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At least 667 records · Page 37Linked to original sources
PubMed1994

The beta D-sheet residues of the Lck-derived SH2 domain determine specificity of the interaction with tyrosine-phosphorylated ligands in Ramos B cells.

Src homology 2 (SH2) domains are noncatalytic regions that are conserved among a group of cellular signaling proteins. SH2 domains share the common property of binding phosphotyrosine-containing peptides. Previously, we showed that SH2 domains expressed as recombinant glutathione S-transferase-fusion proteins (GST-SH2) from GTPase-activating protein, Shc, zeta-chain-associated protein tyrosine kinase Zap-70, and Src-like tyrosine kinases precipitated distinct sets of phospho-proteins from activated B cells. To determine the intrinsic structural motifs responsible for the binding specificity within the different SH2 domains, we created chimeric SH2 domains especially focusing on crystal structure-defined contact residues. Recombinant SH2 domains of Lck, Zap-70, and Shc were tested in Ramos B cell lysates for phosphotyrosine-dependent protein binding. Biomolecular interaction analysis (BIAcore) was used to characterize the interaction between the various recombinant SH2 molecules and defined phosphorylated peptides. In agreement with the crystal structure data from the Src and the Lck SH2 domains, our results show that most of the "specificity information" of the Lck SH2 domain is provided by the beta D-sheet, located downstream of the SH2 conserved consensus motif GTFLVRES. In addition, the overall affinity is critically influenced by residues located at the N terminus of the SH2 domain.

Amino Acid Sequence↗
PubMed1996

The effect of a 1-week administration of cetirizine on the chemotaxis and superoxide anion production of neutrophils from healthy volunteers.

The aim of the present investigation was to examine the effect of a 1-week oral administration of the anti-allergic drug cetirizine on healthy volunteer neutrophil chemotaxis and superoxide anion (O2-) production. Eight male volunteers were selected after clinical examination and laboratory tests. Neutrophils were isolated from peripheral blood using a discontinuous density gradient. Spontaneous migration and platelet activating factor (PAF, 10(-6) and 10(-8) M) or zymosan-activated plasma (ZAP)-induced chemotaxis were studied in a 48-well microchemotaxis chamber. Basal and phorbol 12-myristate 13-acetate (PMA, 30 nM) stimulated O2- production were measured spectro-photometrically. Cetirizine (10 mg per day) was given orally during 1 week. Both neutrophil chemotaxis and O2- production were assessed before and 2 h, 24 h, and 1 week after orally administered cetirizine. Plasma cetirizine levels were monitored by high performance liquid chromatography (HPLC) with ultraviolet detection. Spontaneous neutrophil migration and PAF-or ZAP-induced chemotaxis showed no significant variation before or at various intervals after the initiation of treatment with cetirizine. Basal and PMA-stimulated neutrophil O2-production was also not affected by cetirizine. The maximum concentration attained by cetirizine (Cmax) was 293 +/- 38 ng/ml and generally peaked (Tmax) within 1.9 +/- 0.7 h. We conclude that the administration of cetirizine for 1 week does not alter human neutrophil chemotaxis and O2- production.

Adult↗
PubMed1997

Differential T cell signaling induced by antagonist peptide-MHC complexes and the associated phenotypic responses.

Certain changes in TCR contact residues have been shown to have profound effects on the capacity of a peptide Ag to stimulate a T cell response. Although some of these changes apparently lead to a complete loss of the ability to interact with the TCR, others result in partial agonist activity (e.g., cytokine production without proliferation) or antagonist activity (i.e., the capacity to inhibit the engagement to the TCR by Ag). We show MHC class II-restricted antagonist activity was associated with a differential pattern of early tyrosine phosphorylation events that was characterized by a preponderance of phosphorylation of low molecular mass TCRzeta and the failure to phosphorylate Zap-70. These early tyrosine phosphorylation patterns are the same as those previously described for partial agonists. Thus, a partial agonist phenotype such as anergy induction cannot be ascribed in a causal manner to this pattern of tyrosine phosphorylation. We further extend the studies of signal transduction elicited by agonist and antagonist peptides by characterizing differential recruitment of Zap-70 associated with TCRzeta isoforms and differential phosphorylation of p120 proto-oncogene c-Cbl. Another early event following TCR engagement by Ag, down-modulation of the TCR, was studied with antagonist peptides. We show that antagonist peptides do not cause TCR down-modulation. This failure may represent a mechanism by which antagonists inhibit antigen-mediated stimulation of T cells.

