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PubMed2006

An optimized whole blood method for flow cytometric measurement of ZAP-70 protein expression in chronic lymphocytic leukemia.

BACKGROUND: ZAP-70 protein expression has been proposed as a marker for immunoglobulin heavy chain mutational status, which some studies have correlated with disease course in B-cell chronic lymphocytic leukemia (CLL). Studies published to date measuring levels of expression of ZAP-70 intracellular protein using flow cytometry have demonstrated poor performance, as defined by the difference in signal in known positive and negative lymphocyte populations. METHODS: A recently published method (Chow S, Hedley DW, Grom P, Magari R, Jacobberger JW, Shankey TV, Cytometry A 2005;67:4-17) to measure intracellular phospho-epitopes was optimized using a design of experiments (DOE) approach to provide the best separation of ZAP-70 expression in positive T- or NK-cells as compared to negative B-cells in peripheral blood samples. A number of commercially available anti-ZAP-70 antibody-conjugates were screened using this methodology, and the antibody-conjugate showing the best performance was chosen to develop a four-color, five antibody assays to measure ZAP-70 levels in whole blood specimens. RESULTS: Using the optimized fixation and permeabilization method, improvement in assay performance (signal-to-noise, S/N) was seen in most of the antibodies tested. The custom SBZAP conjugate gave the best S/N when used in conjunction with this optimized fixation /permeabilization method. In conjunction with carefully standardized instrument set-up protocols, we obtained both intra- and interlaboratory reproducibility in the analysis of ZAP-70 expression in whole blood samples from normal and CLL patients. CONCLUSIONS: The development of a sensitive, specific and highly reproducible ZAP-70 assay represents only the first essential step for any clinical assay. The universal implementation of a validated data analysis method and the establishment of methodology-based cutoff points for clinical outcomes must next be established before ZAP-70 protein analysis can be routinely implemented in the clinical laboratory.

Antibodies, Monoclonal↗
PubMed2002

Effect of age and oxidative stress on tyrosine phosphorylation of ZAP-70.

Oxidative stress is believed to be one of the leading contributors to the aging process and loss of cellular function with aging. Although, several age-associated phenomena have been correlated with reactive oxygen species, the role of oxidative stress in the age-related decline of T-cell activity is not yet clear. The present study was carried out to determine the effect of reactive oxygen species on tyrosine phosphorylation of ZAP-70, a key enzyme in the T-cell signal transduction pathway. Our previous as well as present studies have demonstrated an age-related down-regulation of tyrosine phosphorylation of ZAP-70 following activation of freshly isolated T cells with various stimulating agents. Currently, we observe that under mild and chronic oxidative stress, tyrosine phosphorylation of ZAP-70 is impaired following stimulation of the T cells with anti-CD3 antibody. Interestingly, in contrast to our observation using freshly isolated T cells, there is no age-associated impairment of tyrosine phosphorylation of ZAP-70 when T cells, cultured for 2 days in a serum-free medium in the presence or in the absence of oxidative stress, are stimulated with anti-CD3 antibody. The current observation suggests that the age-associated down-regulation of tyrosine phosphorylation of ZAP-70 is not an intrinsic defect inherent of the T cells due to aging and is reversible. We also observed that under oxidative stress in vitro, there was no significant difference in inhibition of tyrosine phosphorylation of ZAP-70 in T cells isolated from elderly and young donors. Finally, it was also noted that the down-regulation of tyrosine phosphorylation of ZAP-70 under oxidative stress in young and elderly donors was not due to impairment in the net expression of ZAP-70 under oxidative stress thereby suggesting that dysregulation in the balance of kinase-phosphatase activities under oxidative stress might have been implicated in the observed phenomenon.

Adult↗
PubMed2006

ZAP-70 is expressed by normal and malignant human B-cell subsets of different maturational stage.

