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At least 685 records · Page 38Linked to original sources
PubMed1998

Activation of p21ras/MAPK signal transduction molecules decreases with age in mitogen-stimulated T cells from rats.

Signal transduction is ubiquitously involved in the initiation of physiological signals that lead to growth and proliferation of cells. The signaling cascade mediated by the mitogen-activated protein kinase (MAPK) is considered essential for T cell growth and function. Therefore, it was of interest to determine the influence of age on the induction of MAPK in mitogen-activated T cells. T cells from young (4-6 months) and old (24-26 months) rats responded to concanavalin A (Con A) stimulation by increasing MAPK, c-jun amino terminal kinase (JNK), and p21ras activities. The time course of induction of MAPK/JNK and p21ras activities was similar in T cells isolated from young and old rats. The induction of JNK activity did not change significantly with age; however, the induction of MAPK and p21ras activities was significantly less (50 to 65%) in T cells from old rats than in T cells from young rats. Although the relative protein levels of p42 and p44 MAPK did not change with age, the proportion of the phosphorylated p44 MAPK decreased with age. In addition, it was found that the in vitro kinase activities of the T cell receptor-associated protein tyrosine kinase Lck (p56Lck) and ZAP-70 but not Fyn (p59Fyn) were lower in T cells from old rats than in T cells from young rats. The decline in activities of these signaling molecules with age was not associated with changes in their corresponding protein levels. Thus, our results demonstrate that aging alters the activation of the signal transduction cascade that leads to T cell activation.

Aging↗
PubMed2001

Dexamethasone modulates TCR zeta chain expression and antigen receptor-mediated early signaling events in human T lymphocytes.

Dexamethasone is a potent anti-inflammatory and immunosupressive agent that has complex, yet incompletely defined, effects on the immune response. Here, we explored the effect of dexamethasone on the expression of TCR zeta chain and TCR/CD3-induced early signaling events in human T lymphocytes. Immunoblotting studies using TCR zeta chain specific mAb showed a dose-dependent biphasic effect of dexamethasone on TCR zeta chain expression, that is, it was increased when cells were incubated with 10 nM, whereas the expression was decreased when incubated with 100 nM dexamethasone. The dose-dependent biphasic effect of dexamethsone on the TCR zeta chain expression was also revealed by FACS analysis of permeabilized cells. Time course studies showed that upregulation of the TCR zeta chain at 10 nM dexamethasone reached maximum levels at 24 h and remained elevated up to 48 h. Other subunits of the TCR/CD3 complex were minimally affected under these conditions. The increased expression of the TCR zeta chain following treatment with 10 nM dexamethasone correlated with increased anti-CD3 antibody-induced tyrosine phosphorylation of the TCR zeta chain and downstream signaling intermediate ZAP-70 and PLC gamma with faster kinetics. Similarly, the induction of TCR zeta chain expression at 10 nM dexamethasone correlated with increased and more sustained TCR/CD3-mediated [Ca(2+)](i) response. Reporter gene assays using TCR zeta chain promoter-driven luciferase gene constructs in Jurkat cells showed that treatment with 10 nM dexamethasone increased TCR zeta chain promoter activity and that the region between -160 and +58 was responsible for the observed effect. These results suggest that dexamethasone primarily acts at the transcriptional level and differentially modulates TCR zeta chain expression and antigen receptor-mediated early signaling events in human peripheral T lymphocytes.

Adolescent↗
PubMed1997

Uncoupling of T-cell antigen receptor and downstream protein tyrosine kinases in common variable immunodeficiency.

Patients with common variable immunodeficiency (CVID) are heterogeneous in the clinical manifestations of the disease and the underlying mechanisms leading to the immunodeficiency. Although the overt defect is an impairment in B-cell function, there is increasing evidence of primary T-cell dysfunctions in a proportion of patients with CVID. We have analyzed T-cells from six CVID patients for activation of both early and late events in response to TCR triggering. The data showed that T-cells from three of six CVID patients were defective in the capacity to initiate the TCR/CD3 signaling pathway by activating intracellular tyrosine kinases, associated with impaired proliferative responses to TCR/CD3 triggering. Since both surface expression of the TCR/CD3 complex and intracellular expression of key tyrosine kinases such as p56lek and ZAP-70 were normal in these patients, our data suggest a defect in the earliest step of TCR signal transduction.

