Abstract
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.
Highlights
From the ‡Graduate Institute of Biomedical Sciences and §Department of Cell and Molecular Biology, College of Medicine, Chang Gung University, Tao-Yuan, Taiwan; ¶Molecular Medicine Research Center, Chang Gung University, Tao-Yuan, Taiwan; ʈDivision of Pulmonary Oncology and Interventional Bronchoscopy, Department of Thoracic Medicine, Chang Gung Memorial Hospital, Tao-Yuan, Taiwan
Identification and Quantification of Differentially Expressed Proteins in Karyopherin subunit alpha-2 (KPNA2) Knockdown Cells with the SILAC Proteomic Approach—To investigate the molecular mechanisms of KPNA2-mediated regulation in lung cancer cells, we applied the SILAC-based quantitative proteomic method combined with subcellular fractionation to systematically analyze KPNA2 siRNA-induced differential expression protein profiles in the CL1-5 adenocarcinoma cell line
We found that deletion of 85RPPVKRRLDLE95 classical nuclear localization signal (cNLS) site (NLS1) but not 161KVQKRRIYDI170 (NLS2) in E2F1 substantially impaired its binding to KPNA2, suggesting that the NLS1 was essential for E2F1 binding to KPNA2 (Fig. 5D-a)
Summary
Cell Culture and Stable Isotope Labeling by Amino Acids in Cell Culture—The CL1-5 human lung cancer cell line was derived from one man with poorly differentiated lung adenocarcinoma and kindly provided by Professor P.C. CL1-5 cells cultured in stable isotope labeling medium were transfected with control siRNA (Heavy) and KPNA2 pooled siRNA (Light) (GAAAUGAGGCGUCGCAGAA, GAAGCUACGUGGACAAUGU, AAUCUUACCUGGACACUUU, GUAAAUUGGUCUGUUGAUG)), respectively using Lipofectamine RNAiMAX reagents (Invitrogen) according to the protocol provided by the manufacturer. Subcellular Fractionation—CL1-5 cells transfected with control siRNA or KPNA2 siRNA were mixed at an equal ratio (1:1) and subjected to a subcellular fractionation using the ProteoJETTM Cytoplasmic and Nuclear Protein Extraction Kit K0311 (Fermentas, Canada), according to the manufacturer’s instructions. The resulting protein complexes were eluted with SDS sample buffer, transferred to polyvinylidene difluoride membranes and analyzed by Western blotting using primary antibodies raised against the following proteins: p53 (DO1, Santa Cruz Biotechnologies, Santa Cruz, CA), E2F1(KH95, Santa Cruz Biotechnologies), c-Myc (9E10, Clontech Laboratories, Inc., Palo Alto, CA) and KPNA2 (Proteintech Group Inc.). Membranes were incubated with the appropriate secondary antibodies and the signals were visualized by enhanced chemiluminescence according to the specifications from the supplier (Millipore)
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