ZAP-70 tyrosine kinase is involved in signalling pathways following T-cell receptor stimulation and was originally described only in T cells and natural killer cells. ZAP-70 expression has been reported in normal mouse B lineage cells and in human malignant B lymphocytes, mainly in chronic lymphocytic leukemia (CLL) where it correlates with clinical outcome. We analyzed several B-cell lines and ex vivo malignant B cells, ranging from acute lymphoblastic leukemia to multiple myeloma and reflecting different stages of B-cell differentiation, and they showed ZAP-70 expression regardless their maturation stage. We then analyzed by Western blot and flow cytometry different human normal B-lymphocyte subpopulations: naïve, germinal center and memory B cells from tonsils, CD19+ CD5+ cells from cord blood and CD19+ lymphocytes from peripheral blood. All expressed ZAP-70 protein, though at different levels depending on their differentiation, activation and tissue localization. In addition, ZAP-70 expression levels could be modulated following stimulation via the B-cell receptor. These findings implicate a potential role of ZAP-70 in the signalling pathway of B lymphocytes at different maturational stages, indicate that ZAP-70 expression is not a CLL-specific feature among B-cell malignancies and suggest that the absence of ZAP-70 rather than its presence should be considered abnormal for malignant B lymphocytes.

Antigens, CD↗
PubMed1999

Role of the Src homology 2 domains and interdomain regions in ZAP-70 phosphorylation and enzymatic activity.

The protein tyrosine kinase ZAP-70, which mediates T-cell antigen receptor (TCR) signalling, contains three distinct functional modules, two tandemly arranged SH2 domains, a kinase domain and a linker region (interdomain B) that connects them. ZAP-70 enzymatic activation is strictly dependent on the binding, via its SH2 domains, to the triggered TCR and on tyrosine phosphorylation. Here we utilized recombinant ZAP-70 and carried out a mutational analysis to understand the structural requirements for its activation. We show that deletion of both SH2 domains corresponding to the first 254 residues moderately increases ZAP-70 enzymatic activity on an exogenous substrate in vitro, results in increased tyrosine phosphorylation and produces subtle conformational changes, as judged by altered SDS/PAGE migration. Mutation of Tyr292, 315 and 319 to Phe in the interdomain B region, which constitute the major phosphorylation sites both in vitro and in vivo, did not affect ZAP-70 enzymatic activity. Moreover, deletion analysis of the interdomain B region established residues 320-619 as a minimal region endowed with full kinase activity. We propose that binding of ZAP-70 to the TCR promotes, through conformational changes, its extensive phosphorylation on tyrosine. However, Tyr292, 315 and 319 do not affect ZAP-70 enzymatic activity and may influence ZAP-70 signalling only indirectly by mediating its association with intracellular transducers.

Animals↗
PubMed2005

[Everything you ever wanted to know about ZAP-70].

The ZAP-70 tyrosine kinase has been described more than ten years ago. Its key role in thymocytes development and mature T lymphocytes activation has been illustrated by the characterization of several human immunodeficiencies presenting with mutations in the zap-70 gene resulting in the absence of ZAP-70 expression. More recently, it has been shown that deregulation of ZAP-70 activity can induce autoimmune diseases. Finally, ZAP-70 expression has been shown in some B chronic lymphocytic leukaemia and correlated with bad prognosis of the disease. The diversity of pathologies associated with deregulation of ZAP-70 demonstrates its key role in immune responses. Research aiming at deciphering the different signalling pathways regulated by ZAP-70 will not only shed some lights on these pathologies, but will also help finding new pharmacological tools, targeting ZAP-70, designed to induce immunosuppression or tolerance.

Animals↗
PubMed1995

Activating and inhibitory mutations in adjacent tyrosines in the kinase domain of ZAP-70.

ZAP-70 is an 70-kDa protein tyrosine kinase, expressed exclusively in T cells and NK cells, and plays a critical role in mediating T cell activation in response to T cell receptor engagement. The strong correlation between tyrosine phosphorylation of ZAP-70 and its acquisition of increased kinase activity suggests that is is positively regulated by tyrosine phosphorylation. Previously, we identified tyrosines 492 and 493 of ZAP-70 as being sites of in vivo phosphorylation in response to T cell receptor engagement. To determine the role of phosphorylation in regulating ZAP-70 activity, we mutated each of these tyrosines individually to phenylalanine. When expressed in COS cells, Y493F-mutated ZAP-70 demonstrated normal basal kinase activity, but, unlike wild type ZAP-70, could not be activated by tyrosine phosphorylation induced by incubation with pervanadate or by co-expression of constitutively activated Lck. This suggests that Tyr-493 phosphorylation is required for the tyrosine phosphorylation-induced activation of ZAP-70. The Y492F mutation resulted in 4-fold higher basal kinase activity, which could be stimulated further by tyrosine phosphorylation. These results reveal that critical tyrosine residues in the kinase domain of ZAP-70 are important in regulation of its catalytic activity.