Adult↗
PubMed1995

The Syk/ZAP-70 protein tyrosine kinase connection to antigen receptor signalling processes.

The T- and B-cell receptor (TCR and BCR) signal transduction processes involve a coordinated interplay between two classes of non-receptor protein tyrosine kinases (PTKs), the Src-family and the Syk/ZAP-70 family of PTKs. Following antigen-receptor stimulation, the Src-family of PTKs mediate the phosphorylation of tyrosine residues contained in a signalling motif localized in the TCR and BCR subunits. The phosphorylation of this signalling motif recruits the Syk/ZAP-70 family of PTKs into the antigen receptor complex. This mechanism requires the tandem SH2 domains in ZAP-70 complexing to two critically spaced phosphotyrosine residues within the signalling motif. The clustering of Syk/ZAP-70 and cross-talk between this family and the Src-PTKs regulates subsequent signalling events that lead to a variety of cellular responses, such as antibody secretion, lymphokine production, cytolytic activity, proliferation, differentiation and cell survival.

Amino Acid Sequence↗
PubMed2005

Chromosome abnormalities with prognostic impact in B-cell chronic lymphocytic leukemia.

The detailed analysis of the biologic features led to a rapid increase in clinically relevant information in CLL. The recognition of the prognostic role of IgVH hypermutation status and related phenotypic changes (CD38, ZAP-70 expression) as well as of chromosome abnormalities defined by cytogenetic analysis enabled a refined classification of the disease. Improvements in karyotyping and the introduction of fluorescence in situ hybridization (FISH) in routine hematological diagnostics raised the detection rate of chromosomal aberrations to approx. 60-80% in CLL. Among them, deletions of 17p and 11q have been associated with unfavorable prognosis. The deletion of the p53 locus (17p13) was described as the strongest independent predictor for aggressive behavior, resistance to chemotherapy and early death. On the contrary, an isolated deletion at 13q14 or a normal karyotype was related with a long survival. Classical and molecular cytogenetic analysis became an important tool for individual risk estimation. Unlike any other approaches, cytogenetic monitoring reflects the genetic heterogeneity and clonal growth dynamics during the course of the disease.

ADP-ribosyl Cyclase 1↗
PubMed2004

Advances in the understanding of biology and prognosis in chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) has a heterogeneous clinical course and outcome. Recent advances in the diagnosis and molecular characterization of CLL permit improved prediction of disease prognosis, which could result in better management. We review the biology and diagnosis of CLL and the role of analysis of immunoglobulin heavy-chain variable region mutation status, molecular cytogenetics (interphase fluorescence in situ hybridization), and the expression of CD38 and ZAP-70 in the evaluation and management of patients with CLL.

ADP-ribosyl Cyclase 1↗
PubMed1996

Analysis of the T-cell activation signaling pathway mediated by tyrosine kinases, protein kinase C, and Ras protein, which is modulated by intracellular cyclic AMP.

