Absence of ZAP-70 prevents signaling through the antigen receptor on peripheral blood T cells but not on thymocytes

Recently, a severe combined immunodeficiency syndrome with a deficiency of CD8+ peripheral T cells and a TCR signal transduction defect in peripheral CD4+ T cells was associated with mutations in ZAP-70. Since TCR signaling is required in developmental decisions resulting in mature CD4 (and CD8) T cells, the presence of peripheral CD4+ T cells expressing TCRs incapable of signaling in these patients is paradoxical. Here, we show that the TCRs on thymocytes, but not peripheral T cells, from a ZAP-70-deficient patient are capable of signaling. Moreover, the TCR on a thymocyte line derived from this patient can signal, and the homologous kinase Syk is present at high levels and is tyrosine phosphorylated after TCR stimulation. Thus, Syk may compensate for the loss of ZAP-70 and account for the thymic selection of at least a subset of T cells (CD4+) in ZAP-70-deficient patients.     

Lck regulates the tyrosine phosphorylation of the T cell receptor subunits and ZAP-70 in murine thymocytes

The Src-family and Syk/ZAP-70 family of protein tyrosine kinases (PTK) are required for T cell receptor (TCR) functions. We provide evidence that the Src-family PTK Lck is responsible for regulating the constitutive tyrosine phosphorylation of the TCR zeta subunit in murine thymocytes. Moreover, ligation of the TCR expressed on thymocytes from Lck-deficient mice largely failed to induce the phosphorylation of TCR- zeta, CD3 epsilon, or ZAP-70. In contrast, we find that the TCR-zeta subunit is weakly constitutively tyrosine phosphorylated in peripheral T cells isolated from Lck-null mice. These data suggest that Lck has a functional role in regulation of TCR signal transduction in thymocytes. In peripheral T cells, other Src-family PTKs such as Fyn may partially compensate for the absence of Lck.     

T cell development and T cell responses in mice with mutations affecting tyrosines 292 or 315 of the ZAP-70 protein tyrosine kinase

After stimulation of the T cell receptor (TCR), the tyrosine residues 292 and 315 in interdomain B of the protein tyrosine kinase ZAP-70 become phosphorylated and plausibly function as docking sites for Cbl and Vav1, respectively. The two latter proteins have been suggested to serve as substrates for ZAP-70 and to fine-tune its function. To address the role of these residues in T cell development and in the function of primary T cells, we have generated mice that express ZAP-70 molecules with Tyr to Phe substitution at position 292 (Y292F) or 315 (Y315F). When analyzed in a sensitized TCR transgenic background, the ZAP-70 Y315F mutation reduced the rate of positive selection and delayed the occurrence of negative selection. Furthermore, this mutation unexpectedly affected the constitutive levels of the CD3-zeta p21 phosphoisoform. Conversely, the ZAP-70 Y292F mutation upregulated proximal events in TCR signaling and allowed more T cells to produce interleukin 2 and interferon gamma in response to a given dose of antigen. The observation that ZAP-70 Y292F T cells have a slower rate of ligand-induced TCR downmodulation suggests that Y292 is likely involved in regulating the duration activated TCR reside at the cell surface. Furthermore, we showed that Y292 and Y315 are dispensable for the TCR-induced tyrosine phosphorylation of Cbl and Vav1, respectively. Therefore, other molecules present in the TCR signaling cassette act as additional adaptors for Cbl and Vav1. The present in vivo analyses extend previous data based on transformed T cell lines and suggest that residue Y292 plays a role in attenuation of TCR signaling, whereas residue Y315 enhances ZAP-70 function.     

