AAK1相互作用蛋白的筛选及其调控细胞内整体翻译水平的研究

Screening of AAK1 interaction proteins and its role in regulating global translation level in cells

目的·探究衔接子相关蛋白激酶1(adaptor-associated protein kinase 1,AAK1)新的相互作用蛋白,以及除网格蛋白介导的内吞作用外AAK1介导的生物学功能。方法·通过在HEK-293T细胞中分别外源性转染带有标签的AAK1载体与空白对照载体,利用标签特异性的琼脂糖凝胶进行免疫共沉淀(co-immunoprecipitation,CoIP),并联合质谱分析的方法获得潜在与AAK1相互作用的蛋白;通过CoIP初步验证质谱结果;通过荧光共聚焦成像观察AAK1与其潜在结合蛋白在细胞内的空间定位;通过体外纯化重组蛋白,利用谷胱甘肽巯基转移酶融合蛋白沉降实验(glutathione-S-transferase pulldown,GST Pulldown)进一步明确蛋白间是否为直接的相互作用;通过嘌呤霉素结合实验观察AAK1对于细胞内整体翻译水平的调控作用。结果·质谱结果提示AAK1可能与以脆性X相关蛋白1(fragile X mental retardation syndrome-related protein 1,FXR1)、FXR2、脆性X智力低下蛋白(fragile X mental retardation protein 1,FMRP)三者为核心的一系列蛋白形成复合体。外源性转染AAK1-3xFLAG及FMRP-MYC质粒,利用抗FLAG琼脂糖凝胶富集AAK1-3xFLAG后,可以检测到FMRPMYC的表达;利用内源性抗体进行CoIP,发现富集AAK1可以检测到FMRP的表达。荧光共聚焦成像显示EGFP-AAK1与mCherry-FMRP在细胞质中存在部分空间共定位。GST Pulldown显示FMRP可以直接沉淀HIS6-AAK1重组蛋白。嘌呤霉素结合实验显示相同时间内嘌呤霉素标记的细胞内新合成肽段数量与AAK1蛋白表达量呈正相关。结论·AAK1与FMRP在细胞质内存在直接的相互作用,且AAK1可以提高细胞内的翻译水平。

Objective·To investigate noval interacting partners for adaptor-associated protein kinase 1(AAK1)and AAK1-mediated biological functions besides clathrin-mediated endocytosis.Methods·The labeled AAK1 vector and the blank control vector were transfected in HEK-293T cells,and the potential AAK1 interacting proteins were obtained by co-immunoprecipitation with agar-specific gel and mass spectrometry.Further verifications were performed by CoIP and fluorescence-based imaging.Recombinant proteins were purified in vitro and the direct interaction between proteins were confirmed by glutathione-S-transferase pulldown(GST Pulldown)assay.The regulation of AAK1 in the global protein synthesis was explored by puromycin incorporation assay.Results·Mass spectrometry results showed that AAK1 was associated with a series of proteins,including fragile X mental retardation syndrome-related protein 1(FXR1),FXR2 and fragile X mental retardation protein 1(FMRP).Enriching with antiFLAG agarose gels after exogenous transfecting of AAK1-3xFLAG and FMRP-MYC plasmids,the expression of FMRP-MYC was detected.The expression of FMRP could also be detected by CoIP with endogenous AAK1 antibodies.Fluorescence-based imaging showed that they were spatially colocalized in the cytoplasm.GST Pulldown assay showed that FMRP could pulldown recombinant HIS6-AAK1 protein.Puromycin incorporation assay showed that in the same amount of time,the number of newly synthesized peptides labeled with puromycin was positively correlated with AAK1 protein expression.Conclusion·AAK1 directly interacts with FMRP in cytoplasm and could up-regulate global protein synthesis level.