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.     

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.     

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 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.     

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.     

Disruption of Lymphocyte Function and Signaling in CD45–associated Protein–null Mice

CD45-AP specifically associates with CD45, a protein tyrosine phosphatase essential for lymphocyte differentiation and antigen receptor–mediated signal transduction. CD45 is thought to mediate antigen receptor signaling by dephosphorylating regulatory tyrosine residues on Src family protein tyrosine kinases such as Lck. However, the mechanism for regulating CD45 protein tyrosine phosphatase activity remains unclear. CD45-AP–null mice were created to examine the role of CD45-AP in CD45-mediated signal transduction. T and B lymphocytes showed reduced proliferation in response to antigen receptor stimulation. Both mixed leukocyte reaction and cytotoxic T lymphocyte functions of T cells were also markedly decreased in CD45-AP–null mice. Interestingly, the interaction between CD45 and Lck was significantly reduced in CD45-AP–null T cells, indicating that CD45-AP directly or indirectly mediates the interaction of CD45 with Lck. Our data indicate that CD45-AP is required for normal antigen receptor signaling and function in lymphocytes.     

Functional Role for Syk Tyrosine Kinase in Natural Killer Cell–mediated Natural Cytotoxicity

Natural killer (NK) cells are named based on their natural cytotoxic activity against a variety of target cells. However, the mechanisms by which sensitive targets activate killing have been difficult to study due to the lack of a prototypic NK cell triggering receptor. Pharmacologic evidence has implicated protein tyrosine kinases (PTKs) in natural killing; however, Lck-deficient, Fyn-deficient, and ZAP-70–deficient mice do not exhibit defects in natural killing despite demonstrable defects in T cell function. This discrepancy implies the involvement of other tyrosine kinases. Here, using combined biochemical, pharmacologic, and genetic approaches, we demonstrate a central role for the PTK Syk in natural cytotoxicity. Biochemical analyses indicate that Syk is tyrosine phosphorylated after stimulation with a panel of NK-sensitive target cells. Pharmacologic exposure to piceatannol, a known Syk family kinase inhibitor, inhibits natural cytotoxicity. In addition, gene transfer of dominant-negative forms of Syk to NK cells inhibits natural cytotoxicity. Furthermore, sensitive targets that are rendered NK-resistant by major histocompatibility complex (MHC) class I transfection no longer activate Syk. These data suggest that Syk activation is an early and requisite signaling event in the development of natural cytotoxicity directed against a variety of cellular targets.The NK cell is a type of lymphocyte that is able to mediate natural cytotoxicity against a variety of tumor cells, virus-infected cells, and hematopoietic targets (1). However, little is known regarding the mechanisms by which these targets trigger natural cytotoxicity. In addition to natural cytotoxicity, the NK cell can mediate antibody-dependent cell-mediated cytotoxicity (ADCC)¹ through its FcR for IgG, FcγRIII. In contrast to natural cytotoxicity, ADCC is well defined with respect to the receptor–ligand interaction as well as intracellular second messenger involvement (for review see reference 2). In spite of this contrast between the well-defined FcR-dependent and the poorly defined natural cytotoxic mechanisms, both killing mechanisms can be inhibited by MHC-recognizing killer cell inhibitory receptors (KIR). Since KIR inhibition seems to target signaling pathways initiated by immunoreceptor tyrosine-based activation motif (ITAM)–containing receptor complexes (e.g., FcγRIII, TCR, and FcεRI; references 3–9), the ability of both FcR-dependent and natural cytotoxicity to be inhibited by KIR suggests that there may be shared signaling elements in the pathways used during the two alternative routes of initiating NK cell–mediated cytotoxicity.Evidence using the PTK inhibitors herbimycin A and genistein demonstrates that PTK activation is necessary for both FcR-mediated and natural cytotoxicity (10–14). However, the available genetic evidence from Lck- and Fyn-deficient mice suggests that neither of these specific Src family tyrosine kinases is necessary for NK cell–mediated cytotoxicity (15, 16). This raises the possibility that another Src family PTK (e.g., Lyn) could subserve this role. Alternatively, the receptors initiating NK cell–mediated killing could use a different kind of PTK that might function in a Src family kinase-independent manner. The Syk family member ZAP-70, which is expressed in NK cells, is unlikely to be this PTK because ZAP-70 appears to require activation by a Src family PTK (17–20); and because NK cells from humans and mice lacking ZAP-70 can mediate normal natural cytotoxicity and ADCC (21–23). In contrast, Syk itself could perform this role. Syk is expressed in NK cells (24) and evidence suggests that Syk is able to function in the absence of Src family kinases (18–20, 25). Although chimeric receptors containing the intracellular portion of ZAP-70 require cross-linking with Src family kinases such as Lck or Fyn to mediate cytotoxicity, chimeric receptors containing Syk can initiate killing in the absence of Src family PTK costimulation (18). In addition, Syk, but not ZAP-70, can induce the tyrosine phosphorylation of ITAM-containing receptor subunits in an Src family PTK-independent manner (19, 20, 25).To test the hypothesis that the Syk tyrosine kinase is functionally involved in natural cytotoxicity, we first stimulated NK cells with a panel of sensitive targets and then characterized their intracellular signaling events. Although stimulation of NK cells with sensitive targets is known to induce increases in intracellular free Ca²⁺ and inositol phosphate release (26–30), more proximal specific signaling events have not been elucidated. In this study, we describe a rapid and reversible increase in the tyrosine phosphorylation of NK cell–derived Syk after target cell stimulation. In addition, either pharmacologic inhibition of Syk kinase activity or expression of dominant-negative, kinase-inactive Syk in NK cells inhibits natural killing. Finally, tumor cells that are made resistant to NK cell–mediated cytotoxicity by MHC class I transfection no longer activate Syk. Together these data emphasize the central contribution of the Syk tyrosine kinase to generation of natural cytotoxicity.     

