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PubMed1995

Analysis of the interaction of ZAP-70 and syk protein-tyrosine kinases with the T-cell antigen receptor by plasmon resonance.

Tyrosine phosphorylation of a 17-amino acid immunoreceptor tyrosine-based activation motif (ITAM), conserved in each of the signaling subunits of the T-cell antigen receptor (TCR), mediates the recruitment of ZAP-70 and syk protein-tyrosine kinases (PTKs) to the activated receptor. The interaction between the two tandemly arranged Src-homology 2 (SH2) domains of this family of PTKs and each of the phosphotyrosine-containing ITAMs was examined by real-time measurements of kinetic parameters. The association rate and equilibrium binding constants for the ZAP-70 and syk SH2 domains were determined for the CD3 epsilon ITAM. Both PTKs bound with ka and Kd values of 5 x 10(6) M-1.sec-1 and approximately 25 nM, respectively. Bindings to the other TCR ITAMs (zeta 1, zeta 2, gamma, and delta ITAMs) were comparable, although the zeta 3 ITAM bound approximately 2.5-fold less well. Studies of the affinity of a single functional SH2 domain of ZAP-70 provided evidence for the cooperative nature of binding of the dual SH2 domains. Mutation of either single SH2 domain decreased the Kd by > 100-fold. Finally, the critical features of the ITAM for syk binding were found to be similar to those required for ZAP-70 binding. These data provide insight into the mechanism by which the multisubunit TCR interacts with downstream effector molecules.

Amino Acid Sequence↗
PubMed1997

The Syk and ZAP-70 SH2-containing tyrosine kinases are implicated in pre-T cell receptor signaling.

An early stage in thymocyte development, after rearrangement of the beta chain genes of the T cell receptor (TCR), involves expression of the pre-TCR complex and accompanying differentiation of CD4(-)CD8(-) double negative (DN) cells to CD4(+)CD8(+) double positive (DP) cells. The ZAP-70 and Syk tyrosine kinases each contain two N-terminal SH2 domains that bind phosphorylated motifs in antigen receptor subunits and are implicated in pre-T receptor signaling. However, mice deficient in either ZAP-70 or Syk have no defect in the formation of DP thymocytes. Here we show that, in mice lacking both Syk and ZAP-70, DN thymocytes undergo beta chain gene rearrangement but fail to initiate clonal expansion and are incapable of differentiating into DP cells after expression of the pre-TCR. These data suggest that the ZAP-70 and Syk tyrosine kinases have crucial but overlapping functions in signaling from the pre-TCR and hence in early thymocyte development.

Animals↗
PubMed1996

A novel phosphotyrosine-binding domain in the N-terminal transforming region of Cbl interacts directly and selectively with ZAP-70 in T cells.

The protooncogene product Cbl has emerged as a novel signal transduction protein downstream of a number of cell surface receptors coupled to tyrosine kinases. Recently, we and others have reported the activation-dependent association of Cbl with the Syk and ZAP-70 tyrosine kinases through presently undefined mechanisms. Potential Src homology 2 and 3 domain binding sites within the C-terminal half of Cbl mediate in vivo interactions with several signaling proteins; however, the N-terminal transforming region (Cbl-N) lacks recognizable catalytic or protein interaction motifs. Here, we show that in vitro Cbl-N (amino acids 1-357) but not Cbl-C (amino acids 358-906) binds to ZAP-70 in a T cell-activation-dependent manner. A point mutation in Cbl-N, G306E, corresponding to a loss-of-function mutation in the Caenorhabditis elegans Cbl homologue, SLI-1, severely compromised Cbl-N/ZAP-70 binding. Cbl-N/ZAP-70 binding was direct and phosphotyrosine-dependent, thus identifying a phosphotyrosine-binding domain within the transforming region of Cbl. In vivo, Cbl-N expressed in T cells selectively associated with the ZAP-70/zeta complex. These results identify a novel mechanism for the direct participation of the N-terminal region of Cbl in ZAP-70 signal transduction, and suggest a biochemical mechanism for the leukemogenicity of the oncogene v-cbl through potential interaction with proliferation-related phosphotyrosyl proteins.

