RESUMO
SRC family kinases (SFKs) are involved in the activation of phosphatidylinositol-3-kinase (PI3K). In addition, the activity of this lipid kinase can be regulated by the DNA repair protein NBS1. Here, we describe a disturbed expression of some members of the non-receptor tyrosine kinase family in lymphoblastoid cell lines generated from cells of Nijmegen breakage syndrome (NBS) patients. Especially, only minor amounts of the kinases LCK and HCK are expressed in the NBS1(-/-) cell lines as compared to the consanguineous NBS1(+/-) cells. We demonstrate that SFK activity is important for a proper activation of PI3K in these cells and that it is reduced in NBS1(-/-) cells. We provide evidence that the observed reduced PI3K activity in NBS lymphoblasts is caused by an impaired expression of the SFKs LCK and/or HCK. Thus, our data establish a new function for the NBS1 protein as a regulator of PI3K activity via SFK members.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Síndrome de Quebra de Nijmegen/enzimologia , Proteínas Nucleares/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Quinases da Família src/metabolismo , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Humanos , Síndrome de Quebra de Nijmegen/genética , Proteínas Nucleares/genética , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/farmacologia , Tirosina/metabolismo , Quinases da Família src/antagonistas & inibidores , Quinases da Família src/genéticaRESUMO
The molecular causes for enhanced radiosensitivity of Nijmegen Breakage Syndrome cells are unclear, especially as repair of DNA damage is hardly impeded in these cells. We clearly demonstrate that radiation hypersensitivity is accompanied by enhanced gamma-radiation-induced apoptosis in NBS1 deficient lymphoblastoid cell lines. Differences in the apoptotic behavior of NBS1 (-/-) and NBS1 (+/-) cells are not due to an altered p53 stabilization or phosphorylation in NBS1 (-/-) cells. gamma-radiation-induced caspase-8 activity is increased and visualization of CD95 clustering by laser scanning microscopy shows a significant higher activation of the death receptor in NBS1 (-/-) cells. Further investigation of the molecular mechanisms reveals a role for reactive oxygen species-triggered activation of CD95. These results demonstrate that NBS1 suppresses the CD95 death receptor-dependent apoptotic pathway after gamma-irradiation and evidence is given that this is achieved by regulation of the PI3-K/AKT survival pathway.
Assuntos
Apoptose/fisiologia , Proteínas de Ciclo Celular/metabolismo , Linfócitos , Proteínas Nucleares/metabolismo , Receptor fas/metabolismo , Apoptose/efeitos da radiação , Caspase 8/metabolismo , Proteínas de Ciclo Celular/genética , Linhagem Celular , Raios gama , Humanos , Linfócitos/fisiologia , Linfócitos/efeitos da radiação , Síndrome de Quebra de Nijmegen , Proteínas Nucleares/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteína Supressora de Tumor p53/metabolismoRESUMO
The involvement of SODD/BAG-4 was studied in TNFR1 signaling, using SODD/BAG-4- overexpressing HeLa cells as a cellular model. Stable transfection of a SODD/BAG-4 cDNA leads to increased levels of the full-length 70-kDa protein and additional C-terminal fragments as well as altered expression of BAG-1. Concomitantly, the protein amounts of both the TNFR1 and, unexpectedly, the CD95 receptors are elevated. These biochemical changes are paralleled and confirmed by a reduced cellular sensitivity to treatment with extracellular TNFalpha and CD95 ligand. Moreover, ATP depletion by oligomycin is less toxic in SODD/BAG-4 overexpressing clones. The radiosensitivity is reduced in some, but not in all of these clones. Our results indicate that SODD/BAG-4 is not only a silencer of TNFR1 but also a modulator of CD95 activity, regulating the balance of both death-promoting and -depressing signals.