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

Engagement of the T cell antigen receptor (TCR) results in activation of several tyrosine kinases leading to tyrosine phosphorylation of protein substrates and activation of multiple biochemical pathways. TCR- mediated activation of the src-family kinases, Lck and Fyn, results in tyrosine phosphorylation of the TCR zeta and CD3 chains. The site of phosphorylation in these chains is the tyrosine-based activation motif (TAM), a 15-16 amino acid module containing two tyrosine residues. Tyrosine-phosphorylated TAMs serve as targets for binding of the zeta- associated protein (ZAP-70) tyrosine kinase via its tandem SH2 domains. This binding correlates with activation of ZAP-70, a critical event in T cell activation. To further define the structural requirements for ZAP-70 interaction with the TCR, we developed a binding assay using immobilized glutathione S-transferase fusion proteins containing the NH2- and/or COOH-terminal SH2 domains of ZAP-70, and soluble synthetic peptides with the sequence of the cytoplasmic region of the TCR zeta chain (TCR zeta cyt) or individual TCR zeta and CD3 epsilon TAM motifs. Direct binding studies demonstrated that the tandem ZAP-70 SH2 domains bind phosphorylated, but not nonphosphorylated, TCR zeta cyt. The NH2- terminal ZAP-70 SH2 domain also binds to TCR zeta cyt but with 100-fold lower affinity. No binding was observed with the COOH-terminal ZAP-70 SH2 domain. Similar studies demonstrated that the ZAP-70 tandem SH2 domain can bind a TCR zeta 3 TAM peptide in which both tyrosine residues are phosphorylated: Little or no binding was observed with peptides phosphorylated at only one tyrosine residue, or a nonphosphorylated peptide. Binding of the tandem SH2 domains to the other two TCR zeta TAM peptides and to a CD3 epsilon TAM peptide was also observed. All four doubly tyrosine phosphorylated TAM peptides cross-compete with each other for binding to the tandem SH2 domains of ZAP-70. The affinity of these peptides for the tandem SH2 construct demonstrated a hierarchy of TAM zeta 1 > or = TAM zeta 2 > TAM epsilon > or = TAM zeta 3. The results provide further evidence that the ZAP-70 interaction with the TCR requires prior phosphorylation of both tyrosine residues within a TAM motif. Binding of ZAP-70 to phospho-TAMs is notable for the high level of cooperativity between the two SH2 domains, which individually demonstrate low affinity interaction with the ligand. The cooperativity ensures higher affinity for the doubly phosphorylated ligand. Affinity differences of as much as 30-fold indicates a significant specificity of interaction of ZAP-70 SH2 domains for different phospho-TAMs.     

p56lck interacts via its src homology 2 domain with the ZAP-70 kinase

p56lck, a member of the src family of protein tyrosine kinases, is an essential component in T cell receptor (TCR) signal transduction. p56lck contains a src homology 2 (SH2) domain found in a number of proteins involved in intracellular signaling. SH2 domains have been implicated in protein-protein interactions by binding to sequences in target proteins containing phosphorylated tyrosine. Using an in vitro assay, we have studied specific binding of tyrosine-phosphorylated proteins to a recombinant p56lck SH2 domain. In nonactivated Jurkat cells, two tyrosine-phosphorylated proteins were detected. Stimulation with anti-CD3 monoclonal antibodies induced the binding of seven additional tyrosine-phosphorylated proteins to the SH2 domain of p56lck. We have identified the zeta-associated tyrosine kinase, ZAP-70, as one of these proteins. Evidence suggests that binding of ZAP-70 to p56lck SH2 is direct and not mediated by zeta. The significance of this interaction was further investigated in vivo. p56lck could be coprecipitated with the zeta/ZAP-70 complex and conversely, ZAP-70 was detected in p56lck immunoprecipitates of activated Jurkat cells. The physical association of p56lck and ZAP-70 during activation supports the recently proposed functional cooperation of these two tyrosine kinases in TCR signaling.     

ZAP-70 protein promotes tyrosine phosphorylation of T cell receptor signaling motifs (ITAMs) in immature CD4(+)8(+) thymocytes with limiting p56(lck)

As a result of interaction with epithelial cells in the thymic cortex, immature CD4(+)8(+) (double positive, DP) thymocytes express relatively few T cell receptors (TCRs) and contain diminished numbers of coreceptor-associated p56(lck) (lck) PTK molecules. As a result, TCR signal transduction in DP thymocytes is significantly impaired, despite its importance for repertoire selection. We report here that, in DP thymocytes, tyrosine phosphorylation of TCR signaling motifs (ITAMs) by lck, an early event in TCR signal transduction, is dependent upon ZAP-70 protein independent of ZAP-70's kinase activity. Furthermore, the dependence on ZAP-70 protein for ITAM phosphorylation diminishes as available lck increases. Importantly, ZAP-70's role in ITAM phosphorylation in DP thymocytes is not limited to protecting phosphorylated ITAMs from dephosphorylation. Rather, this study indicates that ZAP-70 protein augments ITAM phosphorylation in DP thymocytes and so compensates in part for the relative deficiency of coreceptor-associated lck.     

