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PubMed1999

CD28 ligation induces rapid tyrosine phosphorylation of the linker molecule LAT in the absence of Syk and ZAP-70 tyrosine phosphorylation.

CD28 is a T cell surface molecule that is important for T cell activation. CD28-triggered T cell stimulation involves protein tyrosine phosphorylation, a process that is critical for CD28 function. Recently, a linker molecule has been identified as LAT (Linker for Activation of T cells). Studies involving LAT mutants and reconstitution experiments strongly implicate LAT in playing a critical role in T cell activation. We show in the present report that CD28 ligation induces tyrosine phosphorylation of LAT. CD28-induced tyrosine phosphorylation of LAT was rapid, as it was apparent within 1 min of CD28 ligation, reached a peak by 5 min, and declined thereafter. Previous studies implicated the protein tyrosine kinases ZAP-70 and Syk in the TCR-induced tyrosine phosphorylation of LAT. Here, tyrosine phosphorylation of Syk and ZAP-70 was detected after TCR but not after CD28 ligation. Thus, CD28 ligation appears to induce tyrosine phosphorylation of LAT by mechanisms that are independent of ZAP-70 and Syk. The concurrent ligation of CD28 and TCR increased tyrosine phosphorylation of LAT. These results implicate LAT in CD28 signal transduction pathways and in the co-stimulatory process in T cells.

Adaptor Proteins, Signal Transducing↗

PubMed2005

The addition of rituximab to fludarabine improves clinical outcome in untreated patients with ZAP-70-negative chronic lymphocytic leukemia.

BACKGROUND: Clinical trials of monoclonal antibodies in combination with chemotherapy have reported previously unattained response rates in patients with B-cell chronic lymphocytic leukemia (B-CLL); however, the analysis of ZAP-70 protein and/or CD38 may explain better the discordant outcomes independent of treatment. METHODS: The authors conducted a Phase II study, in which rituximab was added to fludarabine for patients with symptomatic, untreated CLL, to evaluate clinical outcomes. Sixty patients with B-CLL received 6 monthly courses of fludarabine (25 mg/m(2) for 5 days) followed by 4 weekly doses of rituximab (375 mg/m(2)). RESULTS: On the basis of National Cancer Institute criteria, 47 of 60 patients (78%) achieved a complete remission, 9 of 60 patients (15%) achieved a partial remission, and 4 of 60 patients (7%) had no response or progressive disease. It is noteworthy that the patients experienced a long progression-free survival (PFS) from treatment (68% at 3 yrs). A significantly shorter PFS was observed in ZAP-70-positive patients (25% vs. 100% at 3 yrs; P = 0.00005), in CD38-positive patients (18% vs. 91% at 3 yrs; P = 0.0002), and in patients who had more minimal residual disease (36% vs. 77% at 2.5 yrs; P = 0.001). CONCLUSIONS: With the addition of rituximab to fludarabine, improved clinical outcomes were obtained, and the stratification of patients by using ZAP-70 and CD38 may help clinicians offer more aggressive and/or experimental approaches to the treatment of patients with high-risk B-CLL subtypes.

Adult↗

PubMed2006

Clinical implications of ZAP-70 expressionin chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is a disease with an extremely heterogeneous prognosis. Because of this and because of the lack of a truly effective therapy, treatment of CLL must be adapted to the individual risk of each patient. Over the last quarter of century, prognosis of patients with CLL has been based on clinical features. In the last few years a number of biological prognostic parameters have been identified. Among them, the expression of ZAP-70 in neoplastic B lymphocytes is increasingly being recognized as of paramount importance to predict outcome. Patients with a low percentage (<20%) of ZAP-70 positive neoplastic B lymphocytes have a much better prognosis that those with a higher proportion (>20%) of ZAP-70 positive neoplastic B lymphocytes.

Biomarkers, Tumor↗

PubMed2003

Unique T cell proliferation associated with PKCmu activation and impaired ZAP-70 phosphorylation in recognition of overexpressed HLA/partially agonistic peptide complexes.

