亲和纯化技术联合质谱分析筛选乳腺癌中p90核糖体S6蛋白激酶4相互作用蛋白

白激酶4相互作用蛋白

刘日强;刘天华;张钦乐;江凯;郭坤;张舒;刘银坤;杨华伟

【期刊名称】《中国癌症杂志》 【年(卷),期】2016(026)007

【摘 要】Background and purpose:As a tumor suppressor gene, ribosomal S6 kinase 4 (RSK4) plays important roles in inhibiting cell proliferation, migration and inducing cell apoptosis. However, the proteins interacting with RSK4 are still unknown. This study aimed to screen proteins interacting with RSK4 in breast cancer cell line MDA-MB-231 by lfag-tag affnity puriifcation and LC-MS/MS (liquid chromatography/mass spectrometry).Methods:The pcDNA3.1/EGFP-RSK4-Flag eukaryotic expression vector was constructed by inserting full lengthRSK4 gene into vector pcDNA3.1/EGFP-Flag. And then the recombinant plasmids were transferred into MDA-MB-231 cells. Real-timelfuorescent quantitative polymerase chain reaction (RTFQ-PCR) and Western blot were used to detect the expression of RSK4 in MDA-MB-231 cells. Affnity puriifcation and LC-MS/MS were applied to screen proteins interacting with RSK4, and the related action mechanism of RSK4 with its interacted proteins was detected based on bioinformatics gene ontology (GO) and ingenuity pathway analysis (IPA).Results:Twenty-four proteins, such as

serine/threonine-pro-tein kinase 38 (STK38)/serine/threonine-protein

kinase 38-like (STK38L), MOB kinase activator 2 (MOB2) and protein arginineN-methyltransferase 5 (PRMT5), were successfully identiifed by Flag-tag affnity puriifcation followed by LC-MS/MS analysis, which probably interacted with RSK4. Bioinformatics analysis of the identiifed proteins suggested the proteins interacting with RSK4 were involved in diverse biological pathways, such as apoptosis and cell migration.

Conclusion:According to bioinformatics results of proteins interacting with RSK4 identiifed by affnity puriifcation and LC-MS/MS, biological networks of RSK4 are involved in apoptosis and migration in breast cancer cells.%背景与目的：p90核糖体S6蛋白激酶4基因(ribosomal S6 kinase 4 gene，RSK4)作为抑癌基因，能够抑制细胞增殖、迁移并诱导细胞的凋亡，但与其配合发挥相应功能的相互作用蛋白尚不明确。该研究利用Flag标签纯化技术和质谱分析筛选鉴定乳腺癌细胞株MDA-MB-231中的RSK4相互作用蛋白。方法：构建含有RSK4全长和Flag亲和标签的真核表达载体pcDNA3.1/EGFP-RSK4-Flag，将其转染至MDA-MB-231乳腺癌细胞株，72 h后收集细胞蛋白。采用实时荧光定量聚合酶链反应(real-time lfuorescent quantitative polymerase chain re-action，RTFQ-PCR)和蛋白[质]印迹法(Western blot)检测RSK4表达情况。运用标签分离纯化RSK4蛋白，液相质谱/质谱分析技术(liquid chromatography mass spectrometry/mass spectrometry，LC-MS/MS)鉴定RSK4相互作用蛋白，通过生物信息学的方法(GO分析和IPA分析)归纳分析互作蛋白以及与RSK4相互作用机制。结果：通过标签纯化联合质谱分析成功鉴定出丝氨酸/苏氨酸蛋白激酶38(serine/threonine-protein kinase 38，STK38)/丝氨酸/苏氨酸蛋白激酶38类(serine/threonine-protein kinase 38-like，STK38L)、MOB激酶致活因子2(MOB kinase activator 2，MOB2)、蛋白精氨酸N甲基转移酶5(protein

arginineN-methyltransferase 5，PRMT5)等24个RSK4相互作用蛋白。对所鉴定出的互作蛋白进行生物信息学分析，提示RSK4互作蛋白组参与包含细胞凋亡和细胞迁移运动等在内的多种生物信号通路。结论：利用亲和纯化技术联合质谱鉴定成功筛选出RSK4相互作用蛋白并通过生物信息学分析获得了RSK4参与乳腺癌细胞的凋亡、迁移相关生物调控网络。 【总页数】8页(P581-588)

【作 者】刘日强;刘天华;张钦乐;江凯;郭坤;张舒;刘银坤;杨华伟

【作者单位】广西医科大学附属肿瘤医院乳腺外科，广西 南宁 530021;复旦大学附属中山医院肝癌研究所，上海 200032; 复旦大学生物医学研究院，上海 200032;复旦大学附属中山医院肝癌研究所，上海 200032; 复旦大学生物医学研究院，上海 200032;复旦大学附属中山医院肝癌研究所，上海 200032; 复旦大学生物医学研究院，上海 200032;复旦大学附属中山医院肝癌研究所，上海 200032;复旦大学附属中山医院肝癌研究所，上海 200032;复旦大学附属中山医院肝癌研究所，上海 200032; 复旦大学生物医学研究院，上海 200032;广西医科大学附属肿瘤医院乳腺外科，广西 南宁 530021 【正文语种】中 文 【中图分类】R737.9 【相关文献】

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