The Vav Binding Site (Y315) in ZAP-70 Is Critical for Antigen Receptor–mediated Signal Transduction

Stimulation of antigen receptors in T and B cells leads to the activation of the Src and Syk families of protein tyrosine kinases (PTK). These PTKs subsequently phosphorylate numerous intracellular substrates, including the 95-kD protooncogene product Vav. Vav is essential for both T and B cell development and T and B cell antigen receptor–mediated signal transduction. After receptor ligation, Vav associates with phosphorylated Syk and ZAP-70 PTKs, an interaction that depends upon its SH2 domain. Here we demonstrate that a point mutation of tyrosine 315 (Y315F) in ZAP-70, a putative Vav SH2 domain binding site, eliminated the Vav– ZAP-70 interaction. Moreover, the Y315 mutation impaired the function of ZAP-70 in antigen receptor signaling. Surprisingly, this mutation also resulted in marked reduction in the tyrosine phosphorylation of ZAP-70, Vav, SLP-76, and Shc. These data demonstrate that the Vav binding site in ZAP-70 plays a critical role in antigen receptor–mediated signal transduction.     

Requirement for tyrosine residues 315 and 319 within zeta chain-associated protein 70 for T cell development

Engagement of the T cell antigen receptor (TCR) induces the transphosphorylation of the zeta chain-associated protein of 70,000 Mr (ZAP-70) protein tyrosine kinase (PTK) by the CD4/8 coreceptor associated Lck PTK. Phosphorylation of Tyr 493 within ZAP-70's activation loop results in the enzymatic activation of ZAP-70. Additional tyrosines (Tyrs) within ZAP-70 are phosphorylated that play both positive and negative regulatory roles in TCR function. Phosphorylation of Tyr residues (Tyrs 315 and 319) within the Interdomain B region of the ZAP-70 PTK plays important roles in the generation of second messengers after TCR engagement. Here, we demonstrate that phosphorylation of these two Tyr residues also play important roles in mediating the positive and negative selection of T cells in the thymus.     

Knock-in mutation of the distal four tyrosines of linker for activation of T cells blocks murine T cell development.

The integral membrane adapter protein linker for activation of T cells (LAT) performs a critical function in T cell antigen receptor (TCR) signal transduction by coupling the TCR to downstream signaling pathways. After TCR engagement, LAT is tyrosine phosphorylated by ZAP-70 creating docking sites for multiple src homology 2-containing effector proteins. In the Jurkat T cell line, the distal four tyrosines of LAT bind PLCgamma-1, Grb2, and Gads. Mutation of these four tyrosine residues to phenylalanine (4YF) blocked TCR-mediated calcium mobilization, Erk activation, and nuclear factor (NF)-AT activation. In this study, we examined whether these four tyrosine residues were essential for T cell development by generating LAT "knock-in" mutant mice that express the 4YF mutant protein under the control of endogenous LAT regulatory sequences. Significantly, the phenotype of 4YF knock-in mice was identical to LAT(-/)- (null) mice; thymocyte development was arrested at the immature CD4(-)CD8(-) stage and no mature T cells were present. Knock-in mice expressing wild-type LAT protein, generated by a similar strategy, displayed a normal T cell developmental profile. These results demonstrate that the distal four tyrosine residues of LAT are essential for preTCR signaling and T cell development in vivo.     

