RESUMO
Reactive oxygen species (ROS) or reactive oxygen intermediates (ROIs) mediate complex signaling involving multiple pathways. In this report, we demonstrate for the first time that endogenous Bruton's tyrosine kinase (Btk) and Akt can interact with each other in DT40 chicken B cells and human Nalm6 B cells and that this interaction is inducible following H2O2 stimulation. This interaction is supported by visualizing the co-localization of Btk and Akt in the perinuclear region and membrane ruffles in COS-7 cells. We have also shown the involvement of phosphatidylinositol 3-kinase (PI 3-K) and Btk in the phosphorylation of Akt following stimulation by hydrogen peroxide (H2O2). Interestingly, Akt phosphorylation was found in the presence of Btk even in the absence of oxidative stress. In addition, we have investigated the involvement of PI 3-K in the MAPKs and ERK and JNK phosphorylation, in the presence or absence of Btk. Phosphorylation of both ERK and JNK increased when the PI 3-K pathway was inhibited and both pathways were modulated positively by Btk. Taken together, based on the study of endogenous conditions, we show the novel interaction of Btk and Akt in H2O2 signaling in B cells.
Assuntos
Linfócitos B/metabolismo , Proteínas Serina-Treonina Quinases , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Transdução de Sinais , Tirosina Quinase da Agamaglobulinemia , Animais , Células COS , Linhagem Celular , Galinhas , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Peróxido de Hidrogênio/farmacologia , Immunoblotting , Microscopia de Fluorescência , Estresse Oxidativo , Oxigênio/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Testes de Precipitina , Ligação Proteica , Proteínas Proto-Oncogênicas c-akt , Fatores de Tempo , TransfecçãoRESUMO
Bruton's tyrosine kinase (Btk) is important for B lymphocyte development. To identify genes that are differentially expressed in primary B cells lacking functional Btk, splenocytes from X-linked immunodeficiency (Xid), Btk knockout (Btk KO) and immunocompetent CBA mice were used in microarrays containing more than 12000 genes and expressed-sequence tags. We found 4515 common transcripts expressed in duplicate experiments in the three strains. Out of these, 38 were differentially expressed genes (21 were up-regulated >2-fold and 17 were down-regulated <-2-fold) between CBA and Btk defective (Xid or Btk KO) mice. Ten out of these genes were selected and quantitative real-time PCR was conducted for validation and further investigation. Real-time experiments correlated nicely with the microarray data. No definitive phenotypic difference has previously been reported between Xid and Btk KO mice. We found 7 genes whose expression differed (>2-fold) between the two strains. Moreover, when the 38 genes that differed between immunocompetent CBA mice and Btk defective mice were ranked according to fold-increase, the levels in Btk KO mice were significantly more altered. This suggests that the defect in Btk KO mice is more severe and demonstrates that the mutant Btk protein in Xid mice does not generally function as dominant-negative form.
Assuntos
Linfócitos B/metabolismo , Perfilação da Expressão Gênica , Síndromes de Imunodeficiência/genética , Proteínas Tirosina Quinases/deficiência , Tirosina Quinase da Agamaglobulinemia , Animais , Antígenos CD/metabolismo , Linfócitos B/efeitos dos fármacos , Western Blotting , Regulação para Baixo , Etiquetas de Sequências Expressas , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Ionomicina/farmacologia , Lipopolissacarídeos/farmacologia , Masculino , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos CBA , Camundongos Knockout , Análise de Sequência com Séries de Oligonucleotídeos , Regiões Promotoras Genéticas/genética , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , RNA Mensageiro/análise , RNA Mensageiro/genética , Reprodutibilidade dos Testes , Baço/citologia , Acetato de Tetradecanoilforbol/farmacologia , Tetraspanina 29RESUMO
We have used both Clontech Atlas Human Hematology/Immunology cDNA microarrays, containing 588 genes, and Affymetrix oligonucleotide U95Av2 human array complementary to more than 12,500 genes to get a global view of genes expressed in Epstein-Barr virus (EBV)-transformed B cells and genes regulated by Bruton's tyrosine kinase (Btk). We compared EBV-transformed wild-type (WT) B cells from a healthy individual, WT1 and an X-linked agammaglobulinemia (XLA) patient cell line, XLA1, using the Clontech filters arrays. Eleven genes were > or =1.9-fold induced in absence of functional Btk. Furthermore, we analyzed a second patient cell line, XLA2, and compared this to two WT cell lines using oligonucleotide arrays. A total of 391 genes were found to be differentially expressed, including kinases and transcriptions factors. Furthermore, one expressed sequence tag and eight complementary DNA clones with unknown function were down-regulated in XLA2, indicating their biological role. Higher-fold inductions, Fyn (39.5), Hck (15.5) and Cyp1B1 (5.8), were observed using oligonucleotide array and were confirmed using real-time PCR for Fyn (20.8), Hck (6.7) and Cyp1B1 (10). Two genes, B cell translocation gene1 (BTG1) and B cell-specific OCT binding factor-1 (OBF-1) were induced > or =1.9-fold in both XLA1 and XLA2 analyzed by Atlas filter arrays andAffymetrix chips, respectively. Data from both filter and oligonucleotide arrays were compared to the gene clusters of a previously published lymphoma expression profile by linking to the UniGene transcript database. Our findings demonstrate for the first time the use of microarray to study the influence of Btk mutations and the use of functional annotation and validation of expression data by comparison of microarray analyses.