Amino Acid Sequence↗
PubMed1995

Regulation of Zap-70 by Src family tyrosine protein kinases in an antigen-specific T-cell line.

To further understand the interactions between Zap-70, Src family kinases, and other T-cell proteins, we have examined the regulation of Zap-70 in the antigen-specific T-cell line BI-141. By analyzing derivatives containing an activated version of either p56lck or p59fynT, it was observed that the two Src-related enzymes augmented T-cell receptor (TCR)-mediated tyrosine phosphorylation of Zap-70, as well as its association with components of the antigen receptor complex. Importantly, the accumulation of TCR.Zap-70 complexes quantitatively and temporally correlated with the induction of tyrosine phosphorylation of the CD3 and zeta chains of TCR. Using a CD4-positive variant of BI-141, we also found that the ability of Zap-70 to undergo tyrosine phosphorylation and associate with TCR was enhanced by aggregation of TCR with the CD4 co-receptor. Further studies allowed the identification of two distinct pools of tyrosine-phosphorylated Zap-70 in activated T-cells. While one population was associated with TCR, the other was co-immunoprecipitated with a 120-kDa tyrosine-phosphorylated protein of unknown identity. In addition to supporting the notion that Src-related enzymes regulate the recruitment of Zap-70 in TCR signaling, these data added further complexity to previous models of regulation of Zap-70. Furthermore, they suggested that p120 may be an effector and/or a regulator of Zap-70 in activated T-lymphocytes.

Animals↗
PubMed2006

ZAP-70 immunoreactivity is a prognostic marker of disease progression in chronic lymphocytic leukemia.

The expression of zeta-associated protein 70 (ZAP-70) in chronic lymphocytic leukemia (CLL) seems to correlate with the mutational status of the immunoglobulin heavy-chain variable-region genes, clinical course and patient prognosis. The aim was to determine the prognostic significance of the immunohistochemical expression of ZAP-70 protein in CLL by means of the long-term follow-up of 108 patients. This study identified 3 patterns of ZAP-70 immunoreactivity: negative (58 patients, 54%), weakly positive (20 patients, 18%) and strongly positive (30 patients, 28%). Overall, ZAP-70 immunoreactivity correlated with an abnormal karyotype ( p = 0.017), a lymphocyte doubling time (LDT) of <6 months ( p = 0.001) and <12 months ( p = 0.01), Rai II - IV and Binet B - C stage ( p = 0.013), the clinical need for chemotherapy ( p < 0.001) and the need for more than 1 chemotherapy line ( p < 0.001). Kaplan-Meier analysis demonstrated that ZAP-70 immunoreactivity closely correlated with a shorter LDT ( p < 0.0001) and time from diagnosis to initial therapy ( p = 0.0001). The same significance was retained when the patients were stratified into the ZAP-70 immunoreactivity groups ( p < 0.0001). This study shows that ZAP-70 immunoreactivity can be a reliable prognostic marker in CLL and proposes a system for evaluating the results. The observations support the inclusion of the immunohistochemical expression of ZAP-70 in clinical trials involving CLL patients.

Adult↗
PubMed1997

ZAP-70 protein tyrosine kinase is constitutively targeted to the T cell cortex independently of its SH2 domains.

