Interactions of p59fyn and ZAP-70 with T-cell receptor activation motifs: defining the nature of a signalling motif.

The tyrosine-based activation motif is a 20- to 25-amino-acid sequence contained in the cytoplasmic domains of many hematopoietic receptors which is sufficient by itself to reconstitute signalling. This motif is characterized by two YXXL/I sequences separated by approximately 10 residues. The molecular basis of signalling by this motif is unknown. Here we demonstrate that the tyrosine-based activation motif is required and sufficient for association with the tyrosine kinases p59fyn and ZAP-70, suggesting that association with these kinases is a general feature of this motif. Focusing on the single activation motif present in epsilon, we analyzed which residues of the motif were critical for binding of p59fyn and ZAP-70. Surprisingly, we found that no single mutation of any residue of epsilon resulted in the loss of p59fyn association. In contrast, single mutations at five residues of the epsilon activating motif abrogated ZAP-70 binding. Both of the tyrosines and the leucine or isoleucine residues that follow them were critical. The spacing between the tyrosines was also important, as deletion of two residues disrupted binding of ZAP-70, although p59fyn binding was not disrupted. Most of the defined features of the tyrosine activation motif are therefore requirements for ZAP-70 binding. Interestingly, the interaction of ZAP-70 with the motif was dependent on the presence of both ZAP-70 SH2 domains and both of the tyrosine residues in the motif, suggesting that ZAP-70 interacts with two phosphotyrosine residues and that the binding of the two SH2 domains is cooperative. In addition, we demonstrate that the interaction between the tyrosine activation motif is direct and requires prior tyrosine phosphorylation of the motif. We propose that the activation of cells by the tyrosine activating motif occurs in four discrete steps: binding of p59fyn, phosphorylation of the motif, binding of ZAP-70, and activation of ZAP-70 kinase activity.     

Structural basis for the inhibition of tyrosine kinase activity of ZAP-70

ZAP-70, a cytoplasmic tyrosine kinase required for T cell antigen receptor signaling, is controlled by a regulatory segment that includes a tandem SH2 unit responsible for binding to immunoreceptor tyrosine-based activation motifs (ITAMs). The crystal structure of autoinhibited ZAP-70 reveals that the inactive kinase domain adopts a conformation similar to that of cyclin-dependent kinases and Src kinases. The autoinhibitory mechanism of ZAP-70 is, however, distinct and involves interactions between the regulatory segment and the hinge region of the kinase domain that reduce its flexibility. Two tyrosine residues in the SH2-kinase linker that activate ZAP-70 when phosphorylated are involved in aromatic-aromatic interactions that connect the linker to the kinase domain. These interactions are inconsistent with ITAM binding, suggesting that destabilization of this autoinhibited ZAP-70 conformation is the first step in kinase activation.     

Rituximab-induced direct inhibition of T-cell activation

Background

Rituximab, an anti-CD20 monoclonal antibody, is reported to increase the T-cell-dependent infection risk. The current study was designed to investigate whether rituximab interferes with T-cell activation.

Patients and methods

Patients with non-Hodgkin lymphoma receiving 4–6 courses of 375?mg/m² rituximab underwent detailed assessment of T-cell activation pre- and post-rituximab. A similar analysis assessed the in vitro effect of rituximab on T-cell activation in response to allogeneic dendritic cells (allo-DCs) and other stimuli.

Results

Patients receiving rituximab exhibited a significant decline in IL-2 and IFN-γ levels in peripheral blood, most prominent after repeated rituximab courses. Evaluation at 3?months after rituximab therapy showed restoration of inflammatory cytokine production. Similarly, in vitro stimulation of peripheral blood mononuclear cells in the presence of rituximab resulted in a significant decrease in T-cell activation markers, inflammatory cytokine production and proliferative capacity. These effects were also observed using B-cell-depleted T cells (CD3⁺CD25^?CD19^?) and were accompanied with disappearance of CD3⁺CD20^dim T-cell population.

Conclusion

Rituximab administration results in transient, dose-dependent T-cell inactivation. This effect is obtained even in B-cell absence and may increase the infection risk.     

Enhancement of lymphocyte responsiveness by a gain-of-function mutation of ZAP-70.

