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PubMedDate not supplied

[Primary immunodeficiency secondary to ZAP-70 deficiency].

The authors present the case of a child with recurrent infections since the age of 4 months, including bilateral pneumonia by Pneumocystis carinii and protracted varicella. Serum immunoglobulin values (when 10 months old), and B cell values were normal. There was persistent lymphocytic leucocytosis, near absence of CD8+ cells, and an increased CD4/CD8 ratio. The percentage of activated T cells and the expression of HLA class I were normal. Proliferation, activation and IL-2 synthesis studies in T cells showed a TCR/CD3-associated signal transduction deficit. ZAP-70 cDNA sequencing showed a mutation, and no ZAP-70 protein was detected in T cells. ZAP-70 deficiency is associated with a rare immune deficiency with absence of CD8+ T cells as well as a functional deficiency in T cells. Seven months after bone marrow transplantation the child is clinically well and immunologically recovered.

Cytoplasmic Granules↗
PubMed1998

Roles of Lck, Syk and ZAP-70 tyrosine kinases in TCR-mediated phosphorylation of the adapter protein Shc.

The adapter protein Shc has been implicated in mitogenic signaling via growth factor receptors, antigen receptors and cytokine receptors. Recent studies have suggested that tyrosine phosphorylation of Shc may play a key role in T lymphocyte proliferation via interaction of phosphorylated Shc with downstream molecules involved in activation of Ras and Myc proteins. However, the sites on Shc that are tyrosine phosphorylated in response to TCR engagement and the ability of different T cell tyrosine kinases to phosphorylate Shc have not been defined. In this report, we show that during TCR signaling, the tyrosines Y239, Y240 and Y317 of Shc are the primary sites of tyrosine phosphorylation. Mutation of all three tyrosines completely abolished tyrosine phosphorylation of Shc following TCR stimulation. Our data also suggest that multiple T cell tyrosine kinases contribute to tyrosine phosphorylation on Shc. In T cells, CD4/Lck-dependent tyrosine phosphorylation on Shc was markedly diminished when Y317 was mutated, suggesting a preference of Lck for the Y317 site. The syk-family kinases (Syk and ZAP-70) were able to phosphorylate the Y239 and Y240 sites, and less efficiently the Y317 site. Moreover, co-expression of Syk or ZAP-70 with Lck resulted in enhanced phosphorylation of Shc on all three sites, suggesting a synergy between the syk-family and scr-family kinases. Of the two potential Grb2 binding sites (Y239 and Y317), Y239 appears to play a greater role in recruiting Sos through Grb2. These studies have implications for Ras activation and mitogenic signaling during T cell activation.

Adaptor Proteins, Signal Transducing↗
PubMed2007

National standardization of ZAP-70 determination by flow cytometry: the French experience.

BACKGROUND: ZAP-70, after being considered as a potential surrogate for VH mutational status, has seen its own prognostic value emerge. We aimed at standardizing a simple, fast, and reproducible flow cytometry method. METHODS: AntiZAP-70 antibody 2F3.2 was used with indirect labeling and secondary anti-IgG2a antibody. The reference values for the expression of the results were determined on 45 normal blood samples. ZAP-70 protein expression was investigated in 192 CLL samples. The indirect technique was compared with FITC-conjugated 2F3.2 clone, and with clone 1E7.2-FITC, -PE or -AlexaFluor 488. RESULTS: Using FITC or PE-conjugated antibodies, 2F3.2 and 1E7.2 clones allowed a much less adequate discrimination between positive and negative cells and discordant cases were most likely true negative cases. Using the AlexaFluor 488 conjugated 1E7.2 clone, the discordant cases were mostly negative with the conjugated antibody and positive with the 2F3.2 clone but Western blotting or RNA microarray confirmed discordant cases were false negative with the conjugated antibody. Subsequently, recommendations were used by 13 centers participating in an interlaboratory quality control protocol. The use of MFI ratio appeared to be more reliable. CONCLUSIONS: Results suggested that slight differences in the procedure had little impact on the interpretation in characteristic cases; however, careful interpretation was required for values close to threshold.

Antibodies↗
PubMed1995

Severe combined immunodeficiency with absence of peripheral blood CD8+ T cells due to ZAP-70 deficiency.