In vivo correction of ZAP-70 immunodeficiency by intrathymic gene transfer

SCID patients have been successfully treated by administration of ex vivo gene-corrected stem cells. However, despite its proven efficacy, such treatment carries specific risks and difficulties. We hypothesized that some of these drawbacks may be overcome by in situ gene correction of T lymphoid progenitors in the thymus. Indeed, in vivo intrathymic transfer of a gene that provides a selective advantage for transduced prothymocytes should result in the generation of functional T lymphocyte progeny, allowing long-term immune reconstitution. We assessed the feasibility of this approach in a murine model of ZAP-70-deficient SCID. A T cell-specific ZAP-70-expressing lentiviral vector was injected into thymi of adult ZAP-70-/- mice without prior conditioning. This resulted in the long-term differentiation of mature TCR-alphabeta+ thymocytes, indicating that the vector had integrated into progenitor cells. Moreover, peripheral ZAP-70-expressing T cells demonstrated a partially diversified receptor repertoire and were responsive to alloantigens in vitro and in vivo. Improved treatment efficacy was achieved in infant ZAP-70-/- mice, in which the thymus is proportionately larger and a higher percentage of prothymocytes are in cycle. Thus, intrathymic injection of a lentiviral vector could represent a simplified and potentially safer alternative to ex vivo gene-modified hematopoietic stem cell transplantation for gene therapy of T cell immunodeficiencies.     

The CD3 chains of the T cell antigen receptor associate with the ZAP-70 tyrosine kinase and are tyrosine phosphorylated after receptor stimulation

Recent work indicates that signaling events resulting from stimulation of the T cell antigen receptor (TCR) can be initiated by the CD3 complex (gamma, delta, epsilon) as well as the zeta chains of the receptor. To help characterize the signaling function of CD3 we examined its associated tyrosine kinase activity since induction of tyrosine phosphorylation is one of the earliest signaling events. Our results indicate that at least two kinases, lck and ZAP-70, contribute to the CD3-associated kinase activity. A likely target of this activity is the CD3 complex itself since we observed that TCR stimulation resulted in rapid tyrosine phosphorylation of the CD3 epsilon and delta chains. To examine the function of the CD3 epsilon chain in particular, we constructed a chimera that fused the extracellular and transmembrane domains of CD8 to the cytoplasmic domain of CD3 epsilon. This chimera demonstrated that CD3 epsilon was independently capable of associating with proteins having tyrosine kinase activity, including ZAP-70. Our results show that the kinase activity that associates with the CD3 complex has characteristics that are quite similar to the previously characterized zeta-associated kinase activity. This finding suggests that both these components of the TCR initiate signaling events using a common mechanism. However, differences in their signaling function could result from recognition of distinct substrates.     

Dominant-negative zeta-associated protein 70 inhibits T cell antigen receptor signaling

Zeta-associated protein (ZAP)-70 is a cytoplasmic protein tyrosine required for T cell antigen receptor (TCR) signaling and development. Mutations in ZAP-70 result in severe combined immunodeficiency in humans. ZAP-70 interacts with the TCR by binding to tyrosine- phosphorylated immunoreceptor tyrosine-based activation motifs (ITAMs) present in the invariant subunits of the TCR complex. Here we report that two ZAP-70 mutants devoid of kinase activity, generated either by a point mutation in the kinase domain to create an inactive kinase, or by truncation of the entire kinase domain (SH2[N+C]), functioned as dominant-negative mutants to specifically suppress TCR-mediated activation of NFAT, a nuclear factor essential for inducible interleukin 2 gene expression. Biochemical studies with the SH2(N+C) mutant showed that it also blocked early TCR signaling events, such as p95vav tyrosine phosphorylation, extracellular signal-regulated kinase 2 activation, and the association of a number of tyrosine- phosphorylated proteins with growth factor receptor-binding protein 2 (GRB2). The inhibitory effects of the SH2(N+C) mutant revealed that it requires an intact phosphotyrosine-binding site in its COOH-terminal SH2 domain. Using a CD8-zeta chimeric receptor to analyze the interaction of the SH2(N+C) mutant with ITAMs of TCR-zeta, we found that this mutant was constitutively bound to the hyperphosphorylated CD8-zeta chimera. These results indicate that tyrosine-phosphorylated ITAM is the target for the action of this dominant-negative mutant, suggesting that the assembly of a functional receptor signaling complex on ITAMs is a critical proximal TCR signaling event leading to downstream activation.     