Cell Line↗
PubMed1998

T cell activation induced by novel gain-of-function mutants of Syk and ZAP-70.

The Syk family tyrosine kinases play a crucial role in antigen receptor-mediated signal transduction, but their regulation and cellular targets remain incompletely defined. Following receptor engagement, phosphorylation of tyrosine residues within ZAP-70 and Syk is thought to control both kinase activity and recruitment of modulatory factors. We report here the characterization of novel mutants of ZAP-70 and Syk, in which conserved C-terminal tyrosine residues have been replaced by phenylalanines (ZAP YF-C, Syk YF-C). Both mutant kinases display a prominent gain-of-function phenotype in Jurkat T cells, as demonstrated by lymphokine promoter activation, tyrosine phosphorylation of potential targets in vivo, and elevated intracellular calcium mobilization. While the presence of p56-Lck was required for ZAP YF-C-induced signaling, Syk YF-C showed enhanced functional activity in Lck-deficient JCaM1 Jurkat cells. Our results implicate the C terminus of Syk family kinases as an important regulatory region modulating T cell activation.

Amino Acid Sequence↗
PubMed2001

Cbl promotes ubiquitination of the T cell receptor zeta through an adaptor function of Zap-70.

Triggering of the T cell antigen receptor (TCR).CD3 complex induces its ubiquitination. However, the molecular events that lead to ubiquitin conjugation to these cell surface molecules have not been defined. Here we report that Cbl, a RING-type E3 ubiquitin-protein ligase, promotes ubiquitination of TCR zeta chain, which requires its functional variant Src homology 2 domain and an intact RING finger. The tyrosine kinase Zap-70, which binds to both TCR zeta and Cbl, plays an adaptor role in these events. Mutations in TCR zeta, Zap-70, or Cbl that disrupt the interaction between TCR zeta and Zap-70 or between Zap-70 and Cbl reduce ubiquitination of TCR zeta. Our results suggest a novel mechanism by which Cbl negatively regulates T cell development and activation by inducing ubiquitination of the TCR.CD3 components.

Cell Line↗
PubMed2000

Alternative antigen receptor (TCR) signaling in T cells derived from ZAP-70-deficient patients expressing high levels of Syk.

ZAP-70-deficient patients present with nonfunctional CD4+ T cells in the periphery. We find that a subset of primary ZAP-70-deficient T cells, expressing high levels of the related protein-tyrosine kinase Syk, can proliferate in vitro. These cells (denoted herein as Syk(hi)/ZAP-70(-) T cells) provide a unique model in which the contribution of Syk to TCR-mediated responses can be explored in a nontransformed background. Importantly, CD3-induced responses, such as tyrosine phosphorylation of cellular substrates (LAT, SLP76, and PLC-gamma1), as well as calcium mobilization, which are defective in T cells expressing neither ZAP-70 nor Syk, are observed in Syk(hi)/ZAP-70(-) T cells. However, Syk(hi)/ZAP-70(-) T cells differ from control T cells with respect to the T cell antigen receptor (TCR)-mediated activation of the MAPK cascades: extracellular signal-regulated kinase activity and recruitment of the JNK and p38 stress-related MAPK pathways are diminished. This distinct phenotype of Syk(hi)/ZAP-70(-) T cells is associated with a profound decrease in CD3-mediated interleukin 2 secretion and proliferation relative to control T cells. Thus, ZAP-70 and Syk appear to play distinct roles in transducing a TCR-mediated signal.

Adaptor Proteins, Signal Transducing↗
PubMed2005

ZAP-70 in B cell malignancies.