Amino Acid Sequence↗
PubMed1998

Peripheral T lymphocytes from women with breast cancer exhibit abnormal protein expression of several signaling molecules.

We examined signaling molecules of peripheral blood T lymphocytes obtained from women with breast cancer. In 6 of 14 patients, T lymphocytes displayed an impaired ability to translocate NFêB p65 (Rel-A) following activation by anti-CD3 and IL-2. This observation was made despite normal cytoplasmic levels of the Rel-A protein. We also detected abnormally low levels of the signaling molecules T-cell receptor (TCR)-zeta, ZAP-70 and p56lck in 4 of 14 breast cancer patients, i.e., defects in T-cell signaling molecules. T lymphocytes from 6 of the 14 patients also exhibited an increased expression of the dual specificity phosphatase, map kinase phosphatase-1 (MKP-1). MKP-1 inactivates MAP kinase and therefore may interfere with the activation of c-jun and c-fos. Abnormalities of I or more signaling molecules were found in 9 of 14 patients; however, only 3 patients had T cells that exhibited all 5 defects. Our data have implications for the detection of potentially dysfunctional T cells in patients with cancer. For example, the analysis of only 1 signaling molecule may allow patients with significant defects in T-cell signaling to go unnoticed. Finally, despite impaired Rel-A translocation, T cells were capable of transcribing IL-2. Impairments in the translocation of Rel-B and c-Rel further suggest that the NFKB family members Rel-A, Rel-B and c-Rel are not required for the transcription of IL-2 in the peripheral T lymphocytes of patients with breast cancer.

Adult↗
PubMed2000

Flow cytometric analysis of immunoprecipitates: high-throughput analysis of protein phosphorylation and protein-protein interactions.

BACKGROUND: Activation-induced protein phosphorylation can be studied by Western blotting, but this method is time consuming and depends on the use of radioactive probes for quantitation. We present a novel assay for the assessment of protein phosphorylation based on latex particles and flow cytometry. METHODS: This method employs monoclonal antibodies coupled to latex particles to immobilize protein kinase substrates. Their phosphorylation status is assessed by reactivity with phosphoepitope-specific antibodies. The amount of immobilized protein on the particles was analyzed by direct or indirect immunofluorescence with antibodies to nonphosphorylated epitopes. RESULTS: The assay allowed measurement of phosphorylation of multiple protein kinase substrates in stimulated T cells, including the zeta chain of the T-cell receptor, ZAP-70, CD3, CD5, SHP-1, and ERK-2, using 1-3 microg of total cell protein per sample. The assay provided high resolution of kinetics of phosphorylation and dephosphorylation. Interactions of protein kinase substrates with associated signaling molecules were demonstrated. CONCLUSIONS: The novel assay allows high-throughput quantitative measurement of protein modifications during signal transduction.

Animals↗
PubMed1996

Differential and multiple binding of signal transducing molecules to the ITAMs of the TCR-zeta chain.

A biotin-streptavidin-based technique was developed for high affinity, unidirectional, and specific immobilization of synthetic peptides to a solid phase. Biotinylated 23-mer carboxamide peptides corresponding to the three immunoreceptor tyrosine-based activation motifs (ITAMs) of the T cell antigen receptor associated zeta-chain (TCR-zeta) in their bis-, mono-, or unphosphorylated forms were used to study the binding of cellular proteins from human Jurkat T cells to these signal transduction motifs. The protein tyrosine kinase ZAP-70 bound specifically to all bisphosphorylated peptides but not to the mono- or unphosphorylated peptides. In contrast, Shc, phosphatidylinositol 3-kinase (Pl3K), Grb2, and Ras-GTPase activating protein (GAP) bound with different affinities to the bis- or monophosphorylated peptides, while the Src family protein tyrosine kinase (PTK) Fyn did not bind specifically to any of the tested peptides. The different preferences of the studied signaling molecules for distinct ITAMs, and in particular the binding of some of them preferentially to monophosphorylated peptides, suggests that the TCR-zeta may bind multiple signaling molecules with each ITAM binding a unique set of such molecules. In addition, partial phosphorylation of the ITAMs may result in recruitment of different proteins compared to double phosphorylation. This may be crucial for coupling of the TCR to various effector functions under different conditions of receptor triggering.

