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1.
Marracci GH Marquardt WE Strehlow A McKeon GP Gross J Buck DC Kozell LB Bourdette DN 《Biochemical and biophysical research communications》2006,344(3):963-971
Lipoic acid is an antioxidant that suppresses and treats a model of multiple sclerosis, experimental autoimmune encephalomyelitis. We now demonstrate that treatment of human PBMC and T cell lines with LA downmodulated CD4 expression in a concentration-dependent manner. LA treatment of Con A stimulated PBMC specifically removed CD4 from the T-cell surface, but not CD3. Epitope masking by LA was excluded by using monoclonal antibodies targeting different domains of CD4. Incubation on ice inhibited CD4 removal following LA treatment, suggesting that endocytosis was involved in its downmodulation. LA is in a unique category of compounds that induce CD4 downmodulation by various mechanisms (e.g., gangliosides). We hypothesized that LA might induce dissociation of p56(Lck) from CD4, thus leading to its downmodulation. Immunoblot analyses demonstrated reduced co-precipitation of p56(Lck) from Jurkat T-cells following LA treatment and precipitation of CD4. This unique immunomodulatory effect of LA warrants further investigation. 相似文献
2.
Lysates from the Jurkat T lymphocyte cell line were immunoblotted with anti-Pyk2, and two major forms of Pyk2 were identified. When lysates from the p56(Lck) negative (J.CaM1/Rep3) and CD45 negative Jurkat cell line derivatives were immunoblotted with anti-Pyk2, only the lower mobility form of Pyk2 was predominant. Transfection of J.CaM1 cells with p56(Lck) restored expression of the multiple forms of Pyk2. Using RT-PCR, we found that both species of the alternatively spliced mRNA for Pyk2 were present in all of the lines regardless of their ability to express CD45 or p56(Lck) protein. When p56(Lck) immunoprecipitates were immunoblotted with anti-Pyk2, only the higher mobility form of Pyk2 immunoprecipitated with p56(Lck). These data demonstrate that certain members of the Src family of kinases interact preferentially with the different isoforms of Pyk2 and may have a role in the regulation of the Pyk2 protein in lymphocytes. 相似文献
3.
J M Trevillyan Y Lin S J Chen C A Phillips C Canna T J Linna 《Biochimica et biophysica acta》1986,888(3):286-295
4.
Chen P Norris D Iwanowicz EJ Spergel SH Lin J Gu HH Shen Z Wityak J Lin TA Pang S De Fex HF Pitt S Shen DR Doweyko AM Bassolino DA Roberge JY Poss MA Chen BC Schieven GL Barrish JC 《Bioorganic & medicinal chemistry letters》2002,12(10):1361-1364
We have identified a novel series of 1,5-imidazoquinoxalines as inhibitors of Lck with excellent potency (IC50s<5 nM) as well as good cellular activity against T-cell proliferation (IC50s<1 microM). Structure-activity studies demonstrate the requirement for the core heterocycle in addition to an optimal 2,6-disubstituted aniline group. 相似文献
5.
6.
Determination of the solution structure of the SH3 domain of human p56 Lck tyrosine kinase 总被引:2,自引:0,他引:2
Summary The solution structure of the SH3 domain of human p56 Lck tyrosine kinase (Lck-SH3) has been determined by multidimensional heteronuclear NMR spectroscopy. The structure was calculated from a total of 935 experimental restraints comprising 785 distance restraints derived from 1017 assigned NOE cross peaks and 150 dihedral angle restraints derived from 160 vicinal coupling constants. A novel combination of the constant-time 1H–13C NMR correlation experiment recorded with various delays of the constant-time refocusing delays and a fractionally 13C-labelled sample was exploited for the stereo-specific assignment of prochiral methyl groups. Additionally, 28 restraints of 14 identified hydrogen bonds were included. A family of 25 conformers was selected to characterize the solution structure. The average root-mean-square deviations of the backbone atoms (N, C, C, O) among the 25 conformers is 0.42 Å for residues 7 to 63. The N- and C-terminal residues, 1 to 6 and 64 to 81, are disordered, while the well-converged residues 7 to 63 correspond to the conserved sequences of other SH3 domains. The topology of the SH3 structure comprises five anti-parallel -strands arranged to form two perpendicular -sheets, which are concave and twisted in the middle part. The overall secondary structure and the backbone conformation of the core -strands are almost identical to the X-ray structure of the fragment containing the SH2-SH3 domains of p56 Lck [Eck et al. (1994) Nature, 368, 764–769]. The X-ray structure of the SH3 domain in the tandem SH2-SH3 fragment is spatially included within the ensemble of the 25 NMR conformers, except for the segment of residues 14 to 18, which makes intermolecular contacts with an adjacent SH2 molecule and the phosphopeptide ligand in the crystal lattice. Local structural differences from other known SH3 domains are also observed, the most prominent of which is the absence in Lck-SH3 of the two additional short -strands in the regions Ser15 to Glu17 and Gly25 to Glu27 flanking the so-called RT-Src loop. This loop (residues Glu17 to Leu24), together with the n-Src loop (residues Gln37 to Ser46) confines the ligand interaction site which is formed by a shallow patch of hydrophobic amino acids (His14, Tyr16, Trp41, Phe54 and Phe59). Both loops are flexible and belong to the most mobile regions of the protein, which is assessed by the heteronuclear 15N,1H-NOE values characterizing the degree of internal backbone motions. The aromatic residues of the ligand binding site are arranged such that they form three pockets for interactions with the polyproline ligand.Abbreviations CT
constant time
- HSQC
heteronuclear single-quantum coherence
- NOE
nuclear Overhauser enhancement
- NOESY
nuclear Overhauser enhancement spectroscopy
- SH2
Src homology domain 2
- SH3
Src homology domain 3 相似文献
7.