ZAP-70 has emerged as a protein of potential prognostic importance in chronic lymphocytic leukemia (CLL) following gene expression profiling which compared the 2 well established prognostic sub-sets, those with unmutated and mutated IgVH genes. This protein tyrosine kinase (PTK), known to be of importance in T and NK cell signaling but absent in normal peripheral B cells, is expressed in the majority of the poorer prognosis unmutated CLL and absent in most cases with mutated IgVH genes. ZAP-70 has been shown to be functionally important in the CLL cases in which it is expressed; it is also important in B cell development in mice and there is preliminary evidence for its expression in human B cell progenitors and activated B cells. Whether its expression in a sub-set of CLL cases is a result of a more activated cell type or a reflection of the stage of maturation of the transforming event(s) in CLL is open to debate. ZAP-70 is expressed in a minority of other B cell tumors but correlation with IgVH gene mutational status is lacking. The problems with ZAP-70 measurement, which has yet to be standardized, are reviewed together with its current status as a prognostic marker in CLL.

B-Lymphocytes↗
PubMed1994

p56lck interacts via its src homology 2 domain with the ZAP-70 kinase.

p56lck, a member of the src family of protein tyrosine kinases, is an essential component in T cell receptor (TCR) signal transduction. p56lck contains a src homology 2 (SH2) domain found in a number of proteins involved in intracellular signaling. SH2 domains have been implicated in protein-protein interactions by binding to sequences in target proteins containing phosphorylated tyrosine. Using an in vitro assay, we have studied specific binding of tyrosine-phosphorylated proteins to a recombinant p56lck SH2 domain. In nonactivated Jurkat cells, two tyrosine-phosphorylated proteins were detected. Stimulation with anti-CD3 monoclonal antibodies induced the binding of seven additional tyrosine-phosphorylated proteins to the SH2 domain of p56lck. We have identified the zeta-associated tyrosine kinase, ZAP-70, as one of these proteins. Evidence suggests that binding of ZAP-70 to p56lck SH2 is direct and not mediated by zeta. The significance of this interaction was further investigated in vivo. p56lck could be coprecipitated with the zeta/ZAP-70 complex and conversely, ZAP-70 was detected in p56lck immunoprecipitates of activated Jurkat cells. The physical association of p56lck and ZAP-70 during activation supports the recently proposed functional cooperation of these two tyrosine kinases in TCR signaling.

Base Sequence↗
PubMed1995

Absence of ZAP-70 prevents signaling through the antigen receptor on peripheral blood T cells but not on thymocytes.

Recently, a severe combined immunodeficiency syndrome with a deficiency of CD8+ peripheral T cells and a TCR signal transduction defect in peripheral CD4+ T cells was associated with mutations in ZAP-70. Since TCR signaling is required in developmental decisions resulting in mature CD4 (and CD8) T cells, the presence of peripheral CD4+ T cells expressing TCRs incapable of signaling in these patients is paradoxical. Here, we show that the TCRs on thymocytes, but not peripheral T cells, from a ZAP-70-deficient patient are capable of signaling. Moreover, the TCR on a thymocyte line derived from this patient can signal, and the homologous kinase Syk is present at high levels and is tyrosine phosphorylated after TCR stimulation. Thus, Syk may compensate for the loss of ZAP-70 and account for the thymic selection of at least a subset of T cells (CD4+) in ZAP-70-deficient patients.

Cell Line↗
PubMed1998

Ex vivo evidence for asymmetric tyrosine phosphorylation of ZAP-70 on double-positive thymocytes in the positive selection process.

Antigen stimulation via TCR in mature T cells provides rapid induction of tyrosine phosphorylation of intracellular substrates including ZAP-70. To study the potential involvement of tyrosine phosphorylation in CD4+CD8+ [double-positive (DP)] thymocytes in the positive selection process in vivo, we isolated and analyzed them in the presence of phosphatase inhibitor. DP thymocytes were obtained from TCR transgenic mice (TCR-Tg) expressing MHC class I- or class II-restricted TCR in selecting and non-selecting MHC backgrounds respectively. The phosphorylation of ZAP-70 in DP thymocytes of class I-restricted TCR-Tg was significantly higher in the positively selecting background than in the non-selecting one. However, such a phosphorylation difference between selecting and non-selecting TCR-Tg was found to be considerably less in class II-restricted TCR-Tg. A similar bias for ZAP-70 phosphorylation was also observed on selecting DP thymocytes when I-A(beta) deficient- and beta2-microglobulin-deficient mice were compared. These ex vivo studies suggest that TCR-mediated signaling on DP thymocytes induces ZAP-70 phosphorylation under a different manner of engagement of TCR to class I and class II molecules in the positive selection process.