Altered peptide ligands (APL) induce T cell responses different from those induced by the original agonistic peptide. As shown for CD4(+) T cells, partial agonists induce partial T cell activation without proliferation because of lower affinities and higher off rates to TCR than those of agonists. To determine whether overexpression of partially agonistic TCR ligands on antigen-presenting cells provides high-avidity TCR ligands, we generated L cell transfectants expressing various numbers of HLA-DR4 covalently linked with APL derived from a streptococcal peptide and observed responses of the cognate T cells. Some overexpressed HLA-DR4/partially agonistic APL complexes induced T cell proliferation in a density-dependent manner. However, tyrosine phosphorylation of zeta-associated protein-70 (ZAP-70) and linker for activation of T cells and kinase activity of ZAP-70 were not detectable. T cell proliferation stimulated with L cell transfectants was sensitive to the PKC inhibitor Gö6976, but to a lesser extent to Gö6983, suggesting the involvement of mu isotype of PKC (PKCmu). In vitro kinase assays revealed that PKCmu activity was up-regulated only in T cells stimulated with L cell transfectants that induced T cell proliferation. Our data suggest the presence of a unique signaling pathway coupling TCR ligation with T cell proliferation associated with PKCmu activation and impaired ZAP-70 activation.

Adaptor Proteins, Signal Transducing↗

PubMed1994

Cloning of the cDNA for the deleted syk kinase homologous to ZAP-70 from human basophilic leukemia cell line (KU812).

Syk kinase is one of the protein tyrosine kinases and forms a family with ZAP-70. We isolated two different sized cDNA clones of syk (Syk11 and Syk41) from a cDNA library of human KU812 (human basophilic leukemia cell line). The obtained two clones carried different messages, that Syk41 had a 69 bp-long insertion between the SH2 domain and the kinase domain compared with Syk11. Alignment of the two human syk predicted polypeptides with those of the porcine syk and ZAP-70 revealed that human syk was 5 amino acid longer than the porcine syk at the N-termini and that the insertion of the 23 amino acid found in Syk41 was present in the porcine syk and absent in ZAP-70. Reverse transcribed polymerase chain reaction targeting this region showed that both forms of the polyA RNA were expressed in Jurkat cells, human peripheral leukocytes and also KU812 cells and that the inserted form was dominant.

Amino Acid Sequence↗

PubMed1995

Expression of ZAP-70 gene in the developing thymus and various nonlymphoid tissues of embryonic and adult mice.

The full length of cDNA encoding murine ZAP-70 was obtained from a cDNA library of embryonic mouse thymus. Northern blot analysis indicated that the expression of ZAP-70 mRNA was most pronounced in the thymus and spleen and also slightly in the brain. Analysis by in situ hybridization revealed that the mRNA was expressed not only in T cells in the thymus and spleen, but also in nerve cells of the brain. In the thymus, the distribution of positive cells was different between newborn and young adult mice. In the newborn thymus, positive signal in thymocytes was localized in the subcapsular layer as well as in the medulla, while in the young adult thymus, it was localized exclusively in the medulla. Furthermore, the in situ hybridization revealed that positive signal was also seen in the liver, intestine, and lung of embryonic mice. These findings have suggested that ZAP-70 plays an important role in growth, differentiation, and function of not only T cells but also nerve cells and several embryonic tissues.

Amino Acid Sequence↗

PubMed1997

Phenotypic features of selective T cell deficiency characterized by absence of CD8+ T lymphocytes and undetectable mRNA for ZAP-70 kinase.