Calcium-induced uncoupling in communicating human lymphocytes 总被引:1,自引:0,他引:1
8.
Wityak J Das J Moquin RV Shen Z Lin J Chen P Doweyko AM Pitt S Pang S Shen DR Fang Q de Fex HF Schieven GL Kanner SB Barrish JC 《Bioorganic & medicinal chemistry letters》2003,13(22):4007-4010
A novel series of 2-amino-5-carboxamidothiazoles were identified as inhibitors of Lck. Structure-activity studies demonstrate the structural requirements for potent Lck activity. Cyclopropylamide 11d is a potent Lck inhibitor having sub-micromolar activity in a PBL proliferation assay. 相似文献
9.
Chen P Norris D Das J Spergel SH Wityak J Leith L Zhao R Chen BC Pitt S Pang S Shen DR Zhang R De Fex HF Doweyko AM McIntyre KW Shuster DJ Behnia K Schieven GL Barrish JC 《Bioorganic & medicinal chemistry letters》2004,14(24):6061-6066
A series of substituted 2-(aminoheteroaryl)-thiazole-5-carboxamide analogs have been synthesized as novel, potent inhibitors of the Src-family kinase p56Lck. Among them, compound 2 displayed superior in vitro potency and excellent in vivo efficacy. 相似文献
10.
Sharif-Askari E Gaucher D Halwani R Ma J Jao K Abdallah A Haddad EK Sékaly RP 《The Journal of biological chemistry》2007,282(49):36048-36056
Although the death-inducing signaling complex (DISC) is rapidly assembled, several lines of evidence suggest that formation of this complex is not the first consequence of cell surface CD95 (Fas) stimulation but rather a later step in this process. Activation of Fas triggers a cascade of signaling events that culminate in cellular apoptosis. Tyrosine kinases are critical effectors in T cell activation. However, their functional involvement in death receptor-mediated apoptosis is unknown. Here, we used p56(Lck)-deficient cells to show that CD95-induced cell death is highly dependent on p56(Lck) activity and its localization within plasma membrane. We found that p56(Lck) acts upstream of the mitochondria; in the absence of p56(Lck), Bid cleavage and the release of cytochrome c were severely impaired. Moreover, p56(Lck)-deficient cells or cells expressing an inactive form of p56(Lck) displayed defective formation of the DISC post CD95 stimulation. In vivo reconstitution of thymocytes from p56(lck)-deficient mice, which are resistant to apoptosis, with p56(Lck) restored Fas-mediated cell death. Our results support a novel model whereby sensitivity to apoptosis is regulated through quantitative changes in the stoichiometry of DISC components triggered by p56(Lck) activation and localization. 相似文献
11.