Animals↗
PubMed2001

The involvement of the proto-oncogene p120 c-Cbl and ZAP-70 in CD2-mediated T cell activation.

The CD2 co-receptor expressed on the surface of T lymphocytes is able to stimulate T cell activation, proliferation and cytokine production in the absence of direct engagement of the antigen-specific TCR. Engagement of human CD2 by mitogenic pairs of anti-CD2 mAb induces tyrosine phosphorylation of a number of intracellular proteins including a 120 kDa phosphoprotein that we identify as the proto-oncogene c-Cbl. Rapidly tyrosine phosphorylated following stimulation of a number of cell surface receptors, c-Cbl is an adaptor protein that has been shown to associate with a complex of intracellular signaling molecules, and to mediate both positive and negative regulatory effects. Here we show that, like TCR-CD3 stimulation, stimulation of CD2 enhanced the association of c-Cbl with both Crk(L) and the p85 subunit of phosphatidylinositol-3 kinase. Overexpression of wild-type c-Cbl protein inhibited both CD2and CD3-induced NF-AT transcriptional activity, suggesting that CD2 signaling is also negatively regulated by c-Cbl. The inhibitory effect of c-Cbl depended upon its N-terminal phosphotyrosine-binding domain, the domain that has been shown to be required for inhibition of the Syk/ZAP-70 family kinases. In Syk(-) Jurkat T cells stably expressing wild-type ZAP-70, CD2 stimulation induced only a minimal increase in ZAP-70 tyrosine phosphorylation. Nevertheless, ZAP-70 kinase was required for CD2-mediated NF-AT transcriptional activity. Thus, CD2-mediated NF-AT transcriptional activity appears to depend upon ZAP-70/Syk kinases and to be negatively regulated by c-Cbl.

Amino Acid Sequence↗
PubMed2001

Superantigen-induced T cell:B cell conjugation is mediated by LFA-1 and requires signaling through Lck, but not ZAP-70.

The formation of a conjugate between a T cell and an APC requires the activation of integrins on the T cell surface and remodeling of cytoskeletal elements at the cell-cell contact site via inside-out signaling. The early events in this signaling pathway are not well understood, and may differ from the events involved in adhesion to immobilized ligands. We find that conjugate formation between Jurkat T cells and EBV-B cells presenting superantigen is mediated by LFA-1 and absolutely requires Lck. Mutations in the Lck kinase, Src homology 2 or 3 domains, or the myristoylation site all inhibit conjugation to background levels, and adhesion cannot be restored by the expression of Fyn. However, ZAP-70-deficient cells conjugate normally, indicating that Lck is required for LFA-1-dependent adhesion via other downstream pathways. Several drugs that inhibit T cell adhesion to ICAM-1 immobilized on plastic, including inhibitors of mitogen-activated protein/extracellular signal-related kinase kinase, phosphatidylinositol-3 kinase, and calpain, do not inhibit conjugation. Inhibitors of phospholipase C and protein kinase C block conjugation of both wild-type and ZAP-70-deficient cells, suggesting that a phospholipase C that does not depend on ZAP-70 for its activation is involved. These results are not restricted to Jurkat T cells; Ag-specific primary T cell blasts behave similarly. Although the way in which Lck signals to enhance LFA-1-dependent adhesion is not clear, we find that cells lacking functional Lck fail to recruit F-actin and LFA-1 to the T cell:APC contact site, whereas ZAP-70-deficient cells show a milder phenotype characterized by disorganized actin and LFA-1 at the contact site.