During ontogeny, T lymphocytes are selected for CD4 or CD8 expression in part by their ability to signal properly through the TCR. Transmission of such signals requires the activation of specific cytoplasmic protein tyrosine kinases (PTKs) which lead to T-cell activation through poorly understood mechanisms. Recently, mutations in one such PTK, called ZAP-70, have been shown to be responsible for a rare, autosomal recessive form of severe combined immunodeficiency (SCID) in humans. This distinctive SCID syndrome is characterized by the selective absence of peripheral CD8+ T cells and by abundant circulating CD4+ T cells that fail to respond to TCR-mediated stimuli in vitro. In this report, we describe in detail the clinical and laboratory findings in one patient with ZAP-70 deficiency and discuss the insights provided by this disorder into the pathways of TCR signal transduction and T-cell development.

B-Lymphocytes↗
PubMed2005

Hodgkin's disease variant of Richter's syndrome clonally related to chronic lymphocytic leukemia arises in ZAP-70 negative mutated CLL.

Chronic lymphocytic leukemia (CLL) may derive from either immunoglobulin V(H) gene unmutated (naïve) or mutated (antigen-experienced) post-germinal center B-cells. Richter's syndrome denotes the transformation of CLL into aggressive B-cell lymphoma. Most Richter's syndrome cases are secondary diffuse large B-cell lymphomas, but some are Hodgkin's disease variants. Hodgkin's lymphoma is thought to originate from germinal center or post-germinal center B-cells with evidence of somatic V(H) hypermutation. Taking into account CLL and Hodgkin's lymphoma histogenesis, hypothetically only CLL derived from V(H) mutated B-cells can clonally progress to Hodgkin's lymphoma variant of Richter's syndrome. To test our hypothesis, we analyzed the CLL ZAP-70 status as a surrogate for the V(H) mutational status in four patients with subsequent Hodgkin's disease variants of Richter's syndrome. In all three cases with proven or suspected clonal relationship between Hodgkin and Reed-Sternberg- and leukemia cells, the CLL samples remained negative for ZAP-70, corresponding to CLL histogenesis from V(H) mutated B-cells. These empirical data suggest that the histological and clinical diversity of Richter's transformation could be to a part explained by the different origin of CLL, with Hodgkin's disease variants arising probably only in V(H) mutated CLL.

Cell Transformation, Neoplastic↗
PubMed1998

Effect of age on mitogen induced protein tyrosine phosphorylation in human T cell and its subsets: down-regulation of tyrosine phosphorylation of ZAP-70.

Several events of T cell activation have been reported to decline in humans with age. Since protein tyrosine phosphorylation is an early critical event of T cell activation, we performed a systematic analysis of the age-associated changes in the mitogen induced protein tyrosine phosphorylation of human T lymphocytes using SDS-PAGE and Western blotting techniques. Following stimulation with Con A and PHA, an identical pattern of protein tyrosine phosphorylation was observed in the lysates of T cells prepared from seven healthy young adults and eight healthy elderly human subjects. Five different high molecular mass proteins (75, 115, 120, 140 and 170 kDa) were consistently tyrosine phosphorylated in all of the donors from both age groups and peaked between 3 and 10 min. Tyrosine phosphorylation of the above substrates was observed in both CD4 and CD8 subsets. When compared for individual donors from both age groups, variations in the T cell response with regard to net tyrosine phosphorylation for all the substrates was observed. However, the mitogen induced level of tyrosine phosphorylation of only p75 was found to be significantly lower in unfractionated T cells as well as CD4 and CD8 subsets of older subjects than that of young subjects. Using immunoblotting, p75 was identified as ZAP-70, a member of the syk family of protein tyrosine kinases. Understanding of the biochemical basis of the reduced level of tyrosine phosphorylation of ZAP-70 will be helpful in delineating the molecular basis of age-associated impairment of T cell activation.

Adult↗
PubMed2001

Analysis of regulatory phosphorylation sites in ZAP-70 by capillary high-performance liquid chromatography coupled to electrospray ionization or matrix-assisted laser desorption ionization time-of-flight mass spectrometry.

A methodology for the rapid and quantitative analysis of phosphorylation sites in proteins is presented. The coupling of capillary high-performance liquid chromatography (HPLC) to electrospray ionization mass spectrometry (ESI-MS) allowed one to distinguish phosphorylation sites based on retention time and mass difference from complex peptide mixtures. The methodology was first evaluated and validated for a mixture of non-, mono-, and dityrosine-phosphorylated synthetic peptides, corresponding to the tryptic fragment 485-496 (ALGADDSYYTAR) of the human protein tyrosine kinase ZAP-70. The limits of detection for the non-, mono- and diphosphorylated peptides were about 15, 40 and 100 fmol, respectively, when using a 300 microm I.D. column. Application of the method was extended to identify phosphopeptides generated from a trypsin digest of recombinant autophosphorylated ZAP-70, in particular with respect to quantifying the status at the regulatory phosphorylation sites Tyr-492 and Tyr-493. Combination of chromatographic and on-line tandem mass spectrometry data allowed one to ascertain the identity of the detected peptides, a prerequisite to analyses in more complex biological samples. As an extension to the methodology described above, we evaluated the feasibility of interfacing capillary HPLC to matrix assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF-MS), using a micromachined piezoelectric flow-through dispenser as the interface. This enabled direct arraying of chromatographically separated components onto a target plate that was precoated with matrix for subsequent analysis by MALDI-TOF-MS without further sample handling.