Amino Acid Sequence↗
PubMed2000

Autonomous induction of proliferation, JNK and NF-alphaB activation in primary resting T cells by mobilized CD28.

Induction of proliferation in primary resting T cells requires engagement of both the antigen-specific TCR and the co-stimulatory receptor CD28. Here we report that CD28 functions as an autonomous mitogenic receptor which is mobilized by TCR signaling through cytoskeletal rearrangement. Shortcutting of TCR-dependent CD28 recruitment by stimulation with monoclonal antibodies specific for mobilized CD28 results in maximum proliferation and IL-2 secretion in primary resting T cells without activation of ZAP-70, a central component of the TCR's signal transduction machinery. Engagement of mobilized CD28 fully activates the c-Jun N-terminal kinase cascade and translocation of NF-alphaB, two key targets of signal integration in co-stimulation. We propose a two-step activation model for co-stimulation in primary resting T cells in which antigen recognition recruits co-stimulatory receptors which then autonomously transduce signals promoting T cell proliferation.

Animals↗
PubMed2000

Two YxxL segments of a single immunoreceptor tyrosine-based activation motif in the CD3zeta molecule differentially activate calcium mobilization and mitogen-activated protein kinase family pathways.

Immunoreceptor tyrosine-based activation motifs (ITAM), consisting of two YxxL segments, transmit signals leading to IL-2 gene activation in T cells. We investigated here the functional difference in signal transduction between these two YxxL segments in the CD3zeta membrane-proximal ITAM. N-terminal YxxL mutants failed to induce ZAP-70 phosphorylation, elevation of intracellular Ca2+ concentration ([Ca2+]i) or extracellular signal-regulated kinase (ERK) activation even in the presence of CD28 co-stimulation, whereas a mutant of the leucine residue at the C-terminal YxxL segment retained the ability to induce these events although this mutation abrogated the ability to induce IL-2 gene activation. In marked contrast to ERK activation, c-Jun N-terminal kinase (JNK) activation was observed in all mutants when co-stimulated with CD28. The mutant of the leucine residue at the C-terminal YxxL segment had a defect in the transcriptional activation at the NF-AT cis-element, which was restored to wild-type level by addition of a Ca2+ ionophore, suggesting that the intensity and/or duration of [Ca2+]i elevation defines the threshold of T cell activation in this mutant. Our data collectively indicate that the activation pathways of ERK, JNK and Ca2+ mobilization are differentially regulated through YxxL segments of an ITAM.

Amino Acid Sequence↗
PubMed2001

A regulatory role for cytoplasmic YVKM motif in CTLA-4 inhibition of TCR signaling.

CTLA-4 negatively regulates TCR signaling, although the molecular basis for this effect has yet to be elucidated. The cytoplasmic YVKM motif, while binding to phosphatidylinositol 3-kinase, SHP-2 and the AP-1/AP-2 clathrin adaptor complexes, has been reported to play no role in CTLA-4 function. In contrast, in this study, we demonstrate that, although not essential, the YVKM motif contributes to optimal CTLA-4 blockage of TCRzeta or combined TCRzeta/CD28 signaling. Significantly, dependency on the YVKM motif varied with the mode of anti-receptor presentation, where soluble antibody ligation was more dependent on the presence of the motif than immobilized antibody. Previous studies have mainly relied on the use of immobilized antibody. Neither SHP-2 binding, alterations in TCRzeta chain phosphorylation, nor ZAP-70 recruitment was involved in CTLA-4 wild-type or mutant inhibition. Overall, our findings clearly implicate the YVKM motif in optimal CTLA-4 function.

Abatacept↗
PubMed2002

Perturbation of naive TCR transgenic T cell functional responses and upstream activation events by anti-CD4 monoclonal antibodies.