The protein tyrosine kinase ZAP-70 plays an essential role in T-cell activation and development. After T-cell receptor stimulation, ZAP-70 is associated with the receptor and is phosphorylated on many tyrosine residues, including tyrosine 292 (Y-292), in the region between the C-terminal SH2 domain and the kinase domain (interdomain B). Here we show that a mutation of Y-292 (292F) or deletion of interdomain B enhanced the ability of ZAP-70 to reconstitute B-cell receptor stimulation-dependent NF-AT induction in a B-cell line deficient in Syk. In contrast, in a T-cell line, expression of 292F led to basal NF-AT induction independent of T-cell receptor stimulation. These results demonstrate that the role of Y-292 is to negatively regulate the function of ZAP-70 in lymphocytes. This appears to be a dominant function of interdomain B because deletion of most of interdomain B also resulted in a mutant of ZAP-70 with enhanced ability to reconstitute Syk-deficient DT-40 B cells. Since our biochemical studies did not reveal an effect of the 292F mutation on either the kinase activity of ZAP-70 or on the ability of ZAP-70 to bind to the receptor, we propose a model in which Y-292 interacts with an inhibitory protein to negatively regulate ZAP-70 function.     

The kinase, SH3, and SH2 domains of Lck play critical roles in T-cell activation after ZAP-70 membrane localization.

Antigenic stimulation of the T-cell antigen receptor initiates signal transduction through the immunoreceptor tyrosine-based activation motifs (ITAMs). When its two tyrosines are phosphorylated, ITAM forms a binding site for ZAP-70, one of the cytoplasmic protein tyrosine kinases essential for T-cell activation. The signaling process that follows ZAP-70 binding to ITAM has been analyzed by the construction of fusion proteins that localize ZAP-70 to the plasma membrane. We found that membrane-localized forms of ZAP-70 induce late signaling events such as activation of nuclear factor of activated T cells without any stimulation. This activity was observed only when Lck was expressed and functional. In addition, each mutation that affects the function of Lck in the kinase, Src homology 2 (SH2), and SH3 domains greatly impaired the signaling ability of the chimeric protein. Therefore, Lck functions in multiple manners in T-cell activation for the steps following ZAP-70 binding to ITAM.     

Synergistic activation of eIF4A by eIF4B and eIF4G

eIF4A is a key component in eukaryotic translation initiation; however, it has not been clear how auxiliary factors like eIF4B and eIF4G stimulate eIF4A and how this contributes to the initiation process. Based on results from isothermal titration calorimetry, we propose a two-site model for eIF4A binding to an 83.5 kDa eIF4G fragment (eIF4G-MC), with a high- and a low-affinity site, having binding constants K_D of ∼50 and ∼1000 nM, respectively. Small angle X-ray scattering analysis shows that the eIF4G-MC fragment adopts an elongated, well-defined structure with a maximum dimension of 220 Å, able to span the width of the 40S ribosomal subunit. We establish a stable eIF4A–eIF4B complex requiring RNA, nucleotide and the eIF4G-MC fragment, using an in vitro RNA pull-down assay. The eIF4G-MC fragment does not stably associate with the eIF4A–eIF4B–RNA-nucleotide complex but acts catalytically in its formation. Furthermore, we demonstrate that eIF4B and eIF4G-MC act synergistically in stimulating the ATPase activity of eIF4A.     

A role of kinase inactive ZAP-70 in altered peptide ligand stimulated T cell activation

T cell activation signals induced by altered peptide ligands (APLs) are different from those induced by the original agonistic peptide. The characteristics of the former are partial phosphorylation of TCR-zeta and no tyrosine-phosphorylation of zeta-associated protein-70 (ZAP-70). To analyze further those signaling pathways, we introduced a dominant negative (DN) form of ZAP-70 into a human CD4(+) T cell clone in which fully and partially agonistic peptide ligands have been well characterized. We found that some over-expressed partially agonistic ligands (OPALs) induced T cell responses without tyrosine-phosphorylation and kinase activation of ZAP-70. However, those responses were inhibited in T cells expressing DN ZAP-70, which could associate with partially phosphorylated TCR-zeta. In OPAL-stimulated T cells, PLC-gamma1 was phosphorylated and it was suppressed by DN ZAP-70 expression, suggesting that the ZAP-70-TCR-zeta association mediates the activation of PLC-gamma1 leading to T cell responses even in the absence of kinase activation of ZAP-70.     

ZAP-70 restoration in mice by in vivo thymic electroporation

Viral and non-viral vectors have been developed for gene therapy, but their use is associated with unresolved problems of efficacy and safety. Efficient and safe methods of DNA delivery need to be found for medical application. Here we report a new monopolar system of non-viral electro-gene transfer into the thymus in vivo that consists of the local application of electrical pulses after the introduction of the DNA. We assessed the proof of concept of this approach by correcting ZAP-70 deficient severe combined immunodeficiency (SCID) in mice. The thymic electro-gene transfer of the pCMV-ZAP-70-IRES-EGFP vector in these mice resulted in rapid T cell differentiation in the thymus with mature lymphocytes detected by three weeks in secondary lymphoid organs. Moreover, this system resulted in the generation of long-term functional T lymphocytes. Peripheral reconstituted T cells displayed a diversified T cell receptor (TCR) repertoire, and were responsive to alloantigens in vivo. This process applied to the thymus could represent a simplified and effective alternative for gene therapy of T cell immunodeficiencies.     

