神經(jīng)生長因子（NGF）結合細胞表面受體p75NTR （p75神經(jīng)營養(yǎng)素受體）和TrkA （酪氨酸蛋白激酶A）后介導了細胞分化、細胞生存、凋亡、增殖和侵襲等多個重要的生理病理過程. TrKA能與細胞內多個蛋白質相互作用,但是由于NGF信號通路的復雜性,現(xiàn)在仍有必要發(fā)現(xiàn)與之相互作用的蛋白質以更準確地了解NGF信號通路.本研究中我們通過酵母雙雜交的方法篩選到了一個新的與TrKA相互作用的蛋白質——真核生物翻譯起始因子4A1 （eIF4A1）,然后通過谷胱甘肽巰基轉移酶融合蛋白沉降實驗（GST-pull-down）和免疫共沉淀實驗（Co-IP）證明了TrkA和eIF4A1的相互作用.此外NGF能夠增強TrkA和eIF4A1的相互作用.在鑒定相互作用位點實驗中,我們發(fā)現(xiàn)eIF4A1的氨基端結構域和TrkA的TK結構域參與了相互作用. TrkA和e IF4A1共定位在細胞膜上. NGF能夠引起TrkA與泛素蛋白63位的賴氨酸連接,而eIF4A1與TrkA相互作用后能夠抑制TrkA與泛素蛋白63位的賴氨酸連接.綜上,得出結論 e IF4A1通過與TrkA相互作用抑制其泛素化調控NGF信號通路. 

【文章來源】：生物化學與生物物理進展. 2019,46(08)北大核心SCICSCD

【文章頁數(shù)】：11 頁

【文章目錄】：
1 Methods
    1.1 Yeast two hybrid screening
    1.2 Cell culture, transfection and NGF stimulation
    1.3 Plasmids, antibodies and reagents
    1.4 GST pull-down assay
    1.5 Co-immunoprecipitation and immunoblotting
    1.6 Immunofluorescence assay
    1.7 Cytoplasmic and nuclear extraction
2 Results
    2.1 Identification of the novel association of TrkA and eIF4A1
    2.2 eIF4A1 binds to TrkA-ICD in vivo and in vitro
    2.3 Mapping the interacting domain between TrkA and eIF4A1
    2.4 Membrane co-localization of TrkA and eIF4A1
    2.5 NGF stimulated TrkA Lys 63-linked polyubiquitination and eIF4A1 repressed TrkApolyubiquitination
3 Discussion



本文編號：3134958