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.     

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.     

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.     

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.     

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.     

Physical dissociation of the TCR-CD3 complex accompanies receptor ligation

During antigen recognition by T cells, CD4 and the T-cell receptor (TCR)/CD3/zeta complex are thought to interact with the same major histocompatibility complex II molecule in a stable ternary complex. Evidence has suggested that the association of CD4 with TCR/CD3/zeta requires the interaction of the protein tyrosine kinase p56lck with CD4. We have taken a biochemical approach to understand the mechanism by which p56lck and, in particular, its src homology (SH) 2 domain contributes to the association of CD4 with TCR/CD3/zeta during activation. We have previously shown that the p56lck SH2 domain binds directly to tyrosine-phosphorylated ZAP-70. Here we formally demonstrate the in vivo association of p56lck with the homologous protein tyrosine kinases Syk and ZAP-70 after CD3 stimulation of Jurkat cells. A tyrosine-phosphorylated peptide containing the sequence predicted to be optimal for binding to the SH2 domain of src family kinases specifically competes for this association, indicating that tyrosine-phosphorylated ZAP-70 and Syk bind to p56lck by an SH2- mediated interaction. We also show that the same peptide is able to compete for the activation-dependent TCR/CD4 association in Jurkat cells. Moreover, ZAP-70 and CD4 cocap only after CD3 stimulation in human T lymphoblasts. We propose that the interaction of the p56lck SH2 domain with zeta-associated tyrosine-phosphorylated ZAP-70 and/or Syk enables CD4 to associate with antigen-stimulated TCR/CD3/zeta complexes.     

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.     

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.     

Reconstitution of T cell receptor signaling in ZAP-70-deficient cells by retroviral transduction of the ZAP-70 gene

A variant of severe combined immunodeficiency syndrome (SCID) with a selective inability to produce CD8 single positive T cells and a signal transduction defect in peripheral CD4+ cells has recently been shown to be the result of mutations in the ZAP-70 gene. T cell receptor (TCR) signaling requires the association of the ZAP-70 protein tyrosine kinase with the TCR complex. Human T cell leukemia virus type I- transformed CD4+ T cell lines were established from ZAP-70-deficient patients and normal controls. ZAP-70 was expressed and appropriately phosphorylated in normal T cell lines after TCR engagement, but was not detected in T cell lines from ZAP-70-deficient patients. To determine whether signaling could be reconstituted, wild-type ZAP-70 was introduced into deficient cells with a ZAP-70 retroviral vector. High titer producer clones expressing ZAP-70 were generated in the Gibbon ape leukemia virus packaging line PG13. After transduction, ZAP-70 was detected at levels equivalent to those observed in normal cells, and was appropriately phosphorylated on tyrosine after receptor engagement. The kinase activity of ZAP-70 in the reconstituted cells was also appropriately upregulated by receptor aggregation. Moreover, normal and transduced cells, but not ZAP-70-deficient cells, were able to mobilize calcium after receptor ligation, indicating that proximal TCR signaling was reconstituted. These results indicate that this form of SCID may be corrected by gene therapy.     

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 novel human autoimmune syndrome caused by combined hypomorphic and activating mutations in ZAP-70