It is well established that non-depleting anti-CD4 monoclonal antibodies (mAb) have potent immunomodulatory properties in vivo and as such can induce a profound state of tolerance. Receptor blockade, CD4 modulation, or the transmission of a negative signal have all been proposed to explain their effects, however their precise mechanism of action, particularly at the level of cellular signaling, remains obscure. Experiments were thus carried out to examine the underlying mechanisms of action of two non-depleting anti-mouse CD4 mAb, YTS177 and KT6, which differ in their ability to modulate CD4 expression. The effects of the mAb were examined on CD4(+) T cells derived from D0.11.10 TCR transgenic mice. Functional studies revealed that both mAb could effectively block antigen-driven proliferation and IL-2 production but had only modest effects at higher peptide doses. Importantly, mAb pre-treatment of T cells stimulated by sub-optimal peptide seemed to induce an anergy-like state making them unresponsive to subsequent re-stimulation. Analysis of intracellular signaling demonstrated that certain key upstream events such as the phosphorylation of Zap-70 and LAT were also blocked by mAb pretreatment which may be due to interference with stable T cell-APC conjugate formation.

Adaptor Proteins, Signal Transducing↗
PubMed2005

Chemical inhibitors when timing is critical: a pharmacological concept for the maturation of T cell contacts.

Cellular signal transduction proceeds through a complex network of molecular interactions and enzymatic activities. The timing of these molecular events is critical for the propagation of a signal and the generation of a specific cellular response. To define the timing of signalling events, we introduce the combination of high-resolution confocal microscopy with the application of small-molecule inhibitors at various stages of signal transduction in T cells. Inhibitors of Src-family tyrosine kinases and actin dynamics were employed to dissect the role of the lymphocyte-specific tyrosine kinase Lck in the formation and maintenance of T cell receptor/CD3-dependent contacts. Anti-CD3epsilon-coated coverslips served as a highly defined stimulus. The kinetics of the recruitment of the yellow fluorescent protein-tagged signalling protein ZAP-70 were detected by high-resolution confocal microscopy. The analysis revealed that at 5 min after receptor engagement, Lck activity was required for maintenance of contacts. In contrast, after 20 min of receptor engagement, the contacts were Lck-independent. The relevance of the timing of inhibitor application provides a pharmacological concept for the maturation of T cell-substrate contacts.

Animals↗
PubMed1994

In vitro characterization of major ligands for Src homology 2 domains derived from protein tyrosine kinases, from the adaptor protein SHC and from GTPase-activating protein in Ramos B cells.

Antigen receptors of B lymphocytes transmit their activation signal to the cell interior by associating with and activation of specific non-receptor tyrosine kinases. Most of these kinases as well as other cytoplasmic effectors contain at least one Src homology 2 (SH2) domain, known to bind tyrosine-phosphorylated proteins. We examined the binding specificity of SH2 domains from different signaling molecules in B cells and found that each of the SH2 domains tested bound distinct subsets of stimulation-dependent phosphoproteins in vitro. SH2 domains from Src-like tyrosine kinases bound predominantly to the HS1 phosphoprotein. The tandem SH2 domains of the ZAP-70 tyrosine kinase bound to phosphorylated Ig-beta but only weakly to Ig-alpha. Also the SHC-derived SH2 domain formed complexes with the tyrosine-phosphorylated Ig-alpha/beta heterodimer, while the C- and N-terminal SH2 domains of GTPase-activating protein displayed completely different binding preferences. These results suggest that cytoplasmic effector molecules can be recruited to the activated B cell receptor in an SH2-phosphotyrosine-mediated manner. The data also provide a possible explanation for the notion that Ig-alpha and Ig-beta might couple to different biochemical pathways.

Adaptor Proteins, Signal Transducing↗
PubMed1997

The effects of CD3, CD4 and CD28 signaling on lymphocytes during human immunodeficiency virus-1 infection.

