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At least 1,099 records · Page 61Linked to original sources
PubMed2000

The Src kinase p56(lck) up-regulates VLA-4 integrin affinity. Implications for rapid spontaneous and chemokine-triggered T cell adhesion to VCAM-1 and fibronectin.

In circulating lymphocytes, the VLA-4 integrin preexists in multiple affinity states that mediate spontaneous tethering, rolling, and arrest on its endothelial ligand, vascular cell adhesion molecule-1 (VCAM-1). The regulation and function of VLA-4 affinity in lymphocytes has never been elucidated. We show here that p56(lck), the major Src kinase in T cells, is a key regulator of high affinity VLA-4. This high affinity is essential for the rapid development of firm adhesion of resting T cells to VCAM-1 and to their extracellular matrix ligand, fibronectin. Lck-regulated VLA-4 function does not require intact TCR nor several key components of the TCR signaling pathway, including ZAP-70 and SLP-76. Furthermore, stimulation of p56(lck) by the phosphatase inhibitor, pervanadate, triggers firm VLA-4-dependent adhesion to VCAM-1. Although Lck is not required for chemokine receptor signaling to mitogen-activated protein kinase, the presence of Lck-regulated high affinity VLA-4 also facilitates firm adhesion triggered by the chemokine, SDF-1, at short-lived contacts. Surprisingly, bond formation rates, ability to tether cells to VLA-4 ligand, and VLA-4 tether bond stability under shear flow are not affected by VLA-4 affinity or Lck activity. Thus, the ability of high affinity VLA-4 to arrest cells on VCAM-1 under flow arises from instantaneous post-ligand strengthening rather than from increased kinetic stability of individual VLA-4 bonds. These results suggest that p56(lck) maintains high affinity VLA-4 on circulating lymphocytes, which determines their ability to strengthen VLA-4 adhesion and rapidly respond to proadhesive chemokine signals at endothelial sites.

Blotting, Western↗
PubMed2002

A novel Src homology 2 domain-containing molecule, Src-like adapter protein-2 (SLAP-2), which negatively regulates T cell receptor signaling.

We have cloned a novel adapter protein containing Src homology 2 and Src homology 3 domains similar to the Src family of tyrosine kinases. This molecule lacks a catalytic tyrosine kinase domain and is related to a previously identified protein, Src-like adapter protein (SLAP), and is therefore designated SLAP-2. Northern blot analysis indicates that SLAP-2 is predominantly expressed in the immune system. Jurkat T cells express SLAP-2 protein and overexpression of SLAP-2 in these cells negatively regulates T cell receptor signaling as assessed by interleukin-2 promoter or NF-AT promoter reporter constructs. Mutational analysis revealed that an intact SH2 domain of SLAP-2 is essential for this inhibitory effect, whereas mutation of the SH3 domain alone has no effect. This inhibitory effect is upstream of the activation of Ras and increase of intracellular calcium levels, as no inhibition was observed when the cells were activated by phorbol ester plus ionomycin. SLAP-2 interacts with Cbl in vivo in a phosphorylation independent manner and with ZAP-70 and T cell receptor zeta chain upon T cell receptor activation. Finally, we show that the mutation of a predicted myristoylation site within the NH(2)-terminal of SLAP-2 is essential for its inhibitory effect. This report therefore implicates SLAP and SLAP-2 as a family of adapter proteins that negatively regulate T cell receptor signaling.

Adaptor Proteins, Signal Transducing↗
PubMed2003

Interaction of SAP-1, a transmembrane-type protein-tyrosine phosphatase, with the tyrosine kinase Lck. Roles in regulation of T cell function.

SAP-1 is a transmembrane-type protein-tyrosine phosphatase that is expressed in most tissues but whose physiological functions remain unknown. The cytoplasmic region of SAP-1 has now been shown to bind directly the tyrosine kinase Lck. Overexpression of wild-type SAP-1, but not that of a catalytically inactive mutant of SAP-1, inhibited both the basal and the T cell antigen receptor (TCR)-stimulated activity of Lck in human Jurkat T cell lines. Lck served as a direct substrate for dephosphorylation by SAP-1 in vitro. Overexpression of wild-type SAP-1 in Jurkat cells also: (i) inhibited both the activation of mitogen-activated protein kinase and the increase in cell surface expression of CD69 induced by TCR stimulation; (ii) reduced the extent of the TCR-induced increase in the tyrosine phosphorylation of ZAP-70 or that of LAT; (iii) reduced both the basal level of tyrosine phosphorylation of p62dok, as well as the increase in the phosphorylation of this protein induced by CD2 stimulation; and (iv) inhibited cell migration. These results thus suggest that the direct interaction of SAP-1 with Lck results in inhibition of the kinase activity of the latter and a consequent negative regulation of T cell function.