HIV infection--induced posttranslational modification of T cell signaling molecules associated with disease progression.

In attempt to elucidate the mechanism of the HIV infection induced T cell unresponsiveness, we studied signal-transducing molecules proximal to the T cell receptor (TCR) in T lymphocytes of HIV-infected individuals. Total amounts of protein tyrosine kinases (PTKs) Lck, Fyn, and ZAP-70 and the zeta chain of the TCR were found significantly decreased in T cells of symptomatic/AIDS patients as well as in T cells of individuals in acute and early asymptomatic stages of HIV infection. Unexpectedly, the detection of Lck, Fyn, and ZAP-70 was reversed after the treatment of cell lysates with dithiothreitol. This suggests that PTKs Lck, Fyn, and ZAP-70 were modified by a mechanism altering the status of sulfhydryl groups. Moreover, this mechanism seems to affect selectively T cells of HIV infected patients since B cell PTKs Syk and Lyn were detected structurally and functionally intact. Interestingly, similar alterations of signaling molecules were not detected in T cells of HIV-infected long-term asymptomatic individuals. Modification of T cell PTKs may thus underlie the HIV-induced impairment of lymphocyte function and may potentially predict disease progression.     

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.     

Role of the Syk autophosphorylation site and SH2 domains in B cell antigen receptor signaling

To explore the mechanism(s) by which the Syk protein tyrosine kinase participates in B cell antigen receptor (BCR) signaling, we have studied the function of various Syk mutants in B cells made Syk deficient by homologous recombination knockout. Both Syk SH2 domains were required for BCR-mediated Syk and phospholipase C (PLC)-gamma 2 phosphorylation, inositol 1,4,5-triphosphate release, and Ca2+ mobilization. A possible explanation for this requirement was provided by findings that recruitment of Syk to tyrosine-phosphorylated immunoglobulin (Ig) alpha and Ig beta requires both Syk SH2 domains. A Syk mutant in which the putative autophosphorylation site (Y518/Y519) of Syk was changed to phenylalanine was also defective in signal transduction; however, this mutation did not affect recruitment to the phosphorylated immunoreceptor family tyrosine-based activation motifs (ITAMs). These findings not only confirm that both SH2 domains are necessary for Syk binding to tyrosine-phosphorylated Ig alpha and Ig beta but indicate that this binding is necessary for Syk (Y518/519) phosphorylation after BCR ligation. This sequence of events is apparently required for coupling the BCR to most cellular protein tyrosine phosphorylation, to the phosphorylation and activation of PLC- gamma 2, and to Ca2+ mobilization.     