Distinct tyrosine phosphorylation sites in ZAP-70 mediate activation and negative regulation of antigen receptor function.

Biochemical and genetic evidence has implicated two families of protein tyrosine kinases (PTKs), the Src- and Syk-PTKs, in T- and B-cell antigen receptor signaling. ZAP-70 is a member of the Syk-PTKs that associates with the T-cell antigen receptor and undergoes tyrosine phosphorylation following receptor activation. Three tyrosine residues, Tyr-292, -492, and -493, have been identified as sites of phosphorylation following T-cell antigen receptor engagement. Utilizing ZAP-70- and Syk-deficient lymphocytes (Syk-DT40 cells), we provide biochemical and functional evidence that heterologous trans-phosphorylation of Tyr-493 by a Src-PTK is required for antigen receptor-mediated activation of both the calcium and ras pathways. In contrast, cells expressing mutations at Tyr-292 or -492 demonstrate hyperactive T- and B-cell antigen receptor phenotypes. Thus, phosphorylation of ZAP-70 mediates both activation and inactivation of antigen receptor signaling.     

Differential requirement of ZAP-70 for CD2-mediated activation pathways of mature human T cells

This study addresses the role of the tyrosine kinase ZAP-70 in CD2-mediated T cell activation. Patients lacking ZAP-70 have few mature CD8+ T cells and high numbers of CD4+ T cells that are nonfunctional upon TCR triggering. Such a patient with a homozygous deletion in the zap-70 gene that resulted in the complete absence of ZAP-70 protein expression has been identified. Expression of the tyrosine kinases Lck, Fyn, and Syk was normal. The patient's T cells were activated with two different pairs of mitogenic mAbs. CD2-induced phosphorylation of the zeta-chain and influx of Ca2+ was defective in the ZAP-70-deficient T cells, whereas CD2-induced phosphorylation of several other proteins, including Syk, was not affected. CD2-induced proliferation as well as production of TNF-alpha and IFN-gamma was abrogated in ZAP-70-deficient T cells, whereas PMA plus ionomycin induced normal activation of these cells. Together, this study shows that CD2-activation triggers ZAP-70-dependent and -independent pathways. Deletion of ZAP-70 affected CD2- and CD3-mediated proliferation and cytokine production in a similar way, suggesting that one of the different CD2 pathways converges with a CD3 pathway at or upstream of the activation of ZAP-70.     

Herpesvirus saimiri replaces ZAP-70 for CD3- and CD2-mediated T cell activation.

The protein tyrosine kinase ZAP-70 plays a pivotal role involved in signal transduction through the T cell receptor and CD2. Defects in ZAP-70 result in severe combined immunodeficiency. We report that Herpesvirus saimiri, which does not code for a ZAP-70 homologue, can replace this tyrosine kinase. H. saimiri is an oncogenic virus that transforms human T cells to stable growth based on mutual CD2-mediated activation. Although CD2-mediated proliferation of ZAP-70-deficient uninfected T cells was absent, we could establish H. saimiri-transformed T cell lines from two unrelated patients presenting with ZAP-70 deficiencies. In these cell lines, CD2 and CD3 activation were restored in terms of [Ca(2+)](i), MAPK activation, cytokine production, and proliferation. Activation-induced tyrosine phosphorylation of zeta remained defective. The transformed cells expressed very high levels of the ZAP-70-related kinase Syk. This increased expression was not observed in the primary T cells from the patients and was not due to the transformation by the virus because transformed cell lines established from control T cells did not present this particularity. In conclusion, wild type H. saimiri can restore CD2- and CD3-mediated activation in signaling-deficient human T cells. It extends our understanding of interactions between the oncogenic H. saimiri and the infected host cells.     

