RESUMO
Yeast Fis1p participates in mitochondrial fission, together with Dnm1p and Mdv1p. Recently, human Fis1 (hFis1) was reported to be involved in mitochondrial fission, together with Drp1. We established stable transformants with an hFis1 siRNA expression vector. In the stable hFis1 knockdown cells, hFis1 expression was suppressed to approximately 10%, and mitochondrial fission, induced by cisplatin treatment, was delayed. In addition, mouse Fis1 (mFis1) expression promoted mitochondrial fission and cell death in the hFis1 knockdown cells, suggesting that mFis1 complements the function of hFis1. These hFis1 siRNA expression vectors may be useful for studying the molecular function of mammalian Fis1.
Assuntos
Técnicas Genéticas , Vetores Genéticos , Proteínas Mitocondriais/fisiologia , RNA Interferente Pequeno/metabolismo , Animais , Western Blotting , Cisplatino/farmacologia , DNA Complementar/metabolismo , Teste de Complementação Genética , Células HeLa , Humanos , Proteínas de Membrana , Camundongos , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Interferência de RNA , Fatores de TempoRESUMO
Phospholipase C (PLC) plays important roles in phosphoinositide turnover by regulating the calcium-protein kinase C signaling pathway. PLC-L2 is a novel PLC-like protein which lacks PLC activity, although it is very homologous with PLC delta. PLC-L2 is expressed in hematopoietic cells, but its physiological roles and intracellular functions in the immune system have not yet been clarified. To elucidate the physiological function of PLC-L2, we generated mice which had a genetic PLC-L2 deficiency. PLC-L2-deficient mice grew with no apparent abnormalities. However, mature B cells from PLC-L2-deficient mice were hyperproliferative in response to B-cell receptor (BCR) cross-linking, although B2 cell development appeared to be normal. Molecular biological analysis revealed that calcium influx and NFATc accumulation in nuclei were increased in PLC-L2-deficient B cells. Extracellular signal-regulated kinase activity was also enhanced in PLC-L2-deficient B cells. These mice had a stronger T-cell-independent antigen response. These results indicate that PLC-L2 is a novel negative regulator of BCR signaling and immune responses.
Assuntos
Linfócitos B/metabolismo , Lipase/metabolismo , Proteínas Nucleares , Fosfolipases/fisiologia , Proteínas Adaptadoras de Transdução de Sinal , Animais , Southern Blotting , Cálcio/metabolismo , Divisão Celular , Membrana Celular/metabolismo , Núcleo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Citometria de Fluxo , Deleção de Genes , Regulação da Expressão Gênica , Células-Tronco Hematopoéticas , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Modelos Genéticos , Mutagênese , Fatores de Transcrição NFATC , Fenótipo , Fosforilação , Transdução de Sinais , Fatores de Tempo , Distribuição Tecidual , Fatores de Transcrição/metabolismoRESUMO
Neural Wiskott-Aldrich syndrome protein (N-WASP) has been implicated in endocytosis; however, little is known about how it interacts functionally with the endocytic machinery. Sucrose gradient fractionation experiments and immunofluorescence studies with anti-N-WASP antibody revealed that N-WASP is recruited together with clathrin and dynamin, which play essential roles in clathrin-mediated endocytosis, to lipid rafts in an epidermal growth factor (EGF)-dependent manner. Endophilin A (EA) binds to dynamin and plays an essential role in the fission step of clathrin-mediated endocytosis. In the present study, we show that the Src homology 3 (SH3) domain of EA associates with the proline-rich domain of N-WASP and dynamin in vitro. Co-immunoprecipitation assays with anti-N-WASP antibody revealed that EGF induces association of N-WASP with EA. In addition, EA enhances N-WASP-induced actin-related protein 2/3 (Arp2/3) complex activation in vitro. Immunofluorescence studies revealed that actin accumulates at sites where N-WASP and EA are co-localized after EGF stimulation. Furthermore, studies of overexpression of the SH3 domain of EA indicate that EA may regulate EGF-induced recruitment of N-WASP to lipid rafts. These results suggest that, upon EGF stimulation, N-WASP interacts with EA through its proline-rich domain to induce the fission step of clathrin-mediated endocytosis.