A brother and sister developed a previously undescribed constellation of autoimmune manifestations within their first year of life, with uncontrollable bullous pemphigoid, colitis, and proteinuria. The boy had hemophilia due to a factor VIII autoantibody and nephrotic syndrome. Both children required allogeneic hematopoietic cell transplantation (HCT), which resolved their autoimmunity. The early onset, severity, and distinctive findings suggested a single gene disorder underlying the phenotype. Whole-exome sequencing performed on five family members revealed the affected siblings to be compound heterozygous for two unique missense mutations in the 70-kD T cell receptor ζ-chain associated protein (ZAP-70). Healthy relatives were heterozygous mutation carriers. Although pre-HCT patient T cells were not available, mutation effects were determined using transfected cell lines and peripheral blood from carriers and controls. Mutation R192W in the C-SH2 domain exhibited reduced binding to phosphorylated ζ-chain, whereas mutation R360P in the N lobe of the catalytic domain disrupted an autoinhibitory mechanism, producing a weakly hyperactive ZAP-70 protein. Although human ZAP-70 deficiency can have dysregulated T cells, and autoreactive mouse thymocytes with weak Zap-70 signaling can escape tolerance, our patients’ combination of hypomorphic and activating mutations suggested a new disease mechanism and produced previously undescribed human ZAP-70–associated autoimmune disease.The adaptive immune system is tightly regulated to allow responses against invading pathogens while avoiding injurious hyperactivity and misdirected responses to self-proteins. Impairment of lymphocyte pathways by genetic defects in mediators of immune signaling and activation can lead to immunodeficiency, but also to immune dysregulation, autoimmunity, and malignancy (Notarangelo, 2014). Essential steps in T cell activation and signaling include antigen recognition by the TCR–CD3 complex; tyrosine phosphorylation of immunoreceptor activation motifs (ITAMs) of the CD3 and ζ-chains by the tyrosine kinase Lck; interaction between phosphorylated ITAMs and the cytoplasmic tyrosine kinase ZAP-70; phosphorylation of ZAP-70 by Lck to relieve its autoinhibition and promote its activation; and ZAP-70–mediated phosphorylation of its adaptor substrates, leading to downstream events, including activation of the Ras–MAPK pathway and increased intracellular calcium.ZAP-70, a critical T cell signaling molecule, is expressed predominantly in T and NK cells. It exists in an autoinhibited state, which is relieved by a two-step process. The first step, binding of the ZAP-70 tandem SH2 domains to doubly phosphorylated ITAMs of the ζ-chain, requires dissociation of the SH2 linker from the back of the kinase domain and repositioning of the SH2 domains to align with ζ-chain ITAMs. This change in structure facilitates a second conformational change whereby ZAP-70 tyrosines Y315 and Y319 in interdomain B are exposed and phosphorylated by Lck, leading to stabilization of the active conformation of the ZAP-70 catalytic domain to permit phosphorylation of downstream signaling molecules (Au-Yeung et al., 2009; Yan et al., 2013; Klammt et al., 2015). The phosphorylation of Y319 is particularly important because, in the nonphosphorylated state, it interacts with the N-lobe of the catalytic domain to maintain its inactive conformation.Deficiency of ZAP-70 in humans causes a profound combined immunodeficiency (CID) in which CD8 T cells are absent and CD4 T cells are defective (Arpaia et al., 1994; Elder et al., 1994; Roifman, 1995). Affected individuals are susceptible to life-threatening infections and require hematopoietic cell transplantation (HCT) to survive (Arpaia et al., 1994; Chan et al., 1994; Katamura et al., 1999; Elder et al., 2001; Turul et al., 2009; Fischer et al., 2010; Roifman et al., 2010). Some ZAP-70–deficient patients also have skin infiltration with dysfunctional CD4 T cells, elevated serum IgE, and eosinophilia (Katamura et al., 1999; Turul et al., 2009).In contrast to humans, mice with complete Zap-70 deficiency manifest developmental arrest of both CD4 and CD8 T lineages. A hypomorphic murine Zap-70 mutation with reduced ζ-chain binding caused attenuated TCR signaling that permitted survival of autoreactive T cells normally deleted in the thymus (Tanaka et al., 2010). In response to innate stimuli, these self-reactive murine T cells contributed to the development of non–tissue-specific autoantibodies (such as rheumatoid factor and antibody to cyclic citrullinated peptide) and autoimmune arthritis (Sakaguchi et al., 2012). Other hypomorphic alleles of Zap-70 in the mouse have also been associated with nonspecific autoantibodies (e.g., antinuclear antibodies; Siggs et al., 2007). In contrast, antibody-mediated autoimmune disease due to hypomorphic ZAP-70 alleles in human patients has not been reported.We present two siblings with unique mutations of ZAP70 who lacked clinical immunodeficiency, but instead had a novel constellation of early onset, severe autoimmune manifestations, including bullous pemphigoid. Compound heterozygosity for hypomorphic and hyperactive mutant ZAP70 alleles in these patients represents a new genetic mechanism underlying inappropriate T cell activation.     