Yasuda K Kosugi A Hayashi F Saitoh S Nagafuku M Mori Y Ogata M Hamaoka T 《Journal of immunology (Baltimore, Md. : 1950)》2000,165(6):3226-3231
Lck is a member of the Src family kinases expressed predominantly in T cells, and plays a pivotal role in TCR-mediated signal transduction. Myristoylation of glysine 2 in the N-terminal Src homology 4 (SH4) domain of Lck is essential for membrane localization and function. In this study, we examined a site within the SH4 domain of Lck regulating myristoylation, membrane localization, and function of Lck. A Lck mutant in which serine 6 (Ser6) was substituted by an alanine was almost completely cytosolic in COS-7 cells, and this change of localization was associated with a drastic inhibition of myristoylation in this mutant. To assess the role of Ser6 of Lck in T cell function, we established stable transfectants expressing various Lck mutants using Lck-negative JCaM1 cells. The Lck mutant of Ser6 to alanine, most of which did not target to the plasma membrane, was not able to reconstitute TCR-mediated signaling events in JCaM1 cells, as analyzed by tyrosine phosphorylation of intracellular proteins and CD69 expression. These results demonstrate that Ser6 is a critical factor for Lck myristoylation, membrane localization, and function in T cells, presumably because the residue is important for N-myristoyl transferase recognition. 相似文献
12.
13.
Distinct intracellular localization of Lck and Fyn protein tyrosine kinases in human T lymphocytes 总被引:17,自引:8,他引:9
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《The Journal of cell biology》1994,125(3):639-649
Two src family kinases, lck and fyn, participate in the activation of T lymphocytes. Both of these protein tyrosine kinases are thought to function via their interaction with cell surface receptors. Thus, lck is associated with CD4, CD8, and Thy-1, whereas fyn is associated with the T cell antigen receptor and Thy-1. In this study, the intracellular localization of these two protein tyrosine kinases in T cells was analyzed by immunofluorescence and confocal microscopy. Lck was present at the plasma membrane, consistent with its proposed role in transmembrane signalling, and was also associated with pericentrosomal vesicles which co-localized with the cation-independent mannose 6- phosphate receptor. Surprisingly, fyn was not detected at the plasma membrane in either Jurkat T cells or T lymphoblasts but was closely associated with the centrosome and to microtubule bundles radiating from the centrosome. In mitotic cells, fyn co-localized with the mitotic spindle and poles. The essentially non-overlapping intracellular distributions of lck and fyn suggest that these kinases may be accessible to distinct regulatory proteins and substrates and, therefore, may regulate different aspects of T cell activation. Anti- phosphotyrosine antibody staining at the plasma membrane increases dramatically after CD3 cross-linking of Jurkat T cells. The localization of lck to the plasma membrane suggests that it may participate in mediating this increase in tyrosine phosphorylation, rather than fyn. Furthermore, the distribution of fyn in mitotic cells raises the possibility that it functions at the M phase of the cell cycle. 相似文献
14.
Effect of human immunodeficiency virus gp120 glycoprotein on the association of the protein tyrosine kinase p56lck with CD4 in human T lymphocytes 总被引:16,自引:0,他引:16
R J Juszczak H Turchin A Truneh J Culp S Kassis 《The Journal of biological chemistry》1991,266(17):11176-11183
The human immunodeficiency virus binds to CD4+ T lymphocytes through the interaction of its envelope glycoprotein (gp120) with the CD4 molecule. The src-related protein tyrosine kinase p56lck is physically associated with CD4 and is co-immunoprecipitated by CD4 monoclonal antibody (mAb). Activators of protein kinase C (PKC) cause the dissociation of p56lck from CD4. Here we report that gp120 mAb immunoprecipitated the p56lck.CD4.gp120 complex after short term treatment (20 min) of human T lymphocytes with gp120. The p56lck that was associated with the CD4.gp120 complex was dissociated by activators of PKC. This effect was abolished by pretreatment of cells with PKC inhibitors. Thus the p56lck.CD4.gp120 immune complex immunoprecipitated by gp120 mAb behaves in a similar manner, with respect to PKC activation or inhibition, to the p56lck.CD4 complex immunoprecipitated by CD4 mAb. Short term treatment of cells with gp120, followed by gp120 mAb, resulted in an increase in the tyrosine kinase activity of p56lck associated with CD4. However, the amount of enzyme associated with CD4 remained unchanged. Long term treatment (20 h) of human T lymphocytes with gp120 resulted in the down-regulation of cell surface CD4 molecules. A parallel decrease in CD4-associated gp120 was also observed. In addition, gp120 caused the dissociation of p56lck and CD4. However, the dissociation of the p56lck from CD4 occurred at much faster rate than the down-regulation of surface CD4 molecules. Such mechanisms may account for the down-regulation of cell surface CD4 molecules and the depletion of functional CD4+ T lymphocytes which are characteristic of human immunodeficiency virus infections and acquired immune deficiency syndrome pathogenesis. 相似文献
15.