The Src homology 2 domain of Vav is required for its compartmentation to the plasma membrane and activation of c-Jun NH(2)-terminal kinase 1

Vav is a hematopoietic cell-specific guanine nucleotide exchange factor (GEF) whose activation is mediated by receptor engagement. The relationship of Vav localization to its function is presently unclear. We found that Vav redistributes to the plasma membrane in response to Fcin receptor I (FcinRI) engagement. The redistribution of Vav was mediated by its Src homology 2 (SH2) domain and required Syk activity. The FcinRI and Vav were found to colocalize and were recruited to glycosphingolipid-enriched microdomains (GEMs). The scaffold protein, linker for activation of T cells (LAT), and Rac1 (a target of Vav activity) were constitutively present in GEMs. Expression of an SH2 domain-containing COOH-terminal fragment of Vav inhibited Vav phosphorylation and movement to the GEMs but had no effect on the tyrosine phosphorylation of the adaptor protein, SLP-76 (SH2 domain-containing leukocyte protein of 76 kD), and LAT. However, assembly of the multiprotein complex containing these proteins was inhibited. In addition, FcinRI-dependent activation of c-Jun NH(2)-terminal kinase 1 (JNK1) was also inhibited. Thus, Vav localization to the plasma membrane is mediated by its SH2 domain and may serve to regulate downstream effectors like JNK1.     

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.     

The role of p21ras in CD28 signal transduction: triggering of CD28 with antibodies, but not the ligand B7-1, activates p21ras

CD28 is a 44-kD homodimer expressed on the surface of the majority of human T cells that provides an important costimulus for T cell activation. The biochemical basis of the CD28 accessory signals is poorly understood. Triggering of the T cell antigen receptor (TCR) activates the p21ras proteins. Here we show that ligation of CD28 by a monoclonal antibody (mAb) also stimulates p21ras and induces Ras- dependent events such as stimulation of the microtubule-associated protein (MAP) kinase ERK2 and hyperphosphorylation of Raf-1. One physiological ligand for CD28 is the molecule B7-1. In contrast to the effect of CD28 mAb, the present studies show that interactions between CD28 and B7-1 do not stimulate p21ras signaling pathways. Two substrates for TCR-regulated protein tyrosine kinases (PTKs) have been implicated in p21ras activation in T cells: p95vav and a 36-kD protein that associates with a complex of Grb2 and the Ras exchange protein Sos. Triggering CD28 with both antibodies and B7-1 activates cellular PTKs, and we have exploited the differences between antibodies and B7-1 for p21ras activation in an attempt to identify critical PTK-controlled events for Ras activation in T cells. The data show that antibodies against TCR or CD28 induce tyrosine phosphorylation of both Vav and p36. B7-1 also induces Vav tyrosine phosphorylation but has no apparent effect on tyrosine phosphorylation of the Grb2-associated p36 protein. The intensity of the Vav tyrosine phosphorylation is greater in B7-1 than in TCR-stimulated cells. Moreover the kinetics of Vav tyrosine phosphorylation is prolonged in the B7-1-stimulated cells. These studies show that for CD28 signaling, the activation of p21ras correlates more closely with p36 tyrosine phosphorylation than with Vav tyrosine phosphorylation. However, the experiments demonstrate that Vav is a major substrate for B7-activated PTKs and hence could be important in CD28 signal transduction pathway.     

A mouse with a loss-of-function mutation in the c-Cbl TKB domain shows perturbed thymocyte signaling without enhancing the activity of the ZAP-70 tyrosine kinase

The unique tyrosine kinase binding (TKB) domain of Cbl targets phosphorylated tyrosines on activated protein tyrosine kinases (PTKs); this targeting is considered essential for Cbl proteins to negatively regulate PTKs. Here, a loss-of-function mutation (G304E) in the c-Cbl TKB domain, first identified in Caenorhabditis elegans, was introduced into a mouse and its effects in thymocytes and T cells were studied. In marked contrast to the c-Cbl knockout mouse, we found no evidence of enhanced activity of the ZAP-70 PTK in thymocytes from the TKB domain mutant mouse. This finding contradicts the accepted mechanism of c-Cbl-mediated negative regulation, which requires TKB domain targeting of phosphotyrosine 292 in ZAP-70. However, the TKB domain mutant mouse does show aspects of enhanced signaling that parallel those of the c-Cbl knockout mouse, but these involve the constitutive activation of Rac and not enhanced PTK activity. Furthermore, the enhanced signaling in CD4(+)CD8(+) double positive thymocytes appears to be compensated by the selective down-regulation of CD3 on mature thymocytes and peripheral T cells from both strains of mutant c-Cbl mice.     