Dissociation of Intracellular Signaling Pathways in Response to Partial Agonist Ligands of the T Cell Receptor

The T cell receptor (TCR) is a versatile receptor able to generate different signals that result in distinct T cell responses. The pattern of early signals is determined by the TCR binding kinetics that control the ability of the ligand to coengage TCR and coreceptor. Coengagement of TCR and CD4 results in an agonist signaling pattern with complete tyrosine phosphorylation of TCR subunits, and recruitment and activation of ZAP-70. In contrast, TCR engagement without CD4 coengagement causes a partial agonist type of signaling, characterized by distinct phosphorylation of TCR subunits and recruitment but no activation of ZAP-70. The pathways triggered by partial agonist signaling are unknown. Here, we show that agonists cause association of active lck and active ZAP-70 with p120-GTPase–activating protein (p120-GAP). These associations follow engagement of CD4 or CD3, respectively. In contrast, partial agonists do not activate lck or ZAP-70, but induce association of p120-GAP with inactive ZAP-70. Despite these differences, both agonist and partial agonist signals activate the mitogen-activated protein kinase (MAPK) pathway. However, MAPK activation by partial agonists is transient, supporting a kinetic, CD4-dependent model for the mechanism of action of variant TCR ligands. Transient MAPK activation may explain some of the responses to TCR partial agonists and antagonists.     

Retrovirus-mediated transduction of primary ZAP-70-deficient human T cells results in the selective growth advantage of gene-corrected cells: implications for gene therapy

Humans lacking the ZAP-70 protein tyrosine kinase present with an absence of CD8+ T cells and defective CD4+ T cells in the periphery. This severe combined immunodeficiency is fatal unless treated by allogeneic bone marrow transplantation. However, in the absence of suitable marrow donors, the development of alternative forms of therapy is desirable. Because lymphocytes are long-lived, it is possible that introduction of the wild-type ZAP-70 gene into CD4+ ZAP-70-deficient T cells will restore their immune function in vivo. Initial investigations evaluating the feasibility of gene therapy for ZAP-70 deficiency were performed using HTL V-I-transformed lymphocytes. Although transformation was useful in circumventing problems associated with the maintenance of ZAP-70-deficient T cells and low gene transfer levels, the presence of HTL V-I precluded any biological studies. Here, we investigated a retrovirus-mediated approach for the correction of primary T cells derived from two ZAP-70-deficient patients. Upon introduction of the wild-type ZAP-70 gene, TCR-induced MAPK activation, IL-2 secretion and proliferation were restored to approximately normal levels. Importantly, this gain-of-function was associated with a selective growth advantage of gene-corrected cells, thereby indicating the feasibility of a gene therapy-based strategy.     

Regulation of ZAP-70 Intracellular Localization: Visualization with the Green Fluorescent Protein

To investigate the cellular dynamics of ZAP-70, we have studied the distribution and regulation of its intracellular location using a ZAP-70 green fluorescent protein chimera. Initial experiments in epithelial cells indicated that ZAP-70 is diffusely located throughout the quiescent cell, and accumulates at the plasma membrane upon cellular activation, a phenotype enhanced by the coexpression of Lck and the initiation of ZAP-70 kinase activity. Subsequent studies in T cells confirmed this phenotype. Intriguingly, a large amount of ZAP-70, both chimeric and endogenous, resides in the nucleus of quiescent and activated cells. Nuclear ZAP-70 becomes tyrosine phosphorylated upon stimulation via the T cell receptor, indicating that it may have an important biologic function.     