E Monostori D Desai M H Brown D A Cantrell M J Crumpton 《Journal of immunology (Baltimore, Md. : 1950)》1990,144(3):1010-1014
The phosphorylation of the invariant chains associated with the human TCR has been investigated after the stimulation of T lymphocytes with CD2 mAb T11(2) and T11(3), PHA, or phorbol 12,13-dibutyrate. As described previously, stimulation of T cells with either CD2 mAb or phorbol 12,13-dibutyrate resulted in the phosphorylation of the CD3 gamma-chain. The combination of T11(2) and T11(3) mAb also induced phosphorylation of the TCR zeta-chain. The phosphorylated zeta-polypeptide of CD2-activated cells was immunoprecipitated with antiphosphotyrosine antibodies and migrated to a 21- to 23-kDa position during SDS/PAGE. These results indicate that stimulation of human T cells via the CD2 Ag with the T11(2) and T11(3) mAb activates not only protein kinase C but also tyrosine kinase(s), resulting in the phosphorylation of the CD3 gamma-chain and the tyrosine phosphorylation of the zeta-chain, respectively. 相似文献
16.
Inhibition of p56(lck) tyrosine kinase by isothiazolones 总被引:1,自引:0,他引:1
Trevillyan JM Chiou XG Ballaron SJ Tang QM Buko A Sheets MP Smith ML Putman CB Wiedeman P Tu N Madar D Smith HT Gubbins EJ Warrior UP Chen YW Mollison KW Faltynek CR Djurić SW 《Archives of biochemistry and biophysics》1999,364(1):19-29
Lck encodes a 56-kDa protein-tyrosine kinase, predominantly expressed in T lymphocytes, crucial for initiating T cell antigen receptor (TCR) signal transduction pathways, culminating in T cell cytokine gene expression and effector functions. As a consequence of a high-throughput screen for selective, novel inhibitors of p56(lck), an isothiazolone compound was identified, methyl-3-(N-isothiazolone)-2-thiophenecarboxylate(A-125800), which inhibits p56(lck) kinase activity with IC50 = 1-7 microM. Under similar assay conditions, the isothiazolone compound was equipotent in blocking the ZAP-70 tyrosine kinase activity but was 50 to 100 times less potent against the catalytic activities of p38 MAP kinase and c-Jun N-terminal kinase 2alpha. A-125800 blocked activation-dependent TCR tyrosine phosphorylation and intracellular calcium mobilization in Jurkat T cells (IC50 = 35 microM) and blocked T cell proliferation in response to alloantigen (IC50 = 14 microM) and CD3/CD28-induced IL-2 secretion (IC50 = 2.2 microM) in primary T cell cultures. Inhibition of p56(lck )by A-125800 was dose- and time-dependent and was irreversible. A substitution of methylene for the sulfur atom in the isothiazolone ring of the compound completely abrogated the ability to inhibit p56(lck) kinase activity and TCR-dependent signal transduction. Incubation with thiols such as beta-ME or DTT also blocked the ability of the isothiazolone to inhibit p56(lck) kinase activity. LC/MS analysis established the covalent modification of p56(lck) at cysteine residues 378, 465, and 476. Together these data support an inhibitory mechanism, whereby cysteine -SH groups within the p56(lck) catalytic domain react with the isothiazolone ring, leading to ring opening and disulfide bond formation with the p56(lck) enzyme. Loss of p56(lck) activity due to -SH oxidation has been suggested to play a role in the pathology of AIDS. Consequently, a similar mechanism of sulfhydryl oxidation leading to p56(lck) inhibition, described in this report, may occur in the intact T cell and may underlie certain T cell pathologies. 相似文献
17.
Goldmann WH 《Cell biology international》2002,26(6):567-571
Transformation of cells by src -like kinases leads to altered cell morphology associated with the disassembly of focal contacts and concomitant increase in tyrosine phosphorylation of pp125(FAK) x p56(lck) is a lymphocyte-specific member of the src family of protein tyrosine kinases that associates with cell surface glycoproteins such as CD4 and CD8. It phosphorylates and activates pp125(FAK) and increases its autokinase activity, thus pretreatment of pp125(FAK) with protein kinase C (PKC) markedly attenuates its phosphorylation and activation, suggesting a potential regulatory pathway of pp125(FAK) activation in focal contacts. p56(lck) further phosphorylates and activates actin binding protein (ABP-280; filamin) and controls its association with cell surface receptors such as beta-2 integrins, actin filament cross-linking, and possibly lipid membrane insertion. 相似文献
18.