Analysis of CD3-antibody-mediated inhibition of T-cell activation

In this study the influence of a non-mitogenic anti-CD3 antibody on accessory cell-dependent antigen and mitogen-induced T-lymphocyte proliferation has been investigated. The antibody was found to completely inhibit PHA, Con A, PWM, and tetanus toxoid stimulation, with no effect on the proliferation induced by the calcium ionophore A23187. VIT3 completely abrogated the production of IL-2 by lectin-stimulated T cells. It had no effect, however, on the IL-2-dependent proliferation of preactivated T-cell blasts. In addition, the antibody was able to elevate free cytoplasmic Ca2+ levels within minutes after the addition to T cells. Detailed time kinetic analyses revealed that the time interval critical for inhibition was significantly dependent on the interaction between T cells and accessory cells. Under standard conditions, in the presence of 10% non-T cells as accessory cells 50% inhibition was still achieved when VIT3 was added to PHA-stimulated T cells as late as 8 hr after the onset of culture. Delayed addition or a decrease in the number of added accessory cells significantly prolonged this time period. Lectin-stimulated T cells can thus obviously be inhibited via CD3 as long as they have not received all signals including those delivered by accessory cells. Although the underlying mechanisms are not clear so far, the observation that VIT3 at the same time triggers an early cytoplasmic Ca2+ response might indicate that it thereby actively interferes with antigen and lectin-initiated activation processes.     

Identification of a novel isoform of ZAP-70, truncated ZAP kinase

We identified a novel cDNA encoding truncated ZAP-70, which lacked the SH2 domain and a part of interdomain B, and named it truncated ZAP kinase (TZK). TZK was expressed in the thymus, spleen, and lymph nodes with ZAP-70. TZK was expressed in CD44⁺CD25⁻ thymocytes up to mature T cells, but ZAP-70 was not expressed in CD44⁺CD25⁻ or CD44⁺CD25⁺ thymocytes. ZAP-70 or TZK was transfected into P116 cells derived from a Jurkat T-cell line deficient in ZAP-70. The P116 cells with ZAP-70 induced the T-cell receptor-mediated signal transduction, but the cells expressing TZK did not. While ZAP-70 was accumulated at the immune synapse, TZK was not. Meanwhile, impaired phosphorylation of SLP-76, one of the substrates of ZAP-70, in P116 cells upon pervanadate stimulation was rescued in the cells expressing TZK. These findings show that TZK is a novel isoform of ZAP-70, which is expressed in pre-T-cell receptor-minus thymocytes and functions as a kinase not associated with T-cell receptor.     

TCR/CD3 down-modulation and zeta degradation are regulated by ZAP-70

TCR down-modulation following binding to MHC/peptide complexes is considered to be instrumental for T cell activation because it allows serial triggering of receptors and the desensitization of stimulated cells. We studied CD3/TCR down-modulation and zeta degradation in T cells from two ZAP-70-immunodeficient patients. We show that, at high occupancy of the TCR, down-modulation of the CD3/TCR is comparable whether T cells express or do not express ZAP-70. However, if TCR occupancy was low, we found that CD3/TCR was down-regulated to a lesser extent in ZAP-70-negative than in ZAP-70-positive T cells. We studied CD3/TCR down-modulation in P116 (a ZAP-70-negative Jurkat cell-derived clone) and in P116 transfected with genes encoding the wild-type or a kinase-dead form of ZAP-70. Down-modulation of the TCR at high occupancy did not require ZAP-70, whereas at low TCR occupancy down-modulation was markedly reduced in the absence of ZAP-70 and in cells expressing a dead kinase mutant of ZAP-70. Thus, the presence of ZAP-70 alone is not sufficient for down-modulation; the kinase activity of this molecule is also required. The degradation of zeta induced by TCR triggering is also severely impaired in T cells from ZAP-70-deficient patients, P116 cells, and P116 cells expressing a kinase-dead form of ZAP-70. This defect in TCR-induced zeta degradation is observed at low and high levels of TCR occupancy. Our results identify ZAP-70, a tyrosine kinase known to be crucial for T cell activation, as a key player in TCR down-modulation and zeta degradation.     

Immune synapse formation requires ZAP-70 recruitment by ezrin and CD43 removal by moesin

Immunological synapse (IS) formation involves receptor–ligand pair clustering and intracellular signaling molecule recruitment with a coincident removal of other membrane proteins away from the IS. As microfilament–membrane linkage is critical to this process, we investigated the involvement of ezrin and moesin, the two ezrin/radixin/moesin proteins expressed in T cells. We demonstrate that ezrin and moesin, which are generally believed to be functionally redundant, are differentially localized and have important and complementary functions in IS formation. Specifically, we find that ezrin directly interacts with and recruits the signaling kinase ZAP-70 to the IS. Furthermore, the activation of ezrin by phosphorylation is essential for this process. In contrast, moesin dephosphorylation and removal, along with CD43, are necessary to prepare a region of the cell cortex for IS. Thus, ezrin and moesin have distinct and critical functions in the T cell cortex during IS formation.