We investigated the effects of early human immunodeficiency virus-1 infection (HIV-1) on CD4- and CD28-mediated co-signaling of the T cell receptor (TCR)/CD3 complex in peripheral blood lymphocytes (PBL). CD4 ligation either alone or in conjunction with TCR occupancy resulted in abrogated signaling shown by impaired co-association of the tyrosine kinase ZAP-70 with the CD3-zeta chains in virally infected PBL. In addition, down-regulation of CD4-associated TCR signaling resulted in diminished tyrosine phosphorylation of mitogen-activated protein kinase (MAPK), a serine threonine kinase which is critically involved in the regulation of transcription factors. Furthermore, these aberrant CD4-driven signals rendered HIV-1-infected PBL susceptible to activation-induced cell death. By contrast, cross-linking of the TCR/CD3 complex with the CD28 receptor improved tyrosine phosphorylation of MAPK and salvaged infected PBL from activation-induced cell death. Our data demonstrate the importance of appropriate CD3, CD4 and CD28 co-stimulatory function to prevent apoptosis. The CD4-mediated signaling defects of the TCR could contribute to the loss of immunocompetent cells during HIV-1 infection via activation-induced cell death, whereas stimulation through the CD28 pathway could reverse these detrimental effects.

Apoptosis↗
PubMed2003

Development of T cells expressing an altered TCR complex.

Normal mouse T cells may express alternative TCR complexes containing the FcepsilonR gamma chain (FcRgamma) rather than the zeta homodimer that is present in conventional TCR complexes. While these T cells could play critical roles in regulating immunity, the role of alternative TCR complexes and their requirement for signaling molecules in T cell development remains unknown. We show thatexpression of an FcRgamma transgene in zeta chain-deficient mice (FcRgammaTG, zetaKO mice) reduced the percentage and number of CD4(+) T cells present in these animals, when compared to C57BL/6 mice. Further studies of FcRgammaTG, zetaKO mice expressing the DO11.10 TCR (DOTCR) transgene showed that, when compared to mice expressing conventional TCR complexes, the development of CD4(+), DOTCR(+) thymocytes was altered in mice of different MHC backgrounds and required the presence of zeta-associated protein (ZAP)-70 and lck kinases. The CD4(+), DOTCR(+) T cells bearing alternative TCR complexes have impaired Ca(2+) flux and proliferative response to stimulation. Altogether, these results suggest that the altered development of CD4(+) T cells is not due to qualitative differences in TCR-mediated signals, but more consistent with the hypothesis that it is due to reduced signaling strength mediated through the FcRgamma chain containing only one immunoreceptor tyrosine-based activation motif.

Animals↗
PubMed2006

The negative regulators Foxj1 and Foxo3a are up-regulated by a peptide that inhibits systemic lupus erythematosus-associated T cell responses.

A peptide (hCDR1) based on the complementarity determining region-1 of an anti-DNA antibody ameliorates systemic lupus erythematosus (SLE) in induced and spontaneous lupus models. Our objectives were to determine the effects of hCDR1 on TCR signaling and on its negative regulators, Foxj1 and Foxo3a. BALB/c mice were immunized with the SLE-inducing anti-DNA antibody, designated 16/6Id, and treated with hCDR1. hCDR1 treatment specifically inhibited IFN-gamma secretion by T cells in association with down-regulated T-bet expression and NF-kappaB activation; however, GATA-3 expression was not affected. Furthermore, TCR signaling (ZAP-70 phosphorylation) was inhibited, and the mRNA expression of the two modulators of Th1 activation, Foxj1 and Foxo3a, was significantly up-regulated. The latter were also elevated in SLE-afflicted (NZBxNZW)F1 mice that were treated with hCDR1. Addition of TGF-beta, which was elevated following treatment with hCDR1, to T cells from 16/6Id immunized mice, up-regulated Foxj1 and Foxo3a mRNA expression, similarly to hCDR1. In contrast, anti-TGF-beta antibodies added to hCDR1-treated T cells abrogated its effect. Thus, hCDR1 elevates TGF-beta, which contributes to the up-regulation of T cell Foxj1 and Foxo3a expression, leading to inhibition of NF-kappaB activation and IFN-gamma secretion, which is required for the maintenance of SLE.

Animals↗
PubMed2005

Signature of B-CLL with different prognosis by Shrunken centroids of surface antigen expression profiling.