A protein kinase C pseudosubstrate peptide inhibits phosphorylation of the CD3 antigen in streptolysin-O-permeabilized human T lymphocytes. 总被引:5,自引:0,他引:5
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D R Alexander J M Hexham S C Lucas J D Graves D A Cantrell M J Crumpton 《The Biochemical journal》1989,260(3):893-901
Activation of human T lymphocytes leads to the phosphorylation of the CD3-antigen gamma polypeptide. We have investigated a possible role for protein kinase C (PKC) in mediating this phosphorylation event by using T cells permeabilized with streptolysin-O in the presence of 120 mM-K+ buffers containing Ca2+-EGTA. The gamma-chain was phosphorylated by [gamma-32P]ATP in permeabilized T lymphoblasts in the presence of phorbol 12,13-dibutyrate (Pdbu) or phytohaemagglutinin (PHA). Ca2+ alone in the range 0.5-1.0 microM also induced gamma-chain phosphorylation in some T-lymphoblast preparations; that in Jurkat-6 cells occurred at lower concentrations (50-500 nM). Two experimental approaches were used to investigate the possible involvement of PKC. Firstly, when permeabilization was carried out in buffer lacking free Ca2+, PKC was lost from the cells, and gamma-chain phosphorylation could then no longer be induced on subsequent addition of Pdbu or PHA in 400 nM-Ca2+, or 800 nM-Ca2+ alone, to permeabilized cells. However, when permeabilization was carried out in the presence of these three agents, PKC was translocated to intracellular membranes, and subsequent addition of [gamma-32P]ATP to these cells then resulted in gamma-chain phosphorylation. In the second approach, induction of gamma-chain phosphorylation by Pdbu, 1-oleoyl-2-acetylglycerol, 1,2-diolein, PHA or Ca2+ alone was effectively blocked by permeabilizing T cells in the presence of a PKC pseudosubstrate peptide (50 microM). Pseudosubstrate concentrations in the range 7-20 microM inhibited gamma-chain phosphorylation by 50%. In contrast, addition of four other 'irrelevant' basic peptides (50 microM) did not result in detectable inhibition, and 50 microM-pseudosubstrate did not inhibit the phosphorylation of 17 other polypeptides isolated from permeabilized T cells. These data suggest that Pdbu-, 1,2-diacylglycerol-, PHA- and Ca2+-induced phosphorylation of the CD3-antigen gamma chain in permeabilized T cells is mediated by PKC. 相似文献
19.
M Oddsdottir I M Modlin K A Zucker M J Zdon J R Goldenring 《Biochemical and biophysical research communications》1987,148(3):1390-1397
An enriched population of isolated rabbit gastric parietal cells, from the fundic mucosa of New Zealand White rabbit, contained an active cytosolic calmodulin-dependent protein kinase activity with a prominent 100 kDa substrate (pp100). The latter focused as a doublet with isoelectric point of 6.8-7.0. The pp100 protein was phosphorylated only on threonine residues on a single tryptic peptide. Trifluoperazine inhibited the pp100 kinase activity with a KI of 10-15 microM. Addition of exogenous calmodulin was able to restore activity to uninhibited levels. A protein band with a molecular weight and phosphopeptide map identical to pp100, phosphorylated by calcium-dependent kinase, was also observed in rabbit pancreatic cytosol. The data suggest that a type III calmodulin-dependent kinase is present in parietal cell cytosol. 相似文献
20.
Connie M. Williams Guichang Zhang Marek Michalak D. D. Cass 《Sexual plant reproduction》1997,10(2):83-88
To examine possible calcium (Ca2+)-mediated prefertilization events in male gametes of higher plants, we studied protein phosphorylation and the Ca2+-binding proteins, calmodulin and calreticulin, in sperm cells isolated from maize (Zea mays L.) pollen in the presence and absence of Ca2+. Using immunoblotting, we detected calmodulin and calreticulin and Ca2+-induced variations. Exposure of sperm cells to 1 mM Ca2+ for 1 h increased calmodulin content by 136% compared with the control. Ca2+ had little effect on calreticulin at 1 h, but induced a 34% increase after 3 h. Phosphorylation of proteins was low in 1 h-control
and Ca2+-treated cells. However, a 13-fold increase in phosphorylation of a 18-kDa protein was found at 12 h in the presence of Ca2+. Ca2+-induced changes in calmodulin, calreticulin and protein phosphorylation observed in maize sperm cells may reflect prefertilization
changes in vivo that facilitate sperm cell fusion with egg and central cells.
Received: 26 July 1996 / Revision accepted: 7 February 1997 相似文献