Selective T cell deficiency is a rare immune deficiency characterized by the absence of CD8+ T lymphocytes and depressed/absent T cell function. This syndrome has been associated with mutations in the gene for ZAP-70, a tyrosine kinase that has profound effects on signaling via the T cell receptor. In this paper we describe a patient with selective T cell deficiency and certain phenotypic features that are unique among the small number of patients described. The patient had virtually absent T cell function, hypogammaglobulinemia, and no response to vaccination. The T lymphocytes failed to respond to mitogenic stimuli, even in the presence of exogenous interleukin 2. Similar to other patients with this disorder, the T cells were capable of proliferating when stimulated by pharmacologic agents such as phorbol ester and ionomycin. While peripheral blood T cells had limited capability to increase cytosolic Ca2+ levels in response to mitogenic stimulation, thymocytes responded to a large panel of antibodies and mitogens. This report broadens the spectrum of clinical presentations associated with selective T cell deficiency and, for the first time, compares the responses of both peripheral T cells and thymocytes. The data support the concept that the defect in signal transduction resulting from the absence of ZAP-70 is primarily manifested following export of T lymphocytes from the thymus and that selection of CDS-positive T cells is dependent on the presence of ZAP-70.

CD8-Positive T-Lymphocytes↗

PubMed2003

The Polycomb-group protein ENX-2 interacts with ZAP-70.

Human ENX-2 is a homologue of Drosophila Enhancer of zeste, which is a member of Polycomb-group proteins regulating the expression of homeotic genes as chromatin-associated proteins. In this study, we demonstrate that ENX-2 plays an important role as a signaling molecule involved in T cell receptor-mediated signaling pathway. In immunoprecipitation experiments, ENX-2 and zeta associated protein-70 (ZAP-70) were co-precipitated from T cell lysate. When probed with an anti-phospho-tyrosine antibody, ENX-2 was found to be phosphorylated on tyrosine. On the other hand, ENX-2 was not phosphorylated on tyrosine in the mutant Jurkat cell, J.Cam1.6 lacking the activity of lymphocyte protein tyrosine kinase p56(lck). The interaction between ENX-2 and ZAP-70 was abolished in the mutant cell. Furthermore, in-vitro kinase assay using purified p56(lck) demonstrated that ENX-2 became tyrosine phosphorylated by this kinase. These findings show that the phosphorylation of ENX-2 is responsible for the interaction between ENX-2 and ZAP-70.

Blotting, Western↗

PubMed1999

A direct interaction between the adaptor protein Cbl-b and the kinase zap-70 induces a positive signal in T cells.

Engagement of the T-cell receptor (TCR)-CD3 complex induces a rapid increase in the activities of Src-family and Syk/Zap-70-family kinases [1] [2]. These activated kinases then induce the tyrosine phosphorylation of multiple intracellular proteins, eventually leading to T-cell activation. One of the prominent substrates for these kinases is the adaptor protein Cbl [3] and recent studies suggest that Cbl negatively regulates upstream kinases such as Syk and Zap-70 [4] [5]. Cbl-b, a homologue of Cbl, is widely expressed in many tissues and cells including hematopoietic cells [6] [7]. Cbl-b undergoes rapid tyrosine phosphorylation upon stimulation of the TCR and cytokine receptors [8] [9]. The role of Cbl-b is unclear, however. Here, we show that overexpression of Cbl-b in T cells induced the constitutive activation of the transcription factor nuclear factor of activated T cells (NFAT). A loss-of-function mutation in Cbl-b disrupted the interaction between Cbl-b and Zap-70 and nearly completely abrogated the Cbl-b-mediated activation of NFAT. Unlike the proposed role of Cbl as a negative regulator, our results suggest that the Cbl homologue Cbl-b has a positive role in T-cell signaling, most likely via a direct interaction with the upstream kinase Zap-70.

Adaptor Proteins, Signal Transducing↗

PubMed1997

Regulation of Vav-SLP-76 binding by ZAP-70 and its relevance to TCR zeta/CD3 induction of interleukin-2.

T cell activation stimulates p56(lck), p59(fyn), ZAP-70, Vav-SLP-76 binding, and IL-2 transcription. Major questions concern the tyrosine-kinase and relevant site(s) needed for Vav-SLP-76 complex formation and its role in IL-2 production. Here, we show that of the three kinases, only ZAP-70 phosphorylates SLP-76 at specific sites that allow Vav SH2 domain binding. Therefore, while p56(lck) regulates proximal events, ZAP-70 acts downstream on targets such as SLP-76. We also show by in vitro and in vivo analysis that two SLP-76 pYESP motifs (Y113 and Y128) mediate binding, the first being more efficient. A third pYEPP motif (Y145) failed to bind. Finally, TCR zeta CD3 ligation of T cell hybridoma DC27.10 induces IL-2 production without detectable Vav-SLP-76 binding. Therefore, despite effects of Vav-SLP-76 on IL-2 expression, Vav-SLP-76 binding per se is not essential for IL-2 production in all T cells.