T-cell receptor (TCR) triggering by an anti-CD3 antibody or phytohemagglutinin (PHA) as well as the treatment with phorbol myristate acetate (PMA), a direct activator of protein kinase C (PKC), induces activation of Ras in T-lymphocytes (Downward, J. et al. (1990)) Nature 364, 719-723). In this paper, we studied the role of Ras in the process of TCR-mediated T-cell activation using a human lymphomic Jurkat cell line. The stimulatory effect of TCR cross-linking on Ras activation was inhibited by herbimycin A, a specific inhibitor of protein tyrosine kinases (PTKs), whereas PMA-induced Ras activation was not affected. On the other hand, calphostin C, a specific inhibitor of PKC, blocked not only PMA-induced, but also TCR-mediated formation of Ras.GTP. Furthermore, down-regulation of PMA-sensitive PKC severely impaired the activation of Ras in response to TCR-stimulation. Tyrosine-phosphorylation and translocation to the particulate fraction of phospholipase C-gamma 1 (PLC-gamma 1) were observed upon T-cell activation. Subcellular localization of PKC was also changed when the cells were stimulated with an anti-CD3 antibody or PMA. While TCR-stimulated translocation of PKC was observed only transiently, PMA-induced translocation of PKC was more sustained. These results suggest that the activation of PLC-gamma 1 by PTK and subsequent activation of PKC are important for TCR-mediated Ras activation in Jurkat cells. An activated form of Ras enhanced the activation of interleukin 2 (IL-2) promoter by TCR stimulation or PMA treatment, although the activated Ras by itself was insufficient for IL-2 promoter activation. On the other hand, a dominant-inhibitory Ras diminished almost completely the activation of IL-2 promoter induced by PMA plus calcium ionophore, indicating that Ras is essential for transduction of T-cell activation signals. Cholera toxin (CTX), which directly activates Gs alpha, is shown to inhibit the activation of IL-2 promoter. TCR-mediated Ras activation, tyrosine phosphorylation and translocation of cellular proteins including ZAP-70, PLC-gamma 1 , and PKC. An activated Gs alpha mutant as well as dibutylyl cAMP (dBcAMP) also showed similar inhibitory effects.

Cell Compartmentation↗
PubMed1995

Rapid tyrosine phosphorylation of an 85,000 M(r) protein after phorbol ester stimulation of EL4 thymoma cells.

Early signalling events between protein kinase C (PKC) activation and lymphokine transcription were compared between phorbol ester-sensitive and -resistant EL4 cell lines which do or do not respond with interleukin 2 (IL2) production, respectively. The earliest event detected in the sensitive cell line was a dramatic increase in the tyrosine phosphorylation of an 85,000 M(r) protein (p85; 30 s), followed by mobility shifts of raf-1, mitogen-activated protein kinase kinase (MEK), mitogen-activated protein (MAP) kinase, lck and ZAP-70 (within 5 min). In contrast, p85 was not detected in the resistant cell line and lck and raf-1 mobility shifts exhibited delayed kinetics. Both vanadate and okadaic acid blocked the phorbol ester-stimulated p85 tyrosine phosphorylation in the sensitive cell line, suggesting that a phosphatase activity downstream of PKC activation may be required for p85 tyrosine phosphorylation. Characterization of p85 and its regulation should help elucidate some of the earliest events in this PKC pathway.

Animals↗
PubMed1993

CD4, CD8 and the role of CD45 in T-cell activation.

CD4, CD8 and CD45 regulate the coupling of the T-cell receptor complex (CD3-TCR) to tyrosine kinase activation and phosphorylation of key substrates such as phospholipase C gamma 1. CD4 and CD8 contribute to activation signals through their cytoplasmic association with p56lck. Expression of the zeta-chain is required for functional synergy of the T-cell receptor with CD4 in the activation of phospholipase C gamma 1, which probably reflects an interaction between p56lck and zeta-associated kinase ZAP-70. CD45 expression is required for CD3-TCR signaling. CD45 may positively regulate signaling by dephosphorylating the carboxyl-terminal tyrosine of p56lck and p59fyn, and negatively regulate signaling by dephosphorylation of other TCR-associated substrates directly. One ligand for CD45 receptor has been identified as the B cell CD22 molecule. The positive and negative effects of CD45 are sensitive to the composition of CD45 in receptor complexes, and may be regulated by specific associations of CD45 isoforms with other receptors such as CD3-TCR, CD2 and CD4.

Animals↗
PubMed2004

TCR signaling: another Abl-bodied kinase joins the cascade.

Protein tyrosine kinases have long been recognized as the most proximal actors in T-cell antigen receptor (TCR) signaling. Three non-receptor tyrosine kinase families (Src, ZAP-70 and Tec) are known to be critical, but a new study now shows that room needs to be made in this pathway for yet another protein tyrosine kinase family - Abl/Arg.

Adaptor Proteins, Signal Transducing↗
PubMed2006

Spontaneous development of autoimmune arthritis due to genetic anomaly of T cell signal transduction: Part 1.

A point mutation of the gene encoding ZAP-70, a key signal transduction molecule in T cells, results in spontaneous development of T cell-mediated autoimmune arthritis in mice homozygous for the mutation. The genetic anomaly alters differentiation and selection of T cells in the thymus, leading to thymic production of arthritogenic autoimmune T cells. The arthritogenic T cells persist in the periphery and elicit arthritis when activated by microbial agents that stimulate innate immunity. This model is instrumental in understanding how genetic variations in T cell signal transduction, together with environmental influences, contribute to the development of autoimmune disease.

Animals↗
PubMed2002

A dipalmitoyl peptide that binds SH3 domain, disturbs intracellular signal transduction, and inhibits tumor growth in vivo.

The Src homology 3 (SH3) domain plays a crucial role in protein-protein interactions during intracellular signal transduction. Blocking the SH3-mediated protein binding may inhibit the corresponding signal transduction, and thus, block the cellular functions. In this study, a peptide that specifically binds to SH3 domain could be introduced into the intracellular region when the peptides were conjugated with dipalmitic acid and appeared to disturb intracellular signaling. The dipalmitoyl peptide appeared to inhibit the phosphorylation of ZAP-70, Lck, and T-cell antigen receptor zeta in Jurkat. Mobilization of the intracellular free calcium induced by anti-CD3 antibody was reduced after treatment with the dipalmitoyl peptide. It was also observed that the dipalmitoyl peptide inhibited cancer cell growth both in vitro and in vivo. These results suggest that the dipalmitoyl peptide that presumably disturbs SH3-mediated signal transduction may have a potent anti-proliferative activity, which would be useful as a potential anti-tumor agent.

Animals↗
PubMed2000

Protein identification platform utilizing micro dispensing technology interfaced to matrix-assisted laser desorption ionization time-of-flight mass spectrometry.

An integrated protein microcharacterization/identification platform has been developed. The system has been designed to allow a high flexibility in order to tackle challenging analytical problems. The platform comprises a cooled microautosampler, an integrated system for microcolumn HPLC, and a capillary reversed-phase column that is interfaced to matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) system via a low internal volume flow-through microdispenser. The chromatographic separation is continuously transferred onto a MALDI target plate as discrete spots as the dispenser ejects bursts of droplets of the column effluent in a precise array pattern. A refrigerated microfraction collector was coupled to the outlet of the flow-through microdispenser enabling enrichment and re-analysis of interesting fractions. The use of target plates pre-coated with matrix simplified and increased the robustness of the system. By including a separation step prior to the MALDI-TOF-MS analysis and hereby minimizing suppression effects allowed us to obtain higher sequence coverage of proteins compared to conventional MALDI sample preparation methodology. Additionally, synthetic peptides corresponding to autophosphorylated forms of the tryptic fragment 485-496 (ALGADDSYYTAR) of tyrosine kinase ZAP-70 were identified at sensitivities reaching 150 amol.

Amino Acid Sequence↗
PubMed2000

Characterisation and specificity of two single-chain Fv antibodies directed to the protein tyrosine kinase Syk.

In order to obtain single chain Fv fragments (scFv) specific for the protein tyrosine kinase Syk, we screened a human synthetic phage-display library. Two glutathione S-transferase (GST):Syk fusion proteins containing both SH2 domains of Syk were used to perform three rounds of selection of the library. Among the scFv fragments resulting from the third round of selection, the ones specific for the GST portion of the fusion proteins were eliminated by performing enzyme-linked immunosorbent assay tests on GST:Syk versus GST coated plates, and the monoclonal scFv fragments binding only to the GST:Syk coated plates with high affinities were further analysed. We report here the in vitro characterisation of G4G11 and G6G2 anti-Syk scFvs. G4G11 shows the best performance in immunoprecipitation and immunofluorescence experiments, and G6G2 is able to detect Syk in immunoprecipitation, immunofluorescence and on Western blots. Both scFvs are also able to detect the phosphorylated form of Syk, and neither of them binds to Zap-70, the other member of the Syk family of protein tyrosine kinases.

Animals↗
PubMed2001

Prolactin signaling to pim-1 expression: a role for phosphatidylinositol 3-kinase.