With the aim of identifying the immunophenotypic profile of B-cell chronic lymphocytic leukemia (B-CLL) subsets with different prognosis, we investigated by flow cytometry the expression of 36 surface antigens in 123 cases, all with survivals. By analyzing results with unsupervised (hierarchical and K-means clustering) algorithms, three distinct immunophenotypic groups (I, II, and III) were identified, group I (51/123) with longer survivals, as compared to the group II (36/123) and III (36/123). The immunophenotypic signatures of these groups, as determined by applying the nearest Shrunken centroids method as class predictor, were characterized by the coordinated and differential expression of 12 surface markers, that is, group I: above-average expression of CD62L, CD54, CD49c, and CD25, below-average expression of CD38; group II: above-average expression of CD38, CD49d, CD29, and CD49e; and group III: below-average expression of the above markers, overexpression of CD23, CD20, SmIg, and CD79b. As opposed to groups II-III, group I B-CLLs lacked expression of ZAP-70 and activation-induced cytidine deaminase in the majority of cases, while more frequently had mutated IgV(H) genes and IgV(H) mutations consistent with antigen-driven selection. Our findings contribute to improve the immunophenotypical identification of disease subsets with different prognosis and suggest a set of surface antigens to be employed as prognosticators in routine diagnostic/prognostic procedures.

Adult↗
PubMed2025

Infectious Subgenomic Amplicon Strategies for Japanese Encephalitis and West Nile Viruses.

Classical methods for constructing infectious cDNA clones of flaviviruses are often hindered by instability and toxicity. The Infectious-Subgenomic-Amplicons (ISA) method is an advancement which utilizes overlapping DNA fragments representing viral genomic sequence and in-cell recombination to bypass bacterial plasmid assembly. However, the ISA method has limitations due to the toxicity of some ISA DNA fragments in bacteria during synthetic production. We validated modified ISA strategies for producing toxic ISA Japanese encephalitis virus (JEV) and West Nile virus (WNV) DNA fragments. Three approaches were explored, including subdividing toxic DNA fragments into two sub-fragments for synthetic clonal production, using a low-copy bacterial plasmid, and subdividing the toxic DNA fragments into four short overlapping sub-fragments, each up to 1.8 kb. The latter novel approach in ISA applications enabled the synthesis of entirely bacteria-free ISA DNA fragments. Our results demonstrate that subdividing toxic fragments into sub-fragments smaller than 1.8 kb for synthesis is the efficient strategy, circumventing the need for bacterial plasmids and ensuring rapid production of synthetic flaviviruses. This method also shortens the production timeline. We also compared the efficacy of JEV and WNV ISA in zinc finger antiviral protein 1 (ZAP) wild-type and knockout cells and found that knockout cells may be more effective for ISA rescue of flaviviruses, including CpG-enriched strains for live attenuated vaccines. The validated modified ISA strategies provide an efficient approach for producing synthetic JEV and WNV. This will enable rapid research during outbreaks of emerging flaviviruses by facilitating the quick generation of new virus variants.

West Nile virus↗
PubMed1998

Incorporation of leucocyte GPI-anchored proteins and protein tyrosine kinases into lipid-rich membrane domains of COS-7 cells.

Several human leucocyte surface glycoproteins and two lymphoid protein kinases were transiently expressed in monkey COS-7 fibroblastoid cells. All glycosylphospha-tidylinositol (GPI)-anchored proteins (CD14, CD16B, CD48, CD59, CD87 and GPI-anchored versions of CD2 and CD25) and protein tyrosine kinase (PTK) Lck but not transmembrane proteins (CD2, CD4, CD5, CD6, CD8) and PTK ZAP-70 were in part localized in buoyant, lipid-rich, detergent-resistant membrane GPI-microdomains of the COS cells. Endogenous GPI-microdomains of COS cells appear to be, in contrast to those present in leucocytes, essentially devoid of associated PTKs. Our results indicate that GPI-anchor is sufficient to target proteins to these membrane specializations even if expressed ectopically. Moreover, the N-terminal double acylation of the PTK Lck appears to be functional also in COS cells and targets the enzyme to the membrane GPI-microdomains implicated in receptor signalling.

Acylation↗