Animals↗
PubMed2005

Membrane localization and function of Vav3 in T cells depend on its association with the adapter SLP-76.

The Vav family of guanine exchange factors plays a critical role in lymphocyte proliferation, cytoskeletal reorganization, and gene transcription upon immunoreceptor engagement. Although the role of Vav1 in T cells is well documented, the role of Vav3 is less clear. We investigated the subcellular localization of Vav3 during T cell activation. We report here that phosphorylation of Vav3 on tyrosine residue Tyr(173) is not required for T cell receptor (TCR)-induced Vav3 membrane translocation or immunological synapse (IS) recruitment, but mutation of this residue enhanced TCR-induced nuclear factor of activated T cells (NFAT) activation. However, Vav3 mutants either containing an Src homology 2 (SH2)-disabled point mutation (R697L) or lacking its SH3-SH2-SH3 domains were unable to bind SLP-76 did not translocate to the membrane or to the IS and furthermore failed to activate NFAT. Importantly, the membrane translocation of Vav3 was abrogated in Lck, ZAP-70, LAT, and SLP-76-deficient T cells, where Vav3 binding to SLP-76 was disrupted. Finally, we confirmed and underlined the critical role of Vav3 in NFAT activation by knocking down Vav3 expression in Vav1-deficient T cells. Altogether, our data show that TCR-induced association of Vav3 with SLP-76 is required for its membrane/IS localization and function.

Adaptor Proteins, Signal Transducing↗
PubMed2005

The Epstein-Barr virus protein, latent membrane protein 2A, co-opts tyrosine kinases used by the T cell receptor.

Epstein-Barr virus (EBV) is the causative agent of infectious mononucleosis and is associated with several human malignancies. The EBV protein latent membrane protein 2A (LMP2A) promotes viral latency in memory B cells by interfering with B cell receptor signaling and provides a survival signal for mature B cells that have lost expression of surface immunoglobulin. The latter function has suggested that LMP2A may enhance the survival of EBV-positive tumors. EBV is associated with several T cell malignancies and, since LMP2A has been detected in several of these disorders, we examined the ability of LMP2A to transmit signals and interfere with T cell receptor signaling in T cells. We show that LMP2A is tyrosine-phosphorylated in Jurkat TAg T cells, which requires expression of the Src family tyrosine kinases, Lck and Fyn. Lck and Fyn are recruited to the tyrosine-phosphorylated Tyr112 site in LMP2A, whereas phosphorylation of an ITAM motif in LMP2A creates a binding site for the ZAP-70/Syk tyrosine kinases. LMP2A also associates through its two PPPPY motifs with AIP4, a NEDD4 family E3 ubiquitin ligase; this interaction results in ubiquitylation of LMP2A and serves to regulate the stability of LMP2A and LMP2A-kinase complexes. Furthermore, stable expression of LMP2A in Jurkat T cells down-regulated T cell receptor levels and attenuated T cell receptor signaling. Thus, through recruiting tyrosine kinases involved in T cell receptor activation, LMP2A may provide a survival signal for EBV-positive T cell tumors, whereas LMP2A-associated NEDD4 E3 ligases probably titer the strength of this signal.

Amino Acid Sequence↗
PubMed2006

NADP-dependent mannitol dehydrogenase, a major allergen of Cladosporium herbarum.