Sublines of the lactogen-dependent, rat pre-T Nb2 lymphoma are useful as a model for the investigation of prolactin (PRL) signaling mechanisms, regulation of transcription of target genes, and the immunomodulatory and anti-apoptotic actions of the hormone in T lymphocytes. In the present study, coupling of various tyrosine, serine/threonine, and phospholipid kinase signaling mechanisms to PRL-stimulated Nb2-11 cell proliferation and expression of the protooncogene, pim-1, was investigated utilizing pharmacologic antagonists of a broad spectrum of tyrosine kinases (tyrphostin A25), and the specific enzymes, Jak2 (tyrphostin B42) and ZAP-70 (piceatannol), as well as mitogen-activated protein kinase (MAPK, PD98059), protein kinase C (PKC, calphostin C), and phosphatidylinositol 3-kinase (PI3-kinase, LY294002). Inhibition of each pathway attenuated PRL-stimulated Nb2-11 cell proliferation in a concentration-dependent manner. Blockade of MAPK was the least efficacious; it inhibited proliferation maximally by 60%. Northern blot analysis of pim-1 expression in antagonist-treated cells revealed that MAPK, Jak2 and PI3-kinase appeared to signal to initiation of pim-1 transcription; its expression was attenuated by each of the antagonists. In other experiments, PRL was shown to rapidly activate a downstream effector of PI3-kinase, Akt, and this effect was also blocked by LY294002. It is concluded that PRL-stimulated Nb2 cell proliferation requires participation of each of the signaling pathways investigated. Moreover, hormone-mediated expression of pim-1 appears to reflect signaling by MAPK, Jak2, and PI3-kinase.

Animals↗
PubMed2000

The ITAM-bearing transmembrane adaptor DAP12 in lymphoid and myeloid cell function.

DAP12, an ITAM-bearing transmembrane adaptor protein, associates non-covalently with receptors in natural killer (NK) and myeloid cells, and provides signaling function via the Syk and ZAP-70 tyrosine kinase activation pathways. Humans and mice lacking DAP12 (DAP12(-/-)) show normal development of hematopoietic cells. However, DAP12(-/-) humans develop presenile dementia and bone cysts, and DAP12(-/-) mice show impaired immune responses.

Adaptor Proteins, Signal Transducing↗
PubMed1996

Immunodeficiencies caused by genetic defects in protein kinases.

The recognition that defects of ZAP-70 and, more recently, of JAK3 kinase in humans result in severe combined immunodeficiency, and the demonstration that targeting of these and other protein-kinase genes in mice also leads to immunodeficiency, have highlighted the crucial role that these proteins play in T-cell differentiation and activation.

Animals↗
PubMed2001

Activation of Syk protein tyrosine kinase through interaction with integrin beta cytoplasmic domains.

Syk protein tyrosine kinase is essential for immune system development and function [1]and for the maintenance of vascular integrity [2,3]. In leukocytes, Syk is activated by binding to diphosphorylated immune receptor tyrosine-based activation motifs (pITAMs)[1]. Syk can also be activated by integrin adhesion receptors [4,5], but the mechanism of its activation is unknown. Here we report a novel mechanism for Syk's recruitment and activation, which requires that Syk bind to the integrin beta3 cytoplasmic tail. We found that both Syk and the related kinase ZAP-70 bound the beta3 cytoplasmic tail through their tandem SH2 domains. However, unlike Syk binding to pITAMs, this interaction was independent of tyrosine phosphorylation and of the phosphotyrosine binding function of Syk's tandem SH2 domains. Deletion of the four C-terminal residues of the beta3 cytoplasmic tail [beta3(759X)] decreased Syk binding and disrupted its physical association with integrin alphaIIbbeta3. Furthermore, cells expressing alphaIIbbeta3(759X) failed to exhibit Syk activation or lamellipodia formation upon cell adhesion to the alphaIIbbeta3 ligand, fibrinogen. In contrast, FAK phosphorylation and focal adhesion formation were unimpaired by this mutation. Thus, the direct binding of Syk kinase to the integrin beta3 cytoplasmic tail is a novel and functionally significant mechanism for the regulation of this important non-receptor tyrosine kinase.

Amino Acid Sequence↗