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.     

Different Protein Tyrosine Kinases Are Required for B Cell Antigen Receptor–mediated Activation of Extracellular Signal–Regulated kinase,c-Jun NH2-terminal Kinase 1, and p38 Mitogen-activated Protein Kinase

B cell antigen receptor (BCR) cross-linking activates three distinct families of nonreceptor protein tyrosine kinases (PTKs): src-family kinases, Syk, and Btk; these PTKs are responsible for initiating downstream events. BCR cross-linking in the chicken DT40 B cell line also activates three distinct mitogen-activated protein kinases (MAPKs): extracellular signal–regulated kinase (ERK)2, c-jun NH₂-terminal kinase (JNK)1, and p38 MAPK. To dissect the functional roles of these PTKs in MAPK signaling, activation of MAPKs was examined in various PTK-deficient DT40 cells. BCR-mediated activation of ERK2, although maintained in Lyn-deficient cells, was abolished in Syk-deficient cells and partially inhibited in Btk-deficient cells, indicating that BCR-mediated ERK2 activation requires Syk and that sustained ERK2 activation requires Btk. BCR-mediated JNK1 activation was maintained in Lyn-deficient cells but abolished in both Syk- and Btk-deficient cells, suggesting that JNK1 is activated via a Syk- and Btk-dependent pathway. Consistent with this, BCR-mediated JNK1 activation was dependent on intracellular calcium and phorbol myristate acetate–sensitive protein kinase Cs. In contrast, BCR-mediated p38 MAPK activation was detected in all three PTK-deficient cells, suggesting that no single PTK is essential. However, BCR-mediated p38 MAPK activation was abolished in Lyn/Syk double deficient cells, demonstrating that either Lyn or Syk alone may be sufficient to activate p38 MAPK. Our data show that BCR-mediated MAPK activation is regulated at the level of the PTKs.     

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.     

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.     

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.     

CD22 associates with protein tyrosine phosphatase 1C, Syk, and phospholipase C-gamma(1) upon B cell activation

Cross-linking B cell antigen receptor (BCR) elicits early signal transduction events, including activation of protein tyrosine kinases, phosphorylation of receptor components, activation of phospholipase C- gamma (PLC-gamma), and increases in intracellular free Ca2+. In this article, we report that cross-linking the BCR led to a rapid translocation of cytosolic protein tyrosine phosphatase (PTP) 1C to the particulate fraction, where it became associated with a 140-150-kD tyrosyl-phosphorylated protein. Western blotting analysis identified this 140-150-kD protein to be CD22. The association of PTP-1C with CD22 was mediated by the NH2-terminal Src homology 2 (SH2) domain of PTP-1C. Complexes of either CD22/PTP-1C/Syk/PLC-gamma(1) could be isolated from B cells stimulated by BCR engagement or a mixture of hydrogen peroxidase and sodium orthovanadate, respectively. The binding of PLC- gamma(1) and Syk to tyrosyl-phosphorylated CD22 was mediated by the NH2- terminal SH2 domain of PLC-gamma(1) and the COOH-terminal SH2 domain of Syk, respectively. These observations suggest that tyrosyl- phosphorylated CD22 may downmodulate the activity of this complex by dephosphorylation of CD22, Syk, and/or PLC-gamma(1). Transient expression of CD22 and a null mutant of PTP-1C (PTP-1CM) in COS cells resulted in an increase in tyrosyl phosphorylation of CD22 and its interaction with PTP-1CM. By contrast, CD22 was not tyrosyl phosphorylated or associated with PTP-1CM in the presence of wild-type PTP-1C. These results suggest that tyrosyl-phosphorylated CD22 may be a substrate for PTP-1C regulates tyrosyl phosphorylation of CD22.