Cladosporium herbarum is an important allergenic fungal species that has been reported to cause allergic diseases in nearly all climatic zones. 5-30% of the allergic population displays IgE antibodies against molds. Sensitization to Cladosporium has often been associated with severe asthma and less frequently with chronic urticaria and atopic eczema. However, no dominant major allergen of this species has been found so far. We present cloning, production, and characterization of NADP-dependent mannitol dehydrogenase of C. herbarum (Cla h 8) and show that this protein is a major allergen that is recognized by IgE antibodies of approximately 57% of all Cladosporium allergic patients. This is the highest percentage of patients reacting with any Cladosporium allergen characterized so far. Cla h 8 was purified to homogeneity by standard chromatographic methods, and both N-terminal and internal amino acid sequences of protein fragments were determined. Enzymatic analysis of the purified natural protein revealed that this allergen represents a NADP-dependent mannitol dehydrogenase that interconverts mannitol and d-fructose. It is a soluble, non-glycosylated cytoplasmic protein. Two-dimensional protein analysis indicated that mannitol dehydrogenase is present as a single isoform. The cDNA encoding Cla h 8 was cloned from a lambda-ZAP library constructed from hyphae and spores. The recombinant non-fusion protein was expressed in Escherichia coli and purified to homogeneity. Its immunological and biochemical identity with the natural protein was shown by enzyme activity tests, CD spectroscopy, IgE immunoblots with sera of patients, and by skin prick testing of Cladosporium allergic patients. This protein therefore is a new major allergen of C. herbarum.

Alcohol Oxidoreductases↗
PubMed2000

Signaling via the T cell antigen receptor induces phosphorylation of Stat1 on serine 727.

The Stat1 transcription factor plays a pivotal role in both, the antiviral and antigrowth actions of interferons. Stat1 acquires the ability to bind DNA by becoming phosphorylated on Tyr(701). However, to effectively stimulate gene transcription, it must also be phosphorylated on Ser(727). We show that engagement of T cell antigen receptor (TCR)/CD3 complex in either Jurkat cells or peripheral blood lymphocytes stimulates phosphorylation of Ser(727) but not Tyr(701) of Stat1. This process does not require the expression of tyrosine kinases Lck and Zap-70. Interestingly, pretreatment of T cells with the Src kinase inhibitor PP1 completely abrogated CD3-mediated serine phosphorylation of Stat1, whereas inhibitors to MEK1 and phosphatidylinositol 3-kinase had no effect. Phosphorylation of Ser(727) of Stat1 in T cells is not restricted to TCR/CD3 but also results when cells are stimulated via the costimulatory molecule CD28. The combination of CD3 and CD28 did not augment phosphorylation of Stat1 Ser(727). Surprisingly, Stat1-mediated transcriptional activity in response to IFN-alpha was enhanced with CD3 stimulation, whereas CD3 alone had little effect. These findings suggest that Stat1 is a signaling molecule in TCR signaling and may play a role in T cell function.

CD28 Antigens↗
PubMed2004

Proteomic characterization of protein phosphatase complexes of the mammalian nucleus.

Our knowledge of the serine/threonine protein phosphatases of the mammalian nucleus is limited compared with their cytosolic counterparts. Microcystin-Sepharose chromatography and mass spectrometry were utilized to affinity purify and identify protein phosphatase-associated proteins from isolated rat liver nuclei. Far Western analysis with labeled protein phosphatase 1 (PP1) showed that many more PP1 binding proteins exist in the nucleus than were previously demonstrated. Mass spectrometry confirmed the presence in the nucleus of the mammalian PP1 isoforms alpha1, alpha2, beta, and gamma1, plus the Aalpha and several of the B and B' subunits that are complexed to PP2A. Other proteins enriched on the microcystin matrix include the spliceosomal proteins known as the U2 snRNPs SAP145 and SAP155 and the U5 snRNPs p116 and p200, myosin heavy chain, and a nuclear PP1 myosin-targeting subunit related to M110. The putative RNA binding protein ZAP was also established as a nuclear PP1 binding protein using the criteria of co-purification with PP1 on microcystin-Sepharose, co-immunoprecipation, binding PP1 in an overlay assay, and presence of a putative PP1 binding site (KKRVRWAD). These results further support a key role for protein phosphatases in several nuclear functions, including the regulation of pre-mRNA splicing.

Animals↗
PubMed2003

Cloning and characterization of an outer membrane protein (Lip18) from Helicobacter bizzozeronii.