B-Lymphocytes↗
PubMed2002

CTLA-4 suppresses proximal TCR signaling in resting human CD4(+) T cells by inhibiting ZAP-70 Tyr(319) phosphorylation: a potential role for tyrosine phosphatases.

The balance between positive and negative signals plays a key role in determining T cell function. CTL-associated Ag-4 is a surface receptor that can inhibit T cell responses induced upon stimulation of the TCR and its CD28 coreceptor. Little is known regarding the signaling mechanisms elicited by CTLA-4. In this study we analyzed CTLA-4-mediated inhibition of TCR signaling in primary resting human CD4(+) T cells displaying low, but detectable, CTLA-4 cell surface expression. CTLA-4 coligation with the TCR resulted in reduced downstream protein tyrosine phosphorylation of signaling effectors and a striking inhibition of extracellular signal-regulated kinase 1/2 activation. Analysis of proximal TCR signaling revealed that TCR zeta-chain phosphorylation and subsequent zeta-associated protein of 70 kDa (ZAP-70) tyrosine kinase recruitment were not significantly affected by CTLA-4 engagement. However, the association of p56(lck) with ZAP-70 was inhibited following CTLA-4 ligation, correlating with reduced actions of p56(lck) in the ZAP-70 immunocomplex. Moreover, CTLA-4 ligation caused the selective inhibition of CD3-mediated phosphorylation of the positive regulatory ZAP-70 Y319 site. In addition, we demonstrate protein tyrosine phosphatase activity associated with the phosphorylated CTLA-4 cytoplasmic tail. The major phosphatase activity was attributed to Src homology protein 2 domain-containing tyrosine phosphatase 1, a protein tyrosine phosphatase that has been shown to be a negative regulator of multiple signaling pathways in hemopoietic cells. Collectively, our findings suggest that CTLA-4 can act early during the immune response to regulate the threshold of T cell activation.

Abatacept↗
PubMed2004

Zeta-associated protein of 70 kDa (ZAP-70), but not Syk, tyrosine kinase can mediate apoptosis of T cells through the Fas/Fas ligand, caspase-8 and caspase-3 pathways.

The TCR zeta-chain-associated protein of 70 kDA (ZAP-70) and Syk tyrosine kinases play critical roles in regulating TCR-mediated signal transduction. They not only share some overlapped functions but also may play unique roles in regulating the function and development of T cells. However, it is not known whether they have different effects on the activation and activation-induced cell death of T cells. To address this question, we generated cDNAs encoding chimeric molecules that a tailless TCR zeta-chain was directly linked to truncated ZAP-70 (Z/ZAP) or Syk (Z/Syk) molecules lacking the two Src homology 2 domains. Transfection of these molecules into zeta-chain-deficient cells restored their TCR expression. In addition, Z/ZAP and Z/Syk transfectants but not control cells demonstrated kinase activities in phosphorylating an exogenous substrate specific for ZAP-70 and Syk kinases. Z/ZAP transfectants activated through TCRs underwent a faster time course of apoptosis and had a greater percentage of apoptotic cells than that of Z/Syk and control cells. Activated Z/ZAP transfectants increased Fas and Fas ligand (FasL) expression 3- and 40-fold, respectively. Blocking of the Fas/FasL interaction could inhibit the apoptosis of Z/ZAP transfectants. In contrast, although activated Z/Syk transfectants could increase FasL expression, their Fas expression actually decreased and the percentage of apoptotic cells did not increase. Further studies of the mechanisms revealed that activation of Z/ZAP but not Z/Syk transfectants resulted in rapid activation of caspase-3 and caspase-8 that could also be inhibited by blocking Fas/FasL interaction. These results demonstrated that ZAP-70 and Syk play distinct roles in T cell activation and activation-induced cell death.

Animals↗
PubMed1995

Selection transduction defect (STD) due to Zap-70 kinase deficiency.

