SH2 domains of ZAP-70, resulting in the recruitment of ZAP-70 to the activated TCR. (c) The binding of ZAP-70 to the ITAM facilitates the phosphorylation of ZAP-70 at Tyr493 by Lck. (d) Phosphorylation of Tyr493 results in the activation of the kinase activity of ZAP-70. (e) Other tyrosine residues on ZAP-70 are subsequently phosphorylated. (f) Tyrosine-phosphorylated ZAP-70 binds to signalling molecules (ZAP-70-binding proteins) containing SH2 domains or a phosphotyrosine-binding domain. (g) ZAP-?O-binding proteins may serve as substrates for ZAP-70 and mediate the activation of downstream pathways.
SLP-76 function in T C R signalling pathways leading to IL-2 gene activation. T h e mechanism by which Vav and SLP-76 regulate T C R signalling is not fully understood. Both Vav and SLP-76 are tyrosine-phosphorylated following T C R stimulation and contain structural domains important in signal transduction. Vav has a putative G E F (guanine nucleotide exchange factor) domain for the Rho/Rac/CDC42 family of small GTPases, a PH (pleckstrin homology) domain,
T C R signalling requires a finely regulated balance between a positive signal that initiates the signalling response and a negative signal that controls the threshold, extent, and termination of T C R activation. In addition to the P T K Csk, two additional classes of molecules have recently been shown to function as negative regulators of T C R signalling, namely, negative regulatory receptors and PTPases (Table 1). Negative regulatory receptors include CD5 [66°], the killer inhibitory receptors (KIRs) [67"',68"], and cytotoxic T lymphocyte antigen-4 (CTLA-4) [69"']. CD5 is constitutively expressed on T cells, whereas CTLA-4 is only expressed late after T C R activation. T h e KIRs are expressed on natural killer cells and a subset of T cells. Thus, these receptors could provide negative signals at different stages of T C R activation and/or in a cell subset specific manner. How CD5 mediates its negative signal is not known, but both CTLA-4 and KIRs have been shown to mediate their effects by interacting with PTPases. CTLA-4 mediates its effect by interacting with SHP-2, consistent with the fact that T cells from CTLA-4--/- mice have hyperactivated P T K and Ras/MAPK pathways [69"]. T h e KIRs interact with SHP-1 through tyrosine-phosphorylated KIR residues and the SH2 domain of SHP-1 [70",71"]. Mutation of t-he tyrosines in KIRs abrogates both the association of KIR with SHP-1 and the ability of KIRs to inhibit T C R signalling [71"]. In addition to its role in KIR function, SHP-1 appears to have a more general negative regulatory function in T C R signalling by interacting with and downregulating ZAP-70 function, as has been mentioned above.
210
Cell regulation
Condusions Recent advances have provided a clearer picture of how proximal T C R signalling is initiated (Fig. 3). In addition, many downstream pathways and negative regulators of TCR signalling have been identified. It will be important in the near future to clearly establish the link between PTKs and the activation of downstream pathways, such as the Ras pathway. Other key questions for the future include: what are the effector pathways for Vav and SLP76? How are different downstream pathways coordinated to control T C R signalling? One unique feature of T-cell activation is the manifestation of many effector functions, including the expression of multiple lymphokine genes and the activation of cytolytic activity. Study of anergic T cells indicates that the Ras/MAPK pathway is critical for IL-2 gene expression. Expression of other lymphokine genes, such as the IFN-y gene, and the activation of cytolytic activity are largely unaffected in the absence of Ras/MAPK activation. Understanding the control of a given T cell effector function by a specific signalling pathway(s) will be an important challenge in the future.
Acknowledgements We wish to thank members of the Weiss laborators, for discussions and critical reading of the manuscript. D Qian is an associate and A Weiss is an investigator of the Howard Hughes Medical Institute.
References and recommended reading
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Van Oers NSC, Lowin-Kropf B, Finlay D, Connolly K, Weiss A: (x13T cell development is abolished in mice lacking both Lck and Fyn protein tyrosine kinases. Immunity 1996, 5:429-436. Combined disruption of the Lck and Fyn genes completely arrests T-cell development before the CD4+CD8 + stage, resulting in the absence of mature CD4 + and CD8 + T cells in peripheral lymphoid organs. This developmental block is more severe than that due to the disruption of Lck alone, which arrests T-cell development at the early CD4+CD8 + stage and still leaves small numbers of T cells in the peripheral lympoid organs, such as spleen and lymph nodes. These findings demonstrate critical roles of Lck and Fyn in T-cell development and also reveal the potential for functional redundancies of Src family PTKs. 12. •
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28. ••
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Zhao Q, Weiss A: Enhancement of lymphocyte responsiveness by a gain-of-function mutation of ZAP-70. Mol Cell Bio11996, 16:6765-6774. This paper, together with [39"-34"], characterizes the functions of three major in vivo tyrosine phosphorylation sites (Y292, Y492, and Y493) in ZAP-70. The authors of [35"] also demonstrate that the interdomain between the carboxy-terminal SH2 domain and the kinase domain has an overall negative regulatory function, as deletion of this region results in a ZAP-70 mutant with enhanced function. •
36. •
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41.
KatzavS, Sutherland M, Packham G, Yi T, Weiss A: The protein tyrosine kinase ZAP-70 can associate with the SH2 domain of proto-vav. J Bio/Chem 1994, 269:32579-32585.
42. ••
Plas DR, Johnson R, Pingel JT, Matthews RJ, Dalton M, Roy G, Chan AC, Thomas ML: Direct regulation of ZAP-70 by SHP-1 in T cell antigen receptor signaling. Science 1996, 272:1173-1176. The authors provide evidence that SHP-1 functions as a negative regulator of TCR signalling by interacting with ZAP-70 and downregulating the kinase activity of ZAP-70. 43.