A recombinant lambda-Zap II phage was selected by screening a genomic library of Helicobacter bizzozeronii (Hb) using antibodies from a naturally infected cat. DNA sequencing resulted an open reading frame containing 172 codons with a predicted molecular mass of 18 kDa (Lip18). The amino acid sequence showed 22.1, 55.2, 56.7 and 57.1% identity to peptidoglycan-associated lipoprotein of Helicobacter pylori, Agrobacterium tumefaciens, Bartonella bacilliformis, respectively. A peptidoglycan associating alpha-helical motif (LALGQRRSVAVRDYLVS) was located in the C-terminal region. H. bizzozeronii contains a potential lipoprotein signal peptide cleavage site (Val-Val-Gly-Cys), and yields a predicted mature protein with 148 amino acids. The Lip18 was localized into the outer membrane of the bacteria. Immunoblot analysis of serum samples from a dog and cat naturally infected with Helicobacter spp was able to recognize the purified recombinant Lip18.

Amino Acid Motifs↗
PubMed2004

Routine analysis of IgVH mutational status in CLL patients using BIOMED-2 standardized primers and protocols.

Current methods for the detection of IgVH mutational status in chronic lymphocytic leukemia (CLL), which identifies 2 subgroups of patients with significantly different outcomes, are laborious, expensive and do not lend themselves to a routine diagnostic setting. With the introduction of BIOMED-2 primers, a rapid protocol is now available. This study evaluated the protocol by examining DNA from 100 CLL patients. Conventional methods using RNA, and fluorescence in-situ hybridization (FISH) analysis for recurring chromosomal abnormalities, were carried out on 30 and 60 of these patients, respectively. There was complete concordance between the BIOMED-2 protocol and the RNA based method, both in mutational status and gene usage, whilst unmutated IgVH genes showed association with 17p13 and 11q23 deletions, and trisomy 12, associated with poor and intermediate outcomes, respectively. This study demonstrates that it is feasible to use the BIOMED-2 protocol in the diagnostic profile of CLL patients, obviating the need for inclusion of surrogate markers such as ZAP-70.

Adult↗
PubMed2006

Signaling molecules and cytokine production in T cells of patients with B-cell chronic lymphocytic leukemia: long-term effects of fludarabine and alemtuzumab treatment.

Fludarabine and alemtuzumab are routinely used for treatment of B-cell chronic lymphocytic leukemia (B-CLL). The present study aimed to compare the expression of signaling molecules and cytokine production by T cells of B-CLL patients in long-term unmaintained remission/plateau phase following fludarabine or alemtuzumab treatment with that of indolent/untreated B-CLL patients and healthy donors. The frequency and intensity of TCR-CD3zeta chain, p56lck, p59fyn, ZAP-70, PI3-kinase and interferon (IFN)-gamma/interleukin (IL)-4 production in CD4 and CD8 T cells was examined by flow cytometry. T-cell function was assessed by stimulation with purified protein derivative (PPD) and phytohemagglutinin (PHA). Despite a reduction in number, the expression of IFN-gamma/IL-4 in T-cells in patients was significantly higher than in healthy donors. The intensity of most signaling molecules in treated patients was relatively unaffected vs. healthy donors but lower than untreated-indolent patients. However, the total number of T cells which expressed each of the signaling molecules was decreased in patients, with no difference between fludarabine- and alemtuzumab-treated patients. The T-cell response to PHA but not PPD was reduced in treated patients. The results suggest that, despite some alterations in signaling molecules and a reduction in T-cell number, overall T-cell functions may be relatively well preserved long-term after treatment with fludarabine and alemtuzumab.

Aged↗
PubMed1998

Use of prokaryotically expressed nucleocapsid protein as positive antigen in ELISA.

A cDNA library of Rinderpest vaccine virus was prepared in Zap Express vector (Stratagene). The Rinderpest 'N' gene specific clones were selected, characterized and thereafter expressed in E. coli XLOLR strain. The expressed protein was found to be immunogenic in western blot with hyperimmune sera. It reacted with rinderpest and 'N' protein specific monoclonal antibodies in Enzyme Linked Immunosorbent Assay (ELISA). Prokaryotically expressed 'N' protein also gave precipitin band in counter immunoelectrophoresis test (CIE). The expression of N protein was sufficient for its utility as positive antigen in CIE and ELISA used for rinderpest diagnosis.

