【摘要】 神经生长因子(NGF)结合细胞表面受体p75NTR (p75神经营养素受体)和TrkA (酪氨酸蛋白激酶A)后介导了细胞分化、细胞生存、凋亡、增殖和侵袭等多个重要的生理病理过程. TrKA能与细胞内多个蛋白质相互作用,但是由于NGF信号通路的复杂性,现在仍有必要发现与之相互作用的蛋白质以更准确地了解NGF信号通路.本研究中我们通过酵母双杂交的方法筛选到了一个新的与TrKA相互作用的蛋白质——真核生物翻译起始因子4A1 (eIF4A1),然后通过谷胱甘肽巯基转移酶融合蛋白沉降实验(GST-pull-down)和免疫共沉淀实验(Co-IP)证明了TrkA和eIF4A1的相互作用.此外NGF能够增强TrkA和eIF4A1的相互作用.在鉴定相互作用位点实验中,我们发现eIF4A1的氨基端结构域和TrkA的TK结构域参与了相互作用. TrkA和e IF4A1共定位在细胞膜上. NGF能够引起TrkA与泛素蛋白63位的赖氨酸连接,而eIF4A1与TrkA相互作用后能够抑制TrkA与泛素蛋白63位的赖氨酸连接.综上,得出结论 e IF4A1通过与TrkA相互作用抑制其泛素化调控NGF信号通路.更多还原

【Abstract】 Nerve growth factor(NGF) can bind to cell surface receptor p75 NTR and TrkA and play a vital role in cell differentiation, survival, apoptosis, proliferation and migration. TrkA interacted with multiple proteins in vivo, but due to the complexity of the NGF signaling pathway, it is still necessary to explore more proteins that interact with TrkA to gain a more accurate understanding of the NGF pathway. Here we report eIF4 A1 is a new partner of TrkA. We found eIF4 A1 interacted with TrkA via the yeast two hybrid assay. And then the association between TrkA and e IF4 A1 was identified by GST pull-down and coimmunoprecipitation assay. Additionally, NGF stimulated this interaction and the associated binding domain is the N-terminus domain(NTD) in e IF4 A1 and the TK domain in TrkA. And eIF4 A1 could colocalize with TrkA at cell membrane. Furthermore,eIF4 A1 also inhibits TrkA polyubiquitination through lysine(Lys)-63-linked polyubiquitin chains which causes its internalization. So eIF4 A1 plays a novel role in NGF signaling pathway.更多还原