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.     

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.     

The four distal tyrosines are required for LAT-dependent signaling in FcepsilonRI-mediated mast cell activation

The linker for activation of T cells (LAT) is an adaptor protein critical for Fc epsilon RI-mediated mast cell activation. LAT is a substrate of the tyrosine kinases activated after TCR and Fc epsilon RI engagement. After phosphorylation of the cytosolic domain of LAT, multiple signaling molecules such as phospholipase C-gamma1, Grb2, and Gads associate with phosphorylated LAT via their SH2 domains. The essential role of the four distal tyrosines in TCR-mediated signaling and T cell development has been demonstrated by experiments using LAT-deficient cell lines and genetically modified mice. To investigate the role of these four tyrosines of LAT in Fc epsilon RI-mediated mast cell activation, bone marrow-derived mast cells from LAT-deficient mice were infected with retroviral vectors designed to express wild-type or mutant LAT. Examination of bone marrow-derived mast cells expressing various tyrosine to phenylalanine mutants in LAT demonstrates a differential requirement for these different binding sites. In these studies, assays of biochemical pathways, degranulation, and cytokine and chemokine release were performed. Finally, the role of these tyrosines was also evaluated in vivo using genetically modified animals. Deletion of all four distal tyrosines, and in particular, loss of the primary phospholipase C-gamma-binding tyrosine had a significant effect on antigen-induced histamine release.     

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.     

Partially phosphorylated T cell receptor zeta molecules can inhibit T cell activation

The T cell receptor complex (TCR) zeta chain is constitutively tyrosine phosphorylated specifically at two of the six zeta immunoreceptor tyrosine-based activation motif (ITAM) tyrosine residues in resting peripheral T cells. Further phosphorylation of zeta is induced by both agonist and antagonist ligands of the TCR, with agonists inducing complete phosphorylation of the zeta ITAM tyrosines. After antagonist stimulation, zeta phosphorylation is incomplete and generates discrete forms of partially phosphorylated ITAMs. Here, we mutate specific tyrosines in chimeric human CD8-zeta molecules to reflect phosphorylation in resting T cells as well as phosphorylation induced by agonist and antagonist ligands. We demonstrate that such partially phosphorylated TCR-zeta species can inhibit IL-2 production in T cell hybridomas and proliferation in T cell clones. This reveals a previously unrecognized, inhibitory function of partially phosphorylated ITAMs. These findings support the concept that TCR antagonism can arise through the generation of an inhibitory signal within the TCR complex and that constitutive zeta phosphorylation in resting T cells is an inhibitory signaling environment.     

Differential association of protein tyrosine kinases with the T cell receptor is linked to the induction of anergy and its prevention by B7 family-mediated costimulation

When stimulated through their antigen receptor, without costimulation, T cells enter a state of antigen-specific unresponsiveness, termed anergy. B7-mediated costimulation, signaling via CD28, is sufficient to prevent the induction of anergy. Here we show that ligation of T cell receptor (TCR) by alloantigen alone, which results in anergy, activates tyrosine phosphorylation of TCR zeta and its association with fyn. In contrast, TCR ligation in the presence of B7 costimulation, which results in productive immunity, activates tyrosine phosphorylation of TCR zeta and CD3 chains, which associate with activated lck and zeta- associated protein (ZAP) 70. Under these conditions, CD28 associates with activated lck and TCR zeta. These data suggest that the induction of anergy is an active signaling process characterized by the association of TCR zeta and fyn. In addition, CD28-mediated costimulation may prevent the induction of anergy by facilitating the effective association of TCR zeta and CD3 epsilon with the critical protein tyrosine kinase lck, and the subsequent recruitment of ZAP-70. Strategies to inhibit or activate TCR-associated, specific protein tyrosine kinase-mediated pathways may provide a basis for drug development with potential applications in the fields of transplantation, autoimmunity, and tumor immunity.     

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.