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(KPNA2) and Its Potential Novel Cargo Proteins in Nonsmall Cell Lung Cancer *□S

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(KPNA2) and Its Potential Novel Cargo Proteins in Nonsmall Cell Lung Cancer *□S Research © 2012 by The American Society for Biochemistry and Molecular Biology, Inc. This paper is available on line at http://www.mcponline.org Quantitative Proteomics Reveals Regulation of Karyopherin Subunit Alpha-2 (KPNA2) and Its Potential Novel Cargo Proteins in Nonsmall Cell Lung Cancer *□S Chun-I Wang‡‡‡, Kun-Yi Chien‡¶, Chih-Liang Wangʈ, Hao-Ping Liu¶, Chia-Chen Cheng§, Yu-Sun Chang‡¶, Jau-Song Yu‡§¶, and Chia-Jung Yu‡§¶**‡‡ The process of nucleocytoplasmic shuttling is mediated cancer. Molecular & Cellular Proteomics 11: 10.1074/ by karyopherins. Dysregulated expression of karyo- mcp.M111.016592, 1105–1122, 2012. pherins may trigger oncogenesis through aberrant distri- bution of cargo proteins. Karyopherin subunit alpha-2 (KPNA2) was previously identified as a potential bio- Transportation of proteins and RNAs into (import) and out of marker for nonsmall cell lung cancer by integration of the (export) the nucleus occurs through the nuclear pore complex cancer cell secretome and tissue transcriptome data sets. and is a vital event in eukaryotic cells. Nucleocytoplasmic Knockdown of KPNA2 suppressed the proliferation and shuttling of the large complex (Ͼ40 kDa) is mediated by an migration abilities of lung cancer cells. However, the pre- evolutionarily conserved family of transport factors, desig- cise molecular mechanisms underlying KPNA2 activity in cancer remain to be established. In the current study, we nated karyopherins (1). The family of karyopherins, including applied gene knockdown, subcellular fractionation, and importins and exportins, share limited sequence identity (15– stable isotope labeling by amino acids in cell culture- 25%) but adopt similar conformations. In human cells, at least based quantitative proteomic strategies to systematically 22 importin ␤ and 6 importin ␣ proteins have been identified to analyze the KPNA2-regulating protein profiles in an ade- date (2, 3). Karyopherins cannot be classified solely based on nocarcinoma cell line. Interaction network analysis re- their cargo repertoires, because many of these proteins are vealed that several KPNA2-regulating proteins are in- targeted by several different members of the karyopherin volved in the cell cycle, DNA metabolic process, cellular family (4–6). The most well-established mechanism of nucle- component movements and cell migration. Importantly, ocytoplasmic shuttling is the classical nuclear import pathway E2F1 was identified as a potential novel cargo of KPNA2 in in which classical nuclear localization signal (cNLS)1-contain- the nuclear proteome. The mRNA levels of potential ef- ing cargo proteins transported into the nucleus are recog- fectors of E2F1 measured using quantitative PCR indi- ␣ ␤ ␤ cated that E2F1 is one of the “master molecule” re- nized by importin /importin heterodimers (7). All importin sponses to KPNA2 knockdown. Immunofluorescence family members contain an N-terminal Ran-GTP-binding mo- staining and immunoprecipitation assays disclosed co- tif and selectively bind nucleoporins of the nuclear pore com- localization and association between E2F1 and KPNA2. plex, whereas unusually, importin ␤ interacts indirectly with An in vitro protein binding assay further demonstrated hundreds of different cNLS-containing proteins via an adap- that E2F1 interacts directly with KPNA2. Moreover, tor protein, importin ␣. Importin ␣ also acts independently knockdown of KPNA2 led to subcellular redistribution of through direct binding to cargo proteins without the require- E2F1 in lung cancer cells. Our results collectively demon- ment for importin ␤ (8). The import and export processes of strate the utility of quantitative proteomic approaches and nucleocytoplasmic shuttle proteins are complicated, and dys- provide a fundamental platform to further explore the regulated expression of karyopherin may have oncogenic ef- biological roles of KPNA2 in nonsmall cell lung fects resulting from the unusual distribution of cargo proteins. For example, aberrant nucleocytoplasmic localization of tu- From the ‡Graduate Institute of Biomedical Sciences and §Depart- ment of Cell and Molecular Biology, College of Medicine, Chang Gung 1 The abbreviations used are: cNLS, classical nuclear localization University, Tao-Yuan, Taiwan; ¶Molecular Medicine Research Center, signal; KPNA2, Karyopherin subunit alpha-2; NSCLC, nonsmall cell Chang Gung University, Tao-Yuan, Taiwan; ʈDivision of Pulmonary lung cancer; MRN, MRE11/RAD50/NBN; DSB, double-strand break; Oncology and Interventional Bronchoscopy, Department of Thoracic SILAC, stable isotope labeling with amino acids in cell culture; 2D Medicine, Chang Gung Memorial Hospital, Tao-Yuan, Taiwan LC-MS/MS, two-dimensional LC-MS/MS; LC, liquid chromatography; Received December 21, 2011, and in revised form, May 31, 2012 SCX, strong cation exchange; RP18, reverse phase 18; FDR, false Published, MCP Papers in Press, July 25, 2012, DOI 10.1074/ discovery rate; RT-PCR, reverse transcription polymerase chain re- mcp.M111.016592 action; qPCR, real-time quantitative PCR; PI, propidium iodide. Molecular & Cellular Proteomics 11.11 1105 E2F1 is a Novel Cargo of KPNA2 mor suppressor proteins, such as PTEN, WT1, Arf, and p53, is one man with poorly differentiated lung adenocarcinoma and kindly involved in the development of several human cancers (9–14). provided by Professor P.C. Yang (Department of Internal Medicine, Karyopherin subunit alpha-2 (KPNA2) belongs to the National Taiwan University Hospital, Taipei, Taiwan). Cells were main- tained in RPMI 1640 with 10% fetal bovine serum plus antibiotics at karyopherin family and delivers numerous cargo proteins to 37 °C at a humidified atmosphere of 95% air/5% CO2 (33). For SILAC the nucleus, followed by translocation back to cytoplasmic experiments, CL1-5 cells were maintained in lysine-depleted RPMI compartments in a Ran-GTP-dependent manner (15). One 1640 (Invitrogen, Grand Island, NY) supplemented with 10% dialyzed proposed hypothesis is that KPNA2 plays opposing roles in fetal bovine serum (Invitrogen, Carlsbad, CA), and 0.1 mg/ml heavy [U-13C ]L-lysine (Invitrogen) or 0.1 mg/ml light L-lysine (Invitrogen). oncogenesis through modulation of proper subcellular local- 6 Every 3–4 days, cells were split and media replaced with the corre- ization of particular cargo proteins (16). For example, KPNA2 sponding light or heavy labeling medium. In approximately six dou- mediates the nuclear transport of NBN (also known as NBS1 bling times, cells achieved almost 100% incorporation of isotopic or Nibrin), a component of the MRE11/RAD50/NBN (MRN) labeling amino acids and were subjected to small interfering RNA complex involved in double-strand break (DSB) repair, DNA treatments. recombination, cell cycle checkpoint control, and mainte- Gene Knockdown of KPNA2 With Small Interfering RNA—Gene knockdown of KPNA2 was performed as described previously (23). nