Quantitative Proteomics Reveals Regulation of Karyopherin Subunit Alpha-2 (KPNA2) and Its Potential Novel Cargo Proteins in Nonsmall Cell Lung Cancer
Quantitative Proteomics Reveals Regulation of Karyopherin Subunit Alpha-2 (KPNA2) and Its Potential Novel Cargo Proteins in Nonsmall Cell Lung Cancer
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DOI：
10.1074/mcp.m111.016592
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发表时间：
2012-11-01
影响因子：
7
通讯作者：
Yu, Chia-Jung
中科院分区：
文献类型：
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作者：
Wang, Chun-I;Chien, Kun-Yi;Yu, Chia-Jung
核质班的过程是由核蛋白介导的。核蛋白的表达失调可能通过货物蛋白的异常分布引发肿瘤发生。核蛋白亚基α-2（KPNA2）先前被鉴定为通过整合癌细胞分泌组和组织转录组数据集的非小细胞肺癌的潜在生物标志物。 KPNA2的敲低抑制了肺癌细胞的增殖和迁移能力。但是，癌症中KPNA2活性的确切分子机制仍有待确定。在当前的研究中，我们在基于细胞培养的定量蛋白质组学策略中应用了基因敲低，亚细胞分级分离和稳定的同位素标记，以系统地分析腺癌细胞系中KPNA2调节蛋白质谱。相互作用网络分析表明，几种KPNA2调节蛋白参与细胞周期，DNA代谢过程，细胞成分运动和细胞迁移。重要的是，E2F1被确定为核蛋白质组中KPNA2的潜在新型货物。使用定量PCR测量的E2F1潜在效应子的mRNA水平表明，E2F1是对KPNA2敲低的“主分子”响应之一。免疫荧光染色和免疫沉淀测定法揭示了E2F1和KPNA2之间的共定位和关联。体外蛋白结合测定进一步表明E2F1与KPNA2直接相互作用。此外，KPNA2的敲低导致肺癌细胞中E2F1的亚细胞重新分布。我们的结果共同证明了定量蛋白质组学方法的实用性，并提供了一个基本平台，以进一步探索KPNA2在非小细胞肺癌中的生物学作用。分子和细胞蛋白质组学11：10.1074/MCP.M111.016592，1105-1122，2012。
The process of nucleocytoplasmic shuttling is mediated by karyopherins. Dysregulated expression of karyopherins may trigger oncogenesis through aberrant distribution of cargo proteins. Karyopherin subunit alpha-2 (KPNA2) was previously identified as a potential biomarker for nonsmall cell lung cancer by integration of the cancer cell secretome and tissue transcriptome data sets. Knockdown of KPNA2 suppressed the proliferation and migration abilities of lung cancer cells. However, the precise molecular mechanisms underlying KPNA2 activity in cancer remain to be established. In the current study, we applied gene knockdown, subcellular fractionation, and stable isotope labeling by amino acids in cell culture-based quantitative proteomic strategies to systematically analyze the KPNA2-regulating protein profiles in an adenocarcinoma cell line. Interaction network analysis revealed that several KPNA2-regulating proteins are involved in the cell cycle, DNA metabolic process, cellular component movements and cell migration. Importantly, E2F1 was identified as a potential novel cargo of KPNA2 in the nuclear proteome. The mRNA levels of potential effectors of E2F1 measured using quantitative PCR indicated that E2F1 is one of the "master molecule" responses to KPNA2 knockdown. Immunofluorescence staining and immunoprecipitation assays disclosed co-localization and association between E2F1 and KPNA2. An in vitro protein binding assay further demonstrated that E2F1 interacts directly with KPNA2. Moreover, knockdown of KPNA2 led to subcellular redistribution of E2F1 in lung cancer cells. Our results collectively demonstrate the utility of quantitative proteomic approaches and provide a fundamental platform to further explore the biological roles of KPNA2 in nonsmall cell lung cancer. Molecular & Cellular Proteomics 11: 10.1074/mcp.M111.016592, 1105-1122, 2012.