Association of the adaptor molecule LAT with CD4 and CD8 coreceptors identifies a new coreceptor function in T cell receptor signal transduction

Linker for activation of T cells (LAT) is an adaptor protein whose tyrosine phosphorylation is critical for transduction of the T cell receptor (TCR) signal. LAT phosphorylation is accomplished by the protein tyrosine kinase ZAP-70, but it is not at all clear how LAT (which is not associated with the TCR) encounters ZAP-70 (which is bound to the TCR). Here we show that LAT associates with surface CD4 and CD8 coreceptors and that its association is promoted by the same coreceptor cysteine motif that mediates Lck binding. In fact, LAT competes with Lck for binding to individual coreceptor molecules but differs from Lck in its preferential association with CD8 rather than CD4 in CD4(+)CD8(+) thymocytes. Importantly, as a consequence of LAT association with surface coreceptors, coengagement of the TCR with surface coreceptors induces LAT phosphorylation and the specific recruitment of downstream signaling mediators to coreceptor-associated LAT molecules. These results point to a new function for CD4 and CD8 coreceptors in TCR signal transduction, namely to promote LAT phosphorylation by ZAP-70 by recruiting LAT to major histocompatibility complex-engaged TCR complexes.     

Distinct roles for Syk and ZAP-70 during early thymocyte development

The spleen tyrosine kinase (Syk) and zeta-associated protein of 70 kD (ZAP-70) tyrosine kinases are both expressed during early thymocyte development, but their unique thymic functions have remained obscure. No specific role for Syk during beta-selection has been established, and no role has been described for ZAP-70 before positive selection. We show that Syk and ZAP-70 provide thymocytes with unique and separable fitness advantages during early development. Syk-deficient, but not ZAP-70-deficient, thymocytes are specifically impaired in initial pre-TCR signaling at the double-negative (DN) 3 beta selection stage and show reduced cell-cycle entry. Surprisingly, and despite overlapping expression of both kinases, only ZAP-70 appears to promote sustained pre-TCR/TCR signaling during the DN4, immature single-positive, and double-positive stages of development before thymic selection occurs. ZAP-70 promotes survival and cell-cycle progression of developing thymocytes before positive selection, as also shown by in vivo anti-CD3 treatment of recombinase-activating gene 1-deficient mice. Our results establish a temporal separation of Syk family kinase function during early thymocyte development and a novel role for ZAP-70. We propose that pre-TCR signaling continues during DN4 and later stages, with ZAP-70 dynamically replacing Syk for continued pre-TCR signaling.     

The pre-T cell receptor (TCR) complex is functionally coupled to the TCR-zeta subunit

The pre-T cell receptor (TCR) complex regulates early T cell development and consists of a heterodimer of the TCR-beta subunit in association with the pre-TCR-alpha chain. Notably, in contrast to alpha/beta-expressing T cells, several studies suggested that the TCR- zeta chain is not stably associated with this pre-TCR complex. To examine the proximal signaling processes mediated by the pre-TCR complex and the role of the TCR-zeta chain in these processes, we stimulated pre-TCR-expressing cells and analyzed the interactions of the TCR/CD3 invariant chains with the Syk/ZAP-70 family of protein tyrosine kinases. Stimulation of the pre-TCR complex led to the tyrosine phosphorylation of the CD3 epsilon and TCR-zeta chains, as well as the phosphorylation and association of ZAP-70 and Syk with phosphorylated CD3 epsilon and TCR-zeta. These results demonstrate that the pre-TCR complex is functionally coupled to the TCR-zeta subunit and to the ZAP-70 and Syk protein tyrosine kinases.     

A hypomorphic allele of ZAP-70 reveals a distinct thymic threshold for autoimmune disease versus autoimmune reactivity