We have previously described a new type of selective T-cell deficiency (STD) characterized by persistent infections reminiscent of severe combined immunodeficiency (SCID). We show here that STD patients carry a mutation of zap-70 resulting in a loss of the activity of this kinase. The thymus of zap-70-/- patients shows the presence of CD4CD8 double positive cells in the cortex, however, only CD4 but not CD8 single positive cells are present in the medulla. Peripheral CD4+ T cells from the zap-70-/- exhibit markedly reduced tyrosine phosphorylation, fail to produce IL-2, and do not proliferate in response to TCR stimulation by mitogens or antigens. Thus Zap-70 kinase appears to be indispensable for the development of CD8 single positive T cells as well as for signal transduction and function of single positive CD4 T cells.

Amino Acid Sequence↗
PubMed1993

ZAP-1 DNA binding activity is first detected at the onset of zona pellucida gene expression in embryonic mouse oocytes.

ZAP-1 (zona pellucida gene activating protein-1) is a putative transcription factor controlling the oocyte-specific expression of mouse and human zona pellucida genes. The DNA binding activity of ZAP-1 first appears in oocytes from 19-day-old mouse embryos and reaches a maximum level at 10 days after birth. This developmental profile closely parallels that of mouse zona pellucida gene transcription, which is detected in oocytes at 19 days of fetal life using a sensitive RT-PCR method and is maximal in 10-day-old animals. DNA binding activity similar to that of ZAP-1 is present in ovarian extracts from rat, human, and opossum, suggesting that the ZAP-1 protein may be conserved among mammals.

Animals↗
PubMed2004

ZAP is a CRM1-dependent nucleocytoplasmic shuttling protein.

The zinc finger antiviral protein (ZAP) is a recently isolated host antiviral factor. It specifically inhibits the replication of Moloney murine leukemia virus (MMLV) and Sindbis virus (SIN) by preventing the accumulation of viral RNA in the cytoplasm. In this report, we demonstrate that ZAP is predominantly localized in the cytoplasm at steady state but shuttles between the nucleus and the cytoplasm in a CRM1-dependent manner. Two nuclear localization sequences (NLS) and one nuclear export sequence (NES) were identified. One NLS was mapped to amino acids 68-RARVCRRK-75 and the other mapped to a region including amino acids K405 and K406. The NES was mapped to amino acids 284-LEDVSVDV-291. These findings help to understand why ZAP specifically prevents the accumulation of viral RNA in the cytoplasm. These findings also suggest possible functions of ZAP in the nucleus.

Active Transport, Cell Nucleus↗
PubMed2000

Passive immunization with antisperm monoclonal antibody ZAP-7 increases preimplantation embryo mortality in the mouse.

OBJECTIVE: To evaluate the effect of VIC-1 and ZAP-7 antihuman sperm monoclonal antibodies on in vivo fertility in the mouse. DESIGN: A randomized blinded study using a mouse model. SETTING: University-based laboratory. ANIMALS: B6CBAF1 mice (n = 6 per experimental group). INTERVENTION(S): Antisperm antibodies were administered intravaginally to female mice before mating. Control mice received no treatment, saline, or nonspecific antibodies. Number and viability of preimplantation embryos were determined by microscopic observation. Mouse sperm, oocytes, and normal preimplantation embryos were used in indirect immunofluorescence assays with antisperm antibodies. The effect of antibody treatment on sperm motility and vitality was evaluated. MAIN OUTCOME MEASURE(S): Antigen expression, sperm motility and vitality, number and viability of embryos. RESULT(S): ZAP-7 antibody recognizes a sperm antigen expressed in zygotes and early preimplantation embryos. Passive immunization with ZAP-7 increases embryo mortality significantly (more than 40% above controls). Passive immunization with VIC-1 has no deleterious effect. CONCLUSION(S): ZAP-7 monoclonal antibody disrupts fertilization and embryogenesis in the mouse.

Animals↗