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46. •
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53. ••
Crompton T, Gilmour KC, Owen MJ: The MAP kinase pathway controls differentiation from double-negative to doublepositive thymocyte. Cell 1996, 86:243-251. This paper, together with [52"], provides evidence that the Ras/MAPK pathway is involved in early T-cell development from the CD4~DS- stage to the CD4+CD8 + stage. 54. •.
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212
Cell regulation
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during TCR signalling. In addition, [62 °] shows that SLP-76 is a substrate for ZAP-70. 64.
JackmanJK, Motto DG, Sun CI, Tanemoto M, Turck CW, Peltz GA, Koretzky GA, Findell PR: Molecular cloning of SLP-76, a 76-kDa tyrosine phosphoprotein associated with Grb2 in T cells. J Biol Chem 1995, 270:7029-7032.
65.
Crespo P, Bustelo XR, Aaronson DS, Coso OA, Lopez-Barahona M, Barbacid M, Gutkind JS: Rac-1 dependent stimulation of the JNK/SAPK signalling pathway by Vav. Oncogene 1996, 13:455-460.
56. o•
57. *•
Wu J, Katzav S, Weiss A: A functional T cell receptor signaling pathway is required for p95ray activity. Mol Cell Biol 1995, 15:4337-4346. The authorsof these papersused Vav-/- mice [54°'-56 °"] or Vawoverexpressing T cells [57 "°] to demonstrate that Vav is an important mediator of TCR signalling. 58. •
Motto DG, Ross SE, Wu J, Hendricks-Taylor LR, Koretzky GA: Implication of the GRB2-associated phosphoprotein SLP-76 in T cell receptor-mediated interleukin 2 production. J Exp Med 1996, 183:1937-1943. Overexpression of SLP-76 resulted in enhanced TCR-mediated IL-2 gene expression, suggesting a role of SLP-76 in TCR signalling. 59. •-
Wu J, Motto DG, Koretzky GA, Weiss A: Vav and SLP-76 interact and functionally cooperate in IL-2 gene activation, immunity 1996, 4:593-602. This study demonstrates a functional cooperation between Vav and SLP76 during TCR signalling, in addition to an activation-dependent physical interaction between these two molecules. 60.
61.
Onodera H, Motto DG, Koretzky GA, Rothstein DM: Differential regulation of activation-induced tyrosine phosphorylation and recruitment of SLP-76 to Vav by distinct isoforms of the CD45 protein-tyrosine phosphatase. J Biol Chem 1996, 271:22225-22230. TuostoL, Michel F, Acuto O: p95vav associates with tyrosine phosphorylated SLP-76 in antigen-stimulated T cells. J Exp Med 1996, 184:1161-1166.
62. •
Wardenburg JB, Fu C, Jackman JK, Flotow H, Wilkinson SE, Williams DH, Johnson R, Kong G, Chan AC, Findell PR: Phosphorylation of SLP-76 by the ZAP-70 protein-tyrosine kinase is required for T-cell receptor function. J Biol Chem 1996, 271:19641-19644. See annotation [63"]. 63. •
FangN, Motto DG, Ross SE, Koretzky GA: Tyrosine 113, 128, and 145 of SLP-76 are required for optimal augmentation of NFAT promoter activity. J Immunol 1996, 157:3769-3773. The authors of [62",63 °] show that the putative Vav SH2 domain binding tyrosine residues in SLP-76 are required for SLP-76 to exhibit its function
66. •
Tarakhovsky A, Kanner SB, Hombach J, Ledbetter JA, Muller W, Killeen N, Rajewsky K: A role for CD5 in TCR-mediated signal transduction and thymocyte selection. Science 1995, 269:535-537. This paper provides evidence that CD5 is a negative regulator of TCR signalling. 67. ••
Phillips JH, Gumperz JE, Parham P, Lanier LL: Superantigendependent, cell-mediated cytotoxicity inhibited by MHC class I receptors on T lymphocytes. Science 1995, 268:403-405. Describes the identification of KIRs as negative regulators of TCR-activated cytotoxicity. 68. •
D'Andrea A, Chang C, Phillips JH, Lanier LL: Regulation of T cell lymphokine production by killer cell inhibitory receptor recognition of self HLA class I alleles. J Exp Med 1996, 184:789-794. This paper shows that KIRs can inhibit TCR-stimulated lymphokine production. 69. •.
Marengere LE, Waterhouse P, Duncan GS, Mittrucker HW, Fang GS, Mak TW: Regulation of T cell receptor signaling by tyrosine phosphatase SYP association with CTLA-4. Science 1996, 272:1170-11 73. This paper demonstrates that CTLA-4 is a negative regulator of TCR signalling. CTLA-4 interacts with SHP-2, and both the PTK pathway and the Ras/MAPK pathway are all hyperactivated in CTLA-4-/- mice. 70. •
Burshtyn DN, Scharenberg AM, Wagtmann N, Rajagopalan S, Berrada K, Yi T, Kinet J-P, Long EO: Recruitment of tyrosine phosphatase HCP by the killer cell inhibitory receptor. Immunity 1996, 4:77-85. See annotation [71"]. 71. •
Fry AM, Lanier LL, Weiss A: Phosphotyrosine in the killer cell inhibitory receptor motif of NKB1 are required for negative signaling and for association with protein tyrosine phosphatase 1C. J Exp Med 1996, 184:295-300. The authors of [70",71"] show that KIRs mediate their negative regulatory functions via interations with SHP-1.