ZAP-70 is critical for T cell receptor (TCR) signaling. Tyrosine to phenylalanine mutations of Y315 and Y319 in ZAP-70 suggest these residues function to recruit downstream effector molecules, but mutagenesis and crystallization studies reveal that these residues also play an important role in autoinhibition ZAP-70. To address the importance of the scaffolding function, we generated a zap70 mutant mouse (YYAA mouse) with Y315 and Y319 both mutated to alanines. These YYAA mice reveal that the scaffolding function is important for normal development and function. Moreover, the YYAA mice have many similarities to a previously identified ZAP-70 mutant mouse, SKG, which harbors a distinct hypomorphic mutation. Both YYAA and SKG mice have impaired T cell development and hyporesponsiveness to TCR stimulation, markedly reduced numbers of thymic T regulatory cells and defective positive and negative selection. YYAA mice, like SKG mice, develop rheumatoid factor antibodies, but fail to develop autoimmune arthritis. Signaling differences that result from ZAP-70 mutations appear to skew the TCR repertoire in ways that differentially influence propensity to autoimmunity versus autoimmune disease susceptibility. By uncoupling the relative contribution from T regulatory cells and TCR repertoire during thymic selection, our data help to identify events that may be important, but alone are insufficient, for the development of autoimmune disease.Signal transduction by the TCR plays a critical role in T cell development and in the protective and pathological responses mediated by mature T cells. The repertoire of mature T cells and the discrimination of self from nonself are largely determined within the thymus through TCR-dependent processes known as positive and negative selection. It is generally thought that quantitative or qualitative differences in TCR signaling determine the binary decision between positive or negative selection (Starr et al., 2003). Likewise, whether a productive mature T cell response will be made is dictated by signaling events induced by the TCR.ZAP-70, a Syk family tyrosine kinase that associates with the TCR CD3 and ζ subunits, plays a critical role in TCR signaling in immature thymocyte selection and in mature T cell responses (Chan et al., 1992). ZAP-70 contains two N-terminal SH2 domains that mediate its association with doubly phosphorylated immunoreceptor tyrosine-based activation motifs (ITAMs) after their phosphorylation by Lck. The ZAP-70 C-terminal catalytic domain phosphorylates the downstream adaptor molecules LAT and SLP-76 that play critical roles in T cell development and in T cell responses (Horejsí et al., 2004). Interdomain B bridges the C-terminal SH2 and the kinase domains and contains three tyrosine residues (Y292, Y315, and Y319) that are inducibly phosphorylated. Based on tyrosine to phenylalanine mutations, we and others have previously shown that phosphorylation of Y292 may exert a negative regulatory effect on ZAP-70, perhaps by functioning as a docking site for c-Cbl (Lupher et al., 1997; Meng et al., 1999; Rao et al., 2000). In contrast, phenylalanine mutations of Y315 and Y319 suggested that these sites positively regulate ZAP-70 function by recruiting Vav1 and Lck, respectively (Straus et al., 1996; Wu et al., 1997; Pelosi et al., 1999). These sites have also been reported to bind c-Crk (Gelkop and Isakov, 1999) and phospholipase Cγ1 (Williams et al., 1999). Thus, in addition to its catalytic activity, ZAP-70 may have an important scaffolding role in recruiting downstream effector molecules.Surprisingly, in addition to this scaffolding role, we recently discovered that Y315 and Y319 play an important role in regulating ZAP-70 kinase activity. Whereas mutation of both residues to phenylalanine potently inhibits ZAP-70 function, deletion of interdomain B or mutation of these sites to alanine relieves an autoinhibitory conformation and renders the kinase relatively Lck-independent (Brdicka et al., 2005). Stabilizing the autoinhibited ZAP-70 conformation with the YYFF mutations (Y315F and Y319F) allowed the crystallization of full-length ZAP-70. The crystal structure, together with targeted mutagenesis studies, suggest that the Y315 and Y319 stabilize the autoinhibited conformation of the ZAP-70 kinase through hydrophobic interactions involving the aromatic rings of the Y315 and Y319 residues with residues in the inter-SH2 domain (Interdomain A) and the C-terminal lobe of the catalytic domain (Deindl et al., 2007). Comparison of the alignment of the SH2 domains in the autoinhibited conformation to the ITAM-bound SH2 domains (Hatada et al., 1995) further suggests that ZAP-70 undergoes a conformational change upon binding to a doubly phosphorylated ITAM, which allows Y315 and Y319 to be more accessible for phosphorylation. Y315 and Y319 appear to be Lck phosphorylation sites and their phosphorylation stabilizes the active “open” ZAP-70 conformation and prevents the kinase from returning to the autoinhibited conformation (Brdicka et al., 2005; Deindl et al., 2007; Levin et al., 2008). What remains unclear is the relative importance of their scaffolding function in effector molecule recruitment.Two independent groups have generated knockin mice (Magnan et al., 2001) and transgenic mice (Gong et al., 2001) to study the in vivo individual contributions of Y315 and Y319 of ZAP-70 to signal transduction and to T cell development. However, those studies involved mutation of either Y315 or Y319 to phenylalanine, which would have helped stabilize the autoinhibited conformation and could have resulted in attenuated T cell signaling and consequent alteration of positive and negative selection. TCR-mediated calcium increase is greatly diminished in ZAP-70^−/− mice expressing the Y319F transgenic mice whereas the Y315F mutation has a modest effect on calcium responses. Consistent with results from transgenic mice, phosphorylation of phospholipase C γ1, but not Vav1, is reduced in the Y315F knockin mice. These results suggest that Y315 and Y319 contribute to efficient generation of second messengers involved in thymic selection. However, these studies did not take into account the notion that the tyrosine to phenylalanine mutations might also stabilize the autoinhibitory conformation of ZAP-70 rather than only affecting the recruitment of downstream effectors.To address the relative importance of the scaffolding function of these residues, we generated a knockin ZAP-70 mutant strain with Y315 and Y319 simultaneously mutated to alanine residues. By creating a YYAA ZAP-70 knockin mouse we were able to study the contribution of the scaffolding function of these sites without the confounding issue of autoinhibition in the context of phenylalanine mutants. These YYAA mice showed many of the features of a previously identified spontaneous mutant mouse called SKG. The SKG mouse has a syndrome that shares features with human rheumatoid arthritis, including autoimmune arthritis and rheumatoid factor production (Sakaguchi et al., 2003). SKG mice have a single point mutation in the C-terminal SH2 domain of ZAP-70 that results in attenuated TCR signaling and aberrant positive and negative selection. Results suggested that the autoimmune arthritis could be caused by expansion of autoreactive CD4⁺ T cells that have escaped negative selection. Interestingly, unlike SKG mice, although the YYAA mice have similar defects in positive and negative selection in TCR transgenic systems and can be induced to develop rheumatoid factor antibodies, they do not develop autoimmune arthritis. Quantitative assessment of negative selection in response to endogenous superantigens suggests that YYAA and SKG mice have distinct TCR repertoires that could account for the increased susceptibility of both lines of mice to develop rheumatoid factor antibodies, but interestingly, only SKG mice develop arthritis. Our findings demonstrate the importance of phosphorylation of Y315 and Y319 in ZAP-70 as binding sites for key effector molecules. As well, these findings stress the importance of these sites for proper regulation of TCR signaling and for normal T cell repertoire selection and peripheral T cell function in resistance to autoimmune disease susceptibility.     