Amino Acid Sequence↗
PubMed1999

Age-related impairment of p56lck and ZAP-70 activities in human T lymphocytes activated through the TcR/CD3 complex.

Cellular immune responses decrease with aging. Lymphocytes of aged individuals do not perform as well as cells from young subjects in a number of in vitro assays including cell proliferation, cytokine production, and protection against apoptosis. Here, we have tested the hypothesis that a decrease in T cell responses in tymphocytes from elderly subjects could parallel a decrease in the activity of protein tyrosine kinases (PTK) associated with signal transduction in T lymphocytes. We report that anti-CD3-triggered T lymphocyte proliferation was significantly decreased in T lymphocytes from elderly subjects, but the decrease was not due to an alteration of the percentage or mean fluorescence intensities of CD3, CD4, and CD45. Of significance, the activities of p56lck and ZAP-70 in vitro were significantly decreased in T lymphocytes from elderly subjects compared to young individuals. However, the level of expression of the two kinases did not change with aging. The activity of p59fyn did not show changes with aging, suggesting that p59fyn did not compensate for the decreased activity of p56lck. We also found that the extent of tyrosine phosphorylation of the adaptor protein p95vav was similar in activated T lymphocytes from elderly and young subjects. Our results suggest that the altered cellular immune responses observed in T lymphocytes with aging may be the result, at least in part, of an alteration in early events associated with signal transduction through the TcR/CD3 complex that translates into decreased activities of p56lck and ZAP-70. Impairment in the activities of these twokey components of T cell signaling may contribute to reduced immune functions associated with aging.

Adult↗
PubMed1998

Solid phase synthesis of a biased mini tetrapeptoid-library for the discovery of monodentate ITAM mimics as ZAP-70 inhibitors.

The biased library was composed of a novel phosphotyrosine mimic fixed in the P1 position of a tetrapeptoid and combined with three lipophilic N-substituents at the remaining positions giving a total of 27 single compounds. Screening for ZAP-70 antagonism identified 8 as a novel selective monodentate ZAP-70 antagonist and lead in the search for new immunosuppressive drugs.

Humans↗
PubMed2003

Unexpected requirement for ZAP-70 in pre-B cell development and allelic exclusion.

ZAP-70, a member of the Syk family of tyrosine kinases, has been reported to be expressed exclusively in T and NK cells. We show here that it is expressed throughout B cell development and that it plays a role in the transition of pro-B to pre-B cells in the bone marrow, a checkpoint controlled by signals from the pre-B cell receptor (pre-BCR), which monitors for successful rearrangement of immunoglobulin heavy chain genes. Whereas mice deficient in Syk show a partial block at this step, mice mutant in both Syk and ZAP-70 show a complete block at the pro-B cell stage and a failure of heavy chain allelic exclusion, hallmarks of defective pre-BCR signaling.

Alleles↗
PubMed1999

Discovery of potent and selective SH2 inhibitors of the tyrosine kinase ZAP-70.

A series of 1,2,4-oxadiazole analogues has been shown to be potent and selective SH2 inhibitors of the tyrosine kinase ZAP-70, a potential therapeutic target for immune suppression. These compounds typically are 200-400-fold more potent than the native, monophosphorylated tetrapeptide sequences. When compared with the high-affinity zeta-1-ITAM peptide (Ac-NQL-pYNELNLGRREE-pYDVLD-NH(2), wherein pY refers to phosphotyrosine) some of the best 1,2, 4-oxadiazole analogues are approximately 1 order of magnitude less active. This series of compounds displays an unprecedented level of selectivity over the closely related tyrosine kinase Syk, as well as other SH2-containing proteins such as Src and Grb2. Gel shift studies using a protein construct consisting only of C-terminal ZAP-70 SH2 demonstrate that these compounds can effectively engage this particular SH2 domain.

Enzyme Inhibitors↗
PubMed1995

Molecular basis for interaction of the protein tyrosine kinase ZAP-70 with the T-cell receptor.