Adaptor Proteins, Signal Transducing↗

PubMed2002

Crystal structure and NMR studies of the apo SH2 domains of ZAP-70: two bikes rather than a tandem.

The protein kinase ZAP-70 is involved in T-cell activation, and interacts with tyrosine-phosphorylated peptide sequences known as immunoreceptor tyrosine activation motifs (ITAMs), which are present in three of the subunits of the T-cell receptor. We have studied the tandem SH2 (tSH2) domains of ZAP-70, by both X-ray and NMR. Here, we present the crystal structure of the apoprotein, i.e., the tSH2 domain in the absence of ITAM. Comparison with the previously reported complex structure reveals that binding to the ITAM peptide induces surprisingly large movements between the two SH2 domains and within the actual binding sites. The conformation of the ITAM-free protein is partly governed by a hydrophobic cluster between the linker region and the C-terminal SH2 domain. Our data suggest that the two SH2 domains are able to undergo large interdomain movements. The proposed relative flexibility of the SH2 domains is further supported by the finding that no NMR signals could be detected for the two helices connecting the SH2 domains; these are likely to be broadened beyond detection due to conformational exchange. It is likely that this conformational reorientation induced by ITAM binding is the main signaling event activating the kinase domain in ZAP-70. Another NMR observation was that the N-terminal SH2 domain could bind tetrapeptides derived from the ITAM sequence, apparently without the need to interact with the C-terminal domain. In contrast, the C-terminal domain has little affinity for tetrapeptides. The opposite situation is true for binding to plain phosphotyrosine, where the C-terminal domain has a higher affinity. Distinct features in the crystal structure, showing the interdependence of both domains, explain these binding data.

Amino Acid Motifs↗

PubMed1999

Structure of the amino-terminal domain of Cbl complexed to its binding site on ZAP-70 kinase.

Cbl is an adaptor protein that functions as a negative regulator of many signalling pathways that start from receptors at the cell surface. The evolutionarily conserved amino-terminal region of Cbl (Cbl-N) binds to phosphorylated tyrosine residues and has cell-transforming activity. Point mutations in Cbl that disrupt its recognition of phosphotyrosine also interfere with its negative regulatory function and, in the case of v-cbl, with its oncogenic potential. In T cells, Cbl-N binds to the tyrosine-phosphorylated inhibitory site of the protein tyrosine kinase ZAP-70. Here we describe the crystal structure of Cbl-N, both alone and in complex with a phosphopeptide that represents its binding site in ZAP-70. The structures show that Cbl-N is composed of three interacting domains: a four-helix bundle (4H), an EF-hand calcium-binding domain, and a divergent SH2 domain that was not recognizable from the amino-acid sequence of the protein. The calcium-bound EF hand wedges between the 4H and SH2 domains and roughly determines their relative orientation. In the ligand-occupied structure, the 4H domain packs against the SH2 domain and completes its phosphotyrosine-recognition pocket. Disruption of this binding to ZAP-70 as a result of structure-based mutations in the 4H, EF-hand and SH2 domains confirms that the three domains together form an integrated phosphoprotein-recognition module.

Amino Acid Sequence↗

PubMed1998

Trichosanthin inhibits T cell activation by interfering with the recruitment of ZAP-70 to CD3 zeta chain.