Animals↗
PubMed2004

Secondary lymphoid-tissue chemokine induced modulation of T cells.

In this study we were interested in investigating the extent to which stimulation through a chemokine receptor could modulate TCR function. We report that splenic T cells exposed to secondary lymphoid-tissue chemokine (SLC, CCL21) for 72, but not 2 or 24 hours, exhibited a decreased ability to produce IFN-gamma following CD3 crosslinking. Similar findings were observed with CCL2 and CCL5. The decrease in IFN-gamma production was not attributed to a decrease in T cell viability, was not accompanied with an increase in IL-4 production, and could be induced using a G protein coupled receptor agonist indicating involvement of chemokine receptors. One explanation for these findings was that following chemokine exposure the T cells were less efficient at TCR capping and exhibited a decrease in ZAP-70 protein expression. Consequently, these data indicate that CCL21 could modulate the function and expression of proteins necessary for T cell activation.

Animals↗
PubMed1999

Laminin 5 in the human thymus: control of T cell proliferation via alpha6beta4 integrins.

Laminin 5 (alpha3beta3gamma2) distribution in the human thymus was investigated by immunofluorescence on frozen sections with anti-alpha3, -beta3, and -gamma2 mAbs. In addition to a linear staining of subcapsular basal laminae, the three mAbs give a disperse staining in the parenchyma restricted to the medullary area on a subset of stellate epithelial cells and vessel structures. We also found that laminin 5 may influence mature human thymocyte expansion; while bulk laminin and laminin 2, when cross-linked, are comitogenic with a TCR signal, cross-linked laminin 5 has no effect. By contrast, soluble laminin 5 inhibits thymocyte proliferation induced by a TCR signal. This is accompanied by a particular pattern of inhibition of early tyrosine kinases, including Zap 70 and p59(fyn) inhibition, but not overall inhibition of p56(lck). Using a mAb specific for alpha6beta4 integrins, we observed that while alpha3beta1 are known to be uniformly present on all thymocytes, alpha6beta4 expression parallels thymocyte maturation; thus a correspondence exists between laminin 5 in the thymic medulla and alpha6beta4 on mature thymocytes. Moreover, the soluble Ab against alpha6beta4 inhibits thymocyte proliferation and reproduces the same pattern of tyrosine kinase phosphorylation suggesting that alpha6beta4 is involved in laminin 5-induced modulation of T cell activation.

Antibodies, Monoclonal↗
PubMed1999

Aggregation of lipid rafts accompanies signaling via the T cell antigen receptor.

The role of lipid rafts in T cell antigen receptor (TCR) signaling was investigated using fluorescence microscopy. Lipid rafts labeled with cholera toxin B subunit (CT-B) and cross-linked into patches displayed characteristics of rafts isolated biochemically, including detergent resistance and colocalization with raft-associated proteins. LCK, LAT, and the TCR all colocalized with lipid patches, although TCR association was sensitive to nonionic detergent. Aggregation of the TCR by anti-CD3 mAb cross-linking also caused coaggregation of raft-associated proteins. However, the protein tyrosine phosphatase CD45 did not colocalize to either CT-B or CD3 patches. Cross-linking of either CD3 or CT-B strongly induced tyrosine phosphorylation and recruitment of a ZAP-70(SH2)(2)-green fluorescent protein (GFP) fusion protein to the lipid patches. Also, CT-B patching induced signaling events analagous to TCR stimulation, with the same dependence on expression of key TCR signaling molecules. Targeting of LCK to rafts was necessary for these events, as a nonraft- associated transmembrane LCK chimera, which did not colocalize with TCR patches, could not reconstitute CT-B-induced signaling. Thus, our results indicate a mechanism whereby TCR engagement promotes aggregation of lipid rafts, which facilitates colocalization of LCK, LAT, and the TCR whilst excluding CD45, thereby triggering protein tyrosine phosphorylation.

Animals↗
PubMed1988

Adenosine promotes neutrophil chemotaxis.