The crystal structure of the tandem SH2 domains of human ZAP-70 in complex with a peptide derived from the zeta-subunit of the T-cell receptor reveals an unanticipated interaction between the two domains. A coiled coil of alpha-helices connects the two SH2 domains, producing an interface that constitutes one of the two critical phosphotyrosine binding sites. These and other unique features provide the molecular basis for highly selective association of ZAP-70 with the T-cell receptor.

Amino Acid Sequence↗
PubMed2003

Altered thymic T-cell selection due to a mutation of the ZAP-70 gene causes autoimmune arthritis in mice.

Rheumatoid arthritis (RA), which afflicts about 1% of the world population, is a chronic systemic inflammatory disease of unknown aetiology that primarily affects the synovial membranes of multiple joints. Although CD4(+) T cells seem to be the prime mediators of RA, it remains unclear how arthritogenic CD4(+) T cells are generated and activated. Given that highly self-reactive T-cell clones are deleted during normal T-cell development in the thymus, abnormality in T-cell selection has been suspected as one cause of autoimmune disease. Here we show that a spontaneous point mutation of the gene encoding an SH2 domain of ZAP-70, a key signal transduction molecule in T cells, causes chronic autoimmune arthritis in mice that resembles human RA in many aspects. Altered signal transduction from T-cell antigen receptor through the aberrant ZAP-70 changes the thresholds of T cells to thymic selection, leading to the positive selection of otherwise negatively selected autoimmune T cells. Thymic production of arthritogenic T cells due to a genetically determined selection shift of the T-cell repertoire towards high self-reactivity might also be crucial to the development of disease in a subset of patients with RA.

Amino Acid Sequence↗
PubMed1995

Ligation of the T-cell antigen receptor (TCR) induces association of hSos1, ZAP-70, phospholipase C-gamma 1, and other phosphoproteins with Grb2 and the zeta-chain of the TCR.

Signaling by the T-cell antigen receptor (TCR) involves both phospholipase C (PLC)-gamma 1 and p21ras activation. While failing to induce Shc/Grb2 association, ligation of the TCR/CD3 receptor in Jurkat T-cells induced hSos1-Grb2 complexes. In addition to hSos1, Grb2 participates in the formation of a tyrosine phosphoprotein complex that includes 145-, 95-, 70-, 54-, and 36-38-kDa proteins. p145 was identified as PLC-gamma 1 and p70 as the protein tyrosine kinase, ZAP-70. Although of the same molecular weight, p95 was not recognized by an anti-serum to p95 Vav. The SH2 domains of Grb2 and PLC-gamma 1 were required for the formation of this protein complex. In anti-CD3-treated cells, Grb2 redistributed from the cytosol to a particulate cell compartment along with p36/p38, ZAP-70, and PLC-gamma 1. Part of the Grb2 complex associated with the particulate compartment could be extracted with Nonidet P-40, while the rest was Nonidet P-40 insoluble. In both the detergent-soluble and -insoluble fractions, Grb2 coimmunoprecipitated with the zeta-chain of the TCR. Taken together, these results indicate that anti-CD3 induces Grb2-hSos1-PLC-gamma 1-p36/p38-ZAP70 complexes, which localize in the vicinity of TCR-zeta.

Adaptor Proteins, Signal Transducing↗
PubMed1993

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.

Antibodies, Monoclonal↗
PubMed1996

RANTES induces tyrosine kinase activity of stably complexed p125FAK and ZAP-70 in human T cells.

The chemokine RANTES is a chemoattractant and activating factor for T lymphocytes. Investigation of the signal transduction mechanisms induced by RANTES in T cells revealed tyrosine phosphorylation of multiple protein species with prominent bands at 70-85 and 120-130 kD. Immunoprecipitation and Western analyses revealed that a protein of 125 kD was identical to the focal adhesion kinase (FAK) pp125FAK. RANTES stimulated phosphorylation of FAK as early as 30 seconds and immunoblots using antiphosphotyrosine monoclonal antibodies revealed that there was consistent phosphorylation of a 68-70 kD species in the pp125FAK immunoprecipitates. Immunoblotting and kinase assays showed this to be two separate proteins, the tyrosine kinase zeta-associated protein (ZAP) 70, and the focal adhesion protein paxillin. These results indicate a potentially important role for RANTES in the generation of T cell focal adhesions and subsequent cell activation via a molecular complex containing FAK, ZAP-70, and paxillin.

Cell Adhesion↗
PubMed2003

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.

Animals↗