Plant protein Trichosanthin (Tk) has been shown in our previous experiments to suppress antigenic response of T cells. Here we explored its inhibitory mechanisms on the proliferation of human Jurkat leukemia T cell triggered by anti-CD3 McAb. By examination of tyrosine phosphorylation of cell lysate, we were able to show that Tk could interfere with the PTK-related activity in the TCR/CD3-initiated signal transduction in addition to blocking the phosphorylation of PKC. As shown in our experiment, the expression intensity of ZAP-70, a kind of protein tyrosine kinase, was not changed but its phosphorylation could be inhibited. When physical link between CD3 zeta chain and ZAP-70 was further examined by using coimmunoprecipitation after pluse-treatment of the cell line with Tk, the anti-CD3 McAb-induced recruitment of ZAP-70 to CD3 zeta chain was observed to be blocked in some extent. This may account for, at least in part, how Trichosanthin was able to inhibit the TCR-triggered T cell proliferation.

Antibodies, Monoclonal↗

PubMed2002

Natural cytotoxicity uncoupled from the Syk and ZAP-70 intracellular kinases.

The intracellular signals that trigger natural cytotoxicity have not been clearly determined. The Syk and ZAP-70 tyrosine kinases are essential for cellular activation initiated by B and T cell antigen receptors and may drive natural killer (NK) cell cytotoxicity via receptors bearing immunoreceptor tyrosine-based activation motifs (ITAMs). However, we found that, unlike B and T cells, NK cells developed in Syk-/-ZAP-70-/- mice and, despite their nonfunctional ITAMs, lysed various tumor targets in vitro and eliminated tumor cells in vivo, including those without NKG2D ligands. The simultaneous inhibition of phosphatidyl inositol 3 kinase and Src kinases abrogated the cytolytic activity of Syk-/-ZAP-70-/- NK cells and strongly reduced that of wild-type NK cells. This suggests that distinct and redundant signaling pathways act synergistically to trigger natural cytotoxicity.

Animals↗

PubMed2004

Engagement of the B-cell antigen receptor (BCR) allows efficient transduction of ZAP-70-positive primary B-CLL cells by recombinant adeno-associated virus (rAAV) vectors.

Engagement of the B-cell antigen receptor (BCR) by crosslinking of the surface immunoglobulin (sIg) homodimer was studied for recombinant adeno-associated virus (rAAV)-mediated gene transfer into B-cell chronic lymphocytic leukaemia (B-CLL) cells. Leukemic cells obtained from 20 patients were stimulated with anti-sIg-directed antibodies and transduced with rAAV vectors coding for enhanced green fluorescent protein (EGFP) (AAV/EGFP) or CD40L (AAV/CD40L). Transduction of B-CLL cells was enhanced after BCR engagement compared to unstimulated controls (P=0.0356). BCR crosslinking induced a significant, dose- and time-dependent upregulation of heparan sulfate proteoglycan (HSPG), the primary receptor for AAV, on B-CLL cells (mean: 38.2 versus 1.7%; P=0.0006). A correlation of HSPG expression after BCR crosslinking with transduction efficiency by AAV/EGFP (P=0.0153) and AAV/CD40L (P=0.0347) was observed. High expression of zeta-associated protein 70 (ZAP-70) in B-CLL cells correlated with a better transduction efficiency by AAV/EGFP (P<0.0001) and AAV/CD40L (P=0.002), respectively: 48 h after transduction of ZAP-70-positive samples, transgene expression was seen in a mean of 33.8% (s.e.m. 3.7%) and 28.9% (s.e.m. 6.7%) of cells, respectively, and could be specifically blocked by heparin, a soluble competitor of HSPG (P<0.0001). In summary, engagement of the BCR on ZAP-70 positive B-CLL cells allows efficient rAAV-mediated gene delivery.

CD40 Ligand↗

PubMed2001

Tyrosine phosphorylation-dependent activation of NF-kappa B. Requirement for p56 LCK and ZAP-70 protein tyrosine kinases.