Inhibition of T cell receptor signal transduction by tyrosine kinase-interacting protein of Herpesvirus saimiri

T cells play a central role in orchestrating immunity against pathogens, particularly viruses. Thus, impairing T cell activation is an important strategy employed by viruses to escape host immune control. The tyrosine kinase-interacting protein (Tip) of the T lymphotropic Herpesvirus saimiri (HVS) is constitutively present in lipid rafts and interacts with cellular Lck tyrosine kinase and p80 endosomal protein. Here we demonstrate that, due to the sequestration of Lck by HVS Tip, T cell receptor (TCR) stimulation fails to activate ZAP70 tyrosine kinase and to initiate downstream signaling events. TCR zeta chains in Tip-expressing T cells were initially phosphorylated to recruit ZAP70 molecule upon TCR stimulation, but the recruited ZAP70 kinase was not subsequently phosphorylated, resulting in TCR complexes that were stably associated with inactive ZAP70 kinase. Consequently, Tip expression not only markedly inhibited TCR-mediated intracellular signal transduction but also blocked TCR engagement with major histocompatibility complexes on the antigen-presenting cells and immunological synapse formation. These results demonstrate that a lymphotropic herpesvirus has evolved a novel mechanism to deregulate T cell activation to disarm host immune surveillance. This process contributes to the establishment and maintenance of viral latency.