ZAP-70 is a nonreceptor protein tyrosine kinase that is essential for signaling via the T cell antigen receptor (TCR). ZAP-70 becomes phosphorylated and activated by LCK protein tyrosine kinase after interaction of its two NH2-terminal SH2 domains with tyrosine-phosphorylated subunits of the activated TCR. In this study, the localization of ZAP-70 was investigated by immunofluorescence and confocal microscopy. ZAP-70 was found to be localized to the cell cortex in a diffuse band under the plasma membrane in unstimulated T cells, and this localization was not detectably altered by TCR stimulation. Analysis of mutants indicated that ZAP-70 targeting was independent of its SH2 domains but required its active kinase domain. The specific compartmentalization of ZAP-70 suggests that it may interact with an anchoring protein in the cell cortex via its hinge or kinase domains. It is likely that the maintenance of high concentrations of ZAP-70 at the cell cortex, that only has to move a short distance to interact with phophorylated TCR subunits, facilitates rapid initiation of signaling by the TCR. In addition, as the major increase in tyrosine phosphorylation induced by the TCR also occurs at the cell cortex (Ley, S.C., M. Marsh, C.R. Bebbington, K. Proudfoot, and P. Jordan. 1994. J. Cell. Biol. 125:639-649), ZAP-70 may be localized close to its downstream targets.

Binding Sites↗
PubMed1996

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.

Gene Expression Regulation↗
PubMed2004

T-cell receptor-induced phosphorylation of the zeta chain is efficiently promoted by ZAP-70 but not Syk.

Engagement of the T-cell receptor (TCR) results in the activation of Lck/Fyn and ZAP-70/Syk tyrosine kinases. Lck-mediated tyrosine phosphorylation of signaling motifs (ITAMs) in the CD3-zeta subunits of the TCR is an initial step in the transduction of signaling cascades. However, zeta phosphorylation is also promoted by ZAP-70, as TCR-induced zeta phosphorylation is defective in ZAP-70-deficient T cells. We show that this defect is corrected by stable expression of ZAP-70, but not Syk, in primary and transformed T cells. Indeed, these proteins are differentially coupled to the TCR with a 5- to 10-fold higher association of ZAP-70 with zeta as compared to Syk. Low-level Syk-zeta binding is associated with significantly less Lck coupled to the TCR. Moreover, diminished coupling of Lck to zeta correlates with a poor phosphorylation of the positive regulatory tyr352 residue of Syk. Thus, recruitment of Lck into the TCR complex with subsequent zeta chain phosphorylation is promoted by ZAP-70 but not Syk. Importantly, the presence of ZAP-70 positively regulates the TCR-induced tyrosine phosphorylation of Syk. The interplay between Syk and ZAP-70 in thymocytes, certain T cells, and B-chronic lymphocytic leukemia cells, in which they are coexpressed, will therefore modulate the amplitude of antigen-mediated receptor signaling.

B-Lymphocytes↗
PubMed2006

Additional genetic high-risk features such as 11q deletion, 17p deletion, and V3-21 usage characterize discordance of ZAP-70 and VH mutation status in chronic lymphocytic leukemia.

PURPOSE: Immunoglobulin heavy chain variable-region (VH) gene mutation status and zeta-associated protein 70 (ZAP-70) expression are correlated in chronic lymphocytic leukemia (CLL), but their concordance is variable. The goal of this study was to elucidate additional factors potentially characterizing their discordance. PATIENTS AND METHODS: We evaluated ZAP-70 expression by flow cytometry, VH status by DNA sequencing, and genomic aberrations by fluorescence in situ hybridization in 148 CLL patients. The parameters were analyzed for their associations and their individual prognostic impact. RESULTS: ZAP-70 expression and VH mutation status were strongly associated in CLL without additional genetic high-risk-features as defined by the absence of 11q or 17p deletion and V3-21 usage (concordance 84%). In contrast, the proportion of discordant cases was significantly higher (39%), if such additional genetic high-risk features were present. Discordant cases with V3-21 usage were almost exclusively ZAP-70 positive and VH mutated (89%), whereas all but one of the discordant cases with high-risk aberrations were ZAP-70 negative and VH unmutated (92%). By multivariate regression analysis, two models were developed, which both include high-risk genomic aberrations and, alternatively, VH mutation status and V3-21 usage or ZAP-70 expression as independent outcome predictors. CONCLUSION: There were characteristic modes of discordance between ZAP-70 and VH mutation status depending on the presence or absence of additional genetic high-risk features such as 11q and 17p deletion or V3-21 usage. Although the biologic background for these findings is yet to be determined, these data have biologic and clinical implications regarding ZAP-70 as a pathogenic factor and outcome predictor, respectively.

Biomarkers, Tumor↗
PubMed2004

ZAP-70 expression in B-cell hematologic malignancy is not limited to CLL/SLL.