Phosphorylation of the N-terminal domain of I kappa B inhibitory subunits induces activation of the transcription factor NF-kappa B. Although serine phosphorylation has been shown to induce ubiquitination and subsequent proteasome-mediated degradation of I kappa B-alpha, little is known about the mechanisms that lead to release of active NF-kappa B in T cells as a consequence of tyrosine phosphorylation of I kappa B-alpha [Imbert, V., Rupec, R.A., Livolsi, A., Pahl, H.L., Traenckner, B.M., Mueller-Dieckmann, C., Farahifar, D., Rossi, B., Auberger, P., Baeuerle, P. & Peyron, J.F. (1996) Cell 86, 787--798]. The involvement of the tyrosine kinases p56(lck) and ZAP-70 in this reaction is demonstrated here using specific pharmacological inhibitors and Jurkat mutants unable to express these kinases. Although the inhibitors prevented both pervanadate-induced phosphorylation of I kappa B-alpha on Tyr42 and NF-kappa B activation, we observed that, in p56(lck)-deficient Jurkat mutants, NF-kappa B could still associate with I kappa B-alpha despite phosphorylation on Tyr42. Furthermore, the SH2 domain of p56(lck) appeared to be required for pervanadate-induced NF-kappa B activation but not for Tyr42 phosphorylation. These results show that p56(lck) and ZAP-70 are key components of the signaling pathway that leads to phosphotyrosine-dependent NF-kappa B activation in T cells and confirm that tyrosine kinases must control at least two different steps to induce activation of NF-kappa B. Finally, we show that H(2)O(2), which stimulates p56(lck) and ZAP-70 in T cells, is an activator of NF-kappa B through tyrosine phosphorylation of I kappa B-alpha.

DNA↗

PubMed1995

A comparison of the interaction of Shc and the tyrosine kinase ZAP-70 with the T cell antigen receptor zeta chain tyrosine-based activation motif.

Tyrosine-based activation motifs (TAMs) define a conserved signaling sequence, EX2YX2L/IX7YX2L/I, that couples the T cell antigen receptor to protein tyrosine kinases and adapter molecules. The present study shows that phosphorylation of both tyrosines within the motif is required for high affinity binding of the tyrosine kinase ZAP-70 whereas phosphorylation of the single COOH-terminal tyrosine within the motif is optimal for the binding of the adapter Shc. There were also quantitative differences in the ZAP-70 and Shc association with the zeta 1-TAM since nM concentrations of the doubly phosphorylated zeta 1-TAM are sufficient for ZAP-70 recruitment whereas micromolar levels of singly phosphorylated TAMs are necessary for Shc binding. Shc is tyrosine phosphorylated in antigen receptor-activated T cells and can potentially form a complex with the adapter molecule Grb2 and could thus recruit the Ras guanine nucleotide exchange protein Sos into the antigen receptor complex. The present data show that Grb2 can bind to the phosphorylated TAM, but this binding is independent of Shc and there is no formation of zeta 1-TAM.Shc.Grb2.Sos complexes in antigen receptor-activated cells. Accordingly, Shc function should not be considered in the context of Grb2/Sos recruitment to the T cell antigen receptor complex.

Adaptor Proteins, Signal Transducing↗

PubMed2000

ZAP-70 is essential for the T cell antigen receptor-induced plasma membrane targeting of SOS and Vav in T cells.

Translocation of the SOS and Vav GDP/GTP exchange factors proximal to Ras and Rac GTPases localized in the plasma membrane glycolipid-enriched microdomains is a pivotal step required for T cell antigen receptor-induced T cell activation. Here we demonstrate that the T cell antigen receptor zeta-chain-associated ZAP-70 kinase and T cell antigen receptor zeta-chain immunoreceptor tyrosine-based activation motifs are essential for the membrane recruitment of SOS and Vav. Plasma membrane targeting of SOS or Vav begins with the assembly of ZAP-70 with Grb-2 and SOS. The subsequent tyrosine phosphorylation of LAT (linker for activation of T cell) by ZAP-70 leads to a shift in equilibrium from the ZAP-70.Grb-2.SOS(Vav) complex to the (Vav)SOS.Grb-2.LAT complex. This shift results in the targeting of SOS and Vav into glycolipid-enriched microdomains and initiation of the Ras and Rac signaling cascades involved in T cell activation, proliferation, and cytokine production.

Adaptor Proteins, Signal Transducing↗