We have previously (1-4) demonstrated that adenosine, by engaging specific receptors on the surface of neutrophils, inhibits generation of toxic oxygen metabolites by activated neutrophils and prevents these activated neutrophils from injuring endothelial cells. We now report the surprising observation that engagement of these same neutrophil adenosine receptors promotes chemotaxis to C5 fragments (as zymosan-activated plasma [ZAP]) or to the bacterial chemoattractant FMLP. When chemotaxis was studied in a modified Boyden chamber, physiologic concentrations of adenosine promoted chemotaxis by as much as 60%. Adenosine receptor analogues, 5'N-ethylcarboxamidoadenosine (NECA) and N6-phenylisopropyladenosine (PIA), also promoted chemotaxis; the order of agonist potency was consistent with that of an A2 adenosine receptor (NECA greater than PIA greater than or equal to adenosine). A potent antagonist at adenosine receptors, 8-p-sulfophenyltheophylline (10 microM), completely reversed NECA enhancement of chemotaxis but did not affect chemotaxis by itself. Neither NECA nor 2-chloroadenosine, a nonselective adenosine receptor agonist, alone was chemotactic or chemokinetic by checkerboard analysis. NECA also promoted chemotaxis quantitated by a different technique, chemotaxis under agarose, to the surrogate bacterial chemoattractant FMLP. These data suggest that engagement of adenosine A2 receptors uniquely modulates neutrophil function so as to promote migration of neutrophils to sites of tissue damage while preventing the neutrophils from injuring healthy tissues en route.

Adenosine↗
PubMed1993

T cell receptor zeta/CD3-p59fyn(T)-associated p120/130 binds to the SH2 domain of p59fyn(T).

Intracellular signaling from the T cell receptor (TCR)zeta/CD3 complex is likely to be mediated by associated protein tyrosine kinases such as p59fyn(T), ZAP-70, and the CD4:p56lck and CD8:p56lck coreceptors. The nature of the signaling cascade initiated by these kinases, their specificities, and downstream targets remain to be elucidated. The TCR-zeta/CD3:p59fyn(T) complex has previously been noted to coprecipitate a 120/130-kD doublet (p120/130). This intracellular protein of unknown identity associates directly with p59fyn(T) within the receptor complex. In this study, we have shown that this interaction with p120/130 is specifically mediated by the SH2 domain (not the fyn-SH3 domain) of p59fyn(T). Further, based on the results of in vitro kinase assays, p120/130 appears to be preferentially associated with p59fyn(T) in T cells, and not with p56lck. Antibody reprecipitation studies identified p120/130 as a previously described 130-kD substrate of pp60v-src whose function and structure is unknown. TCR-zeta/CD3 induced activation of T cells augmented the tyrosine phosphorylation of p120/130 in vivo as detected by antibody and GST:fyn-SH2 fusion proteins. p120/130 represents the first identified p59fyn(T):SH2 binding substrate in T cells, and as such is likely to play a key role in the early events of T cell activation.

Animals↗
PubMed1996

Selective activation of the calcium signaling pathway by altered peptide ligands.

We previously demonstrated that altered peptide ligands (APL) can partially activate T cells, resulting in multiple distinct functional phenotypes, including the induction of anergy. Such APL stimulate a unique pattern of T cell receptor (TCR) phospho-zeta species, and lack associated ZAP-70 kinase activity. While these data suggested that selective signaling pathways downstream of the TCR/CD3 molecules are activated upon APL stimulation, they did not directly demonstrate this. Thus, we pursued intracellular signaling events successfully stimulated by APL. Because our previous studies showed that cyclosporin A (CsA) completely inhibited anergy induction, we assessed whether TCR ligation by APL cause a rise in cytosolic calcium (Ca+2). Our results show that these ligands can induce Ca+2 transients, in contrast to data generated using analogue peptides in other antigen systems. These opposing results may reflect differences in the intracellular signaling pathways utilized by different APL, or may be due to the exquisite sensitivity of the assay used here. Importantly, the APL-stimulated Ca+2 induction is both initiated and sustained at lower levels than that stimulated by a strong agonist signal, but resembles that stimulated by a weaker agonist stimulus. Alone, the less than optimal Ca+2 induction does not cause anergy, because ionomycin treatment together with the APL does not result in a proliferative signal. Instead, we propose that a combination of this and other signaling pathways induces T cell anergy. Overall, these data support the concept of differential signaling in T cells, as a direct consequence of the phosphotyrosine status of the TCR/CD3 molecules.

Animals↗