ZAP-70 is a tyrosine kinase expressed in normal T cells and NK cells. Expression of ZAP-70 has been associated with poor prognosis in B-cell chronic lymphocytic leukemia and might be a surrogate marker for immunoglobin heavy chain (IGH) mutational status in that disease. Little is known about ZAP-70 expression in other hematologic malignant neoplasms. We examined 446 specimens representing a range of hematopoietic malignant neoplasms for ZAP-70 expression by immunohistochemical analysis. As expected, most T cell-lineage disorders and a subset of small lymphocytic lymphomas were positive. IGH mutational status corresponded to ZAP-70 expression in a small subset of small lymphocytic lymphoma cases subjected to sequence analysis. Of note, however, ZAP-70 was expressed in a minority of other types of B-cell lymphomas, including precursor B-cell acute lymphoblastic leukemia, diffuse large B-cell lymphoma, mantle cell lymphoma, and very rare cases of classic Hodgkin lymphoma. Immunohistochemical analysis represents an alternative method for assessing ZAP-70 expression and reveals that other B-cell malignant neoplasms express ZAP-70.

Blotting, Western↗
PubMed2000

Regulation of beta 1 integrin-mediated adhesion by T cell receptor signaling involves ZAP-70 but differs from signaling events that regulate transcriptional activity.

Stimulation of the CD3/TCR results within minutes in an increase in T cell adhesion mediated by beta(1) integrins. The biochemical pathways that control CD3-mediated increases in beta(1) integrin-mediated adhesion remain poorly characterized. In this study, the role of the tyrosine kinase ZAP-70 in the regulation of beta(1) integrin activity by the CD3/TCR was investigated. CD3 stimulation did not increase beta(1) integrin-mediated adhesion of the ZAP-70-deficient Jurkat T cell line, P116, to the beta(1) integrin ligand fibronectin. Reintroduction of wild-type ZAP-70, but not a kinase-inactive variant, K369R, corrected the adhesive defect observed in P116 T cells. In addition, the kinase-inactive ZAP-70 mutant inhibited CD3-induced adhesion of primary human T cell blasts. Interestingly, a ZAP-70 mutant with a tyrosine to phenylalanine substitution at position 319 (Y319F) restored the adhesive defect in P116 T cells, even though Y319F ZAP-70 failed to fully reconstitute CD3-initiated NF-AT-dependent transcription and tyrosine phosphorylation of the LAT adapter protein. Finally, expression of mutants of LAT and the SLP-76 adapter protein that modulate CD3-mediated activation of an NF-AT reporter gene failed to block CD3-induced increases in beta(1) integrin-mediated adhesion. These observations support a model in which the tyrosine kinase activity of ZAP-70 kinase is critical for regulation of beta(1) integrin activity by CD3/TCR. However, the signaling events downstream of ZAP-70 that regulate CD3/TCR-mediated activation of beta(1) integrin function exhibit key differences when compared with the signaling pathways that regulate transcriptional events initiated by CD3/TCR stimulation.

Adaptor Proteins, Signal Transducing↗
PubMed2006

[ZAP-70 expression in 24 patients with B cell chronic lymphocytic leukemia].

To explore the ZAP-70 expression in chronic lymphocytic leukaemia (CLL) and its relationship with other prognostic factors, the expressions of ZAP-70 protein and CD38 in bone marrow or peripheral blood of 24 patients with B-CLL were determined by four-color flow cytometry. The results showed that ZAP-70 was positively expressed in 37.5% of all B-CLL patients, 20% (3/15) patients in Binet A stage, and 66.7% in Binet B + C. The expression of ZAP-70 had significant difference between Binet A and Binet B + C (P < 0.05). CD38 was expressed in 29.1% of B-CLL patients and 3 out of these cases were in stage A, 2 out of 3 cases in stage A co-expressed CD38 and ZAP-70. The expression of CD38 had no significant difference between Binet A and Binet B + C stage (P > 0.05). ZAP-70+ and CD38+ were both expressed in 83.3% of B-CLL patients (P < 0.05). It is concluded that ZAP-70 protein can be routinely measured by flow cytometry in the laboratory. High expression of ZAP-70 is correlated with other prognostic factors such as clinic stage, chromosome abnormalities and CD38 in B-CLL.

