Wilhelm Michael, Xu Zhiheng, Kukekov Nickolay V, Gire Stephen, Greene Lloyd A
Department of Pediatrics, Columbia University Health Sciences, New York, New York 10032, USA.
J Biol Chem. 2007 Jan 12;282(2):1288-95. doi: 10.1074/jbc.M607038200. Epub 2006 Nov 9.
Nix, a pro-apoptotic BH3-only protein, promotes apoptosis of non-neuronal cells, although the mechanisms involved remain incompletely understood. Using a yeast two-hybrid screen with POSH (plenty of SH3 domains, a scaffold involved in activation of the apoptotic JNK/c-Jun pathway) as the bait, we identified an interaction between POSH and Nix. Co-immunoprecipitation and in vitro binding studies confirmed a direct interaction between POSH and Nix in mammalian cells. When overexpressed in HEK293 cells, Nix promotes apoptosis along with enhanced phosphorylation/activation of JNKs and their target c-Jun. These effects appear to be dependent on POSH because Nix does not promote either JNK/c-Jun phosphorylation or apoptosis of 293 cells that do not express POSH. Nix and POSH appear to mutually stabilize one another and this effect could contribute to their promotion of death. Past work showed induction of Nix transcripts in a cellular model of Parkinson disease based on neuronal PC12 cells exposed to 6-hydroxydopamine. Here, we confirm elevation of Nix protein in this model and that Nix over-expression causes apoptotic death of PC12 cells by a mechanism dependent on c-Jun activation. Expression of s-Nix, a dominant-negative form of Nix, protects neuronal PC12 cells from 6-hydroxydopamine but not from nerve growth factor deprivation. These results indicate that Nix promotes cell death via interaction with POSH and activation of the JNK/c-Jun pathway and that Nix protein is induced and contributes to cell death in a cellular model of Parkinson disease.
Nix是一种仅含BH3结构域的促凋亡蛋白，可促进非神经元细胞凋亡，但其具体机制仍未完全明确。我们以POSH（富含SH3结构域，一种参与凋亡性JNK/c-Jun信号通路激活的支架蛋白）为诱饵进行酵母双杂交筛选，鉴定出POSH与Nix之间存在相互作用。免疫共沉淀和体外结合研究证实了在哺乳动物细胞中POSH与Nix之间存在直接相互作用。当在HEK293细胞中过表达时，Nix可促进细胞凋亡，同时增强JNK及其靶标c-Jun的磷酸化/激活。这些效应似乎依赖于POSH，因为Nix不会促进不表达POSH的293细胞的JNK/c-Jun磷酸化或凋亡。Nix和POSH似乎相互稳定，这种效应可能有助于它们促进细胞死亡。过去的研究表明，在基于暴露于6-羟基多巴胺的神经元PC12细胞建立的帕金森病细胞模型中，Nix转录本会被诱导。在此，我们证实了该模型中Nix蛋白水平升高，且Nix过表达通过依赖c-Jun激活的机制导致PC12细胞凋亡性死亡。Nix的显性负性形式s-Nix的表达可保护神经元PC12细胞免受6-羟基多巴胺的损伤，但不能使其免受神经生长因子剥夺的影响。这些结果表明，Nix通过与POSH相互作用并激活JNK/c-Jun信号通路促进细胞死亡，且在帕金森病细胞模型中Nix蛋白被诱导并参与细胞死亡过程。
