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1.
Sci Signal ; 10(478)2017 May 09.
Article in English | MEDLINE | ID: mdl-28487417

ABSTRACT

Activation of B cells by the binding of antigens to the B cell receptor (BCR) requires the protein kinase C (PKC) family member PKCß. Because PKCs must translocate to the plasma membrane to become activated, we investigated the mechanisms regulating their spatial distribution in mouse and human B cells. Through live-cell imaging, we showed that BCR-stimulated production of the second messenger diacylglycerol (DAG) resulted in the translocation of PKCß from the cytosol to plasma membrane regions containing the tetraspanin protein CD53. CD53 was specifically enriched at sites of BCR signaling, suggesting that BCR-dependent PKC signaling was initiated at these tetraspanin microdomains. Fluorescence lifetime imaging microscopy studies confirmed the molecular recruitment of PKC to CD53-containing microdomains, which required the amino terminus of CD53. Furthermore, we showed that Cd53-deficient B cells were defective in the phosphorylation of PKC substrates. Consistent with this finding, PKC recruitment to the plasma membrane was impaired in both mouse and human CD53-deficient B cells compared to that in their wild-type counterparts. These data suggest that CD53 promotes BCR-dependent PKC signaling by recruiting PKC to the plasma membrane so that it can phosphorylate its substrates and that tetraspanin-containing microdomains can act as signaling hotspots in the plasma membrane.


Subject(s)
B-Lymphocytes/metabolism , Cell Membrane/metabolism , Protein Kinase C/metabolism , Receptors, Antigen, B-Cell/metabolism , Tetraspanin 25/physiology , Animals , Cells, Cultured , Female , Humans , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Phosphorylation , Protein Domains , Signal Transduction
2.
PLoS One ; 9(5): e97844, 2014.
Article in English | MEDLINE | ID: mdl-24832104

ABSTRACT

NK cells express several tetraspanin proteins, which differentially modulate NK cell activities. The tetraspanin CD53 is expressed by all resting NK cells and was previously shown to decrease NK cell cytotoxicity upon ligation. Here, we show that CD53 ligation reduced degranulation of rat NK cells in response to tumour target cells, evoked redirected inhibition of killing of Fc-bearing targets, and reduced the IFN-γ response induced by plate-bound antibodies towards several activating NK cell receptors (Ly49s3, NKR-P1A, and NKp46). CD53 induced activation of the ß2 integrin LFA-1, which was further enhanced upon co-stimulation with activating NK cell receptors. Concordant with a role for CD53 in increasing NK cell adhesiveness, CD53 ligation induced a strong homotypic adhesion between NK cells. Further, the proliferative capacity of NK cells to a suboptimal dose of IL-2 was enhanced by CD53 ligation. Taken together, these data suggest that CD53 may shift NK cell responses from effector functions towards a proliferation phase.


Subject(s)
Killer Cells, Natural/metabolism , Lymphocyte Function-Associated Antigen-1/metabolism , Receptors, Natural Killer Cell/metabolism , Tetraspanin 25/physiology , Animals , Cell Adhesion , Cell Degranulation , Cell Line , Cell Proliferation , Interferon-gamma/biosynthesis , Interleukin-2/physiology , Phosphorylation , Protein Processing, Post-Translational , Rats
3.
Biochim Biophys Acta ; 1830(4): 3011-8, 2013 Apr.
Article in English | MEDLINE | ID: mdl-23313165

ABSTRACT

BACKGROUND: In this study, the association of asthma with CD53, a member of the tetraspanin family, was assessed for the first time in a mechanism-based study. METHODS: Genetic polymorphisms of CD53 were analyzed in 591 subjects and confirmed in a replication study of 1001 subjects. CD53 mRNA and protein levels were measured in peripheral blood leukocytes, and the effects of the promoter polymorphisms on nuclear factor binding were examined by electrophoretic mobility shift assay. Cellular functional studies were conducted by siRNA transfections. RESULTS: Among tagging SNPs of CD53, the -1560 C>T in the promoter region was significantly associated with asthma risk. Compared with the CC genotype, the CT and TT genotypes were associated with a higher asthma risk, with odd ratios of 1.74 (P=0.009) and 2.03 (P=0.004), respectively. These findings were confirmed in the replication study with odd ratios of 1.355 (P=0.047) and 1.495 (P=0.039), respectively. The -1560 C>T promoter SNP had functional effects on nuclear protein binding as well as mRNA and protein expression levels in peripheral blood leukocytes. When CD53 was knocked down by siRNA in THP-1 human monocytic cells stimulated with house dust mite, the production of inflammatory cytokines as well as NFκB activity was significantly over-activated, suggesting that CD53 suppresses over-activation of inflammatory responses. CONCLUSIONS: The -1560 C>T SNP is a functional promoter polymorphism that is significantly associated with population asthma risk, and is thought to act by directly modulating nuclear protein binding, thereby altering the expression of CD53, a suppressor of inflammatory cytokine production.


Subject(s)
Asthma/etiology , Cytokines/biosynthesis , Polymorphism, Single Nucleotide , Promoter Regions, Genetic , Tetraspanin 25/physiology , Animals , Asthma/genetics , Genotype , Humans , Inflammation/immunology , Linkage Disequilibrium , Pyroglyphidae/immunology , Tetraspanin 25/genetics
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