ADP-ribosyl Cyclase 1↗
PubMed2002

Tyrosine 315 determines optimal recruitment of ZAP-70 to the T cell antigen receptor.

Recruitment of ZAP-70 protein tyrosine kinase to the T cell antigen receptor (TCR) is mediated by the binding of the SH2 domains of this enzyme to phosphorylated ITAM motifs in the CD3 and TCRzeta subunits. We have previously shown that the efficiency of both positive and negative thymocyte selection was decreased in knock-in mice expressing ZAP-70 mutated at Tyr315 (ZAP-70-Y315F), a residue laying in the interdomain B of this protein. Surprisingly, in these cells the amount of phosphorylated TCRzeta chain co-precipitating with ZAP-70-Y315F was significantly reduced compared to control mice. We report now that the binding affinity of ZAP-70-Y315F to phosphorylated ITAM is reduced as compared to the wild-type protein, whereas the intrinsic catalytic activity is untouched. Consequently, phosphorylated ITAM appear to be more accessible to protein tyrosine phosphatases (PTP) and can be readily dephosphorylated. We provide evidence suggesting that the defective ITAM binding induced by Tyr315 mutation is independent of the putative role of this residue as a binding site for Vav-1. Finally, we found that the extracellular signal-regulated kinase pathway is impaired in ZAP-70-Y315F-expressing mice. Collectively, these results demonstrate that Tyr315 has an unsuspected structural role in ZAP-70 and may allosterically regulate the function of the nearby SH2 domains.

Allosteric Regulation↗
PubMed2006

Kolmogorov-Smirnov statistical test for analysis of ZAP-70 expression in B-CLL, compared with quantitative PCR and IgV(H) mutation status.

BACKGROUND: ZAP-70 has been proposed as a surrogate marker for immunoglobulin heavy-chain variable region (IgV(H)) mutation status, which is known as a prognostic marker in B-cell chronic lymphocytic leukemia (CLL). The flow cytometric analysis of ZAP-70 suffers from difficulties in standardization and interpretation. We applied the Kolmogorov-Smirnov (KS) statistical test to make analysis more straightforward. METHODS: We examined ZAP-70 expression by flow cytometry in 53 patients with CLL. Analysis was performed as initially described by Crespo et al. (New England J Med 2003; 348:1764-1775) and alternatively by application of the KS statistical test comparing T cells with B cells. Receiver-operating-characteristics (ROC)-curve analyses were performed to determine the optimal cut-off values for ZAP-70 measured by the two approaches. ZAP-70 protein expression was compared with ZAP-70 mRNA expression measured by a quantitative PCR (qPCR) and with the IgV(H) mutation status. RESULTS: Both flow cytometric analyses correlated well with the molecular technique and proved to be of equal value in predicting the IgV(H) mutation status. Applying the KS test is reproducible, simple, straightforward, and overcomes a number of difficulties encountered in the Crespo-method. CONCLUSIONS: The KS statistical test is an essential part of the software delivered with modern routine analytical flow cytometers and is well suited for analysis of ZAP-70 expression in CLL.

Adult↗
PubMed1995

Activation of ZAP-70 kinase activity by phosphorylation of tyrosine 493 is required for lymphocyte antigen receptor function.

ZAP-70 is a protein tyrosine kinase (PTK) required for T-cell development and T-cell antigen receptor (TCR) function. ZAP-70 is associated with the phosphorylated antigen receptor and undergoes tyrosine phosphorylation following receptor activation. We demonstrate here that tyrosine phosphorylation of ZAP-70 results in an increase in its catalytic activity and that this activation is mediated by the phosphorylation of tyrosine residue 493 by the src family of PTKs. The activity of baculoviral expressed ZAP-70 was up-regulated 10-fold when ZAP-70 was co-infected and phosphorylated by the src family PTK, lck. Mutation of Y493 alone abrogated the ability of ZAP-70 to be activated by lck. Moreover, we demonstrate that phosphorylation of Y493 and activation of ZAP-70 is required for antigen receptor-mediated induction of interleukin-2 (IL-2) secretion in lymphocytes.

Amino Acid Sequence↗