Mapping the interactome of overexpressed RAF kinase inhibitor protein in a gastric cancer cell line.
Mapping the interactome of overexpressed RAF kinase inhibitor protein in a gastric cancer cell line.
复制标题
绘制胃癌细胞系中过度表达的 RAF 激酶抑制剂蛋白的相互作用组图
DOI：
10.1186/1471-2407-13-536
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发表时间：
2013-11-09
期刊：
影响因子：
3.8
通讯作者：
Chen Z
中科院分区：
文献类型：
--
作者：
Gu H;Zhan X;Zhang G;Yan L;Cho WC;Li M;Liu T;Chen Z
胃癌（Gastric cancer，GC）是一种严重威胁人类健康的恶性肿瘤，其发病率和死亡率在世界范围内呈逐年上升趋势. RAF激酶抑制蛋白（RKIP）表达下调或缺失与胃癌的发生、分化、侵袭和转移有关。方法将重组质粒pcDNA3.1-RKIP-3xFLAG转染人胃癌细胞系SGC 7901，用抗FLAG M2磁珠纯化RKIP融合蛋白，用串联质谱（MS/MS）鉴定RKIP相互作用蛋白，并进行生物信息学分析。结果MS/MS共鉴定出72个与RKIP相互作用的蛋白，这些蛋白在酶代谢、分子伴侣、生物氧化、细胞骨架组装、信号转导、细胞凋亡等方面发挥重要作用。利用Michigan Molecular Interactions、Functional Linage Network和Predictome分析构建了RKIP相互作用蛋白网络图，以阐明RKIP功能活性的分子途径。MS/MS表征的现有的相互作用复合物（RKIP，HSP 90，14-3-3 β，和角蛋白8）的成分被证实通过Western印迹分析和co-immunoprecipitation.ConclusionThis研究是首次发现的RKIP与HSP 90，14-3-3，和角蛋白的相互作用。本研究结果为深入了解RKIP抑制胃癌发生发展的分子机制提供了理论依据。
BackgroundGastric cancer (GC) is a threat to human health with increasing incidence and mortality worldwide. Down-regulation or absence of RAF kinase inhibitor protein (RKIP) was associated with the occurrence, differentiation, invasion, and metastasis of GC. This study aims to investigate the molecular mechanisms and biological functions of RKIP in the GC biology.MethodsThe fusion expression plasmid pcDNA3.1-RKIP-3xFLAG was transfected into SGC7901 cells, the RKIP fusion proteins were purified with anti-flag M2 magnetic beads, and the RKIP-interacting proteins were identified with tandem mass spectrometry (MS/MS), and were analyzed with bioinformatics tools. Western blot and co-immunoprecipitation were used to confirm the interaction complex.ResultsA total of 72 RKIP-interacting proteins were identified by MS/MS. Those proteins play roles in enzyme metabolism, molecular chaperoning, biological oxidation, cytoskeleton organization, signal transduction, and enzymolysis. Three RKIP-interaction protein network diagrams were constructed with Michigan Molecular Interactions, functional linage network, and Predictome analysis to address the molecular pathways of the functional activity of RKIP. The MS/MS-characterized components of the existing interaction complex (RKIP, HSP90, 14-3-3ϵ, and keratin 8) were confirmed by Western blot analysis and co-immunoprecipitation.ConclusionThis study is the first discovery of the interaction of RKIP with HSP90, 14-3-3, and keratin. The present data would provide insight into the molecular mechanisms of how RKIP inhibits the occurrence and development of GC.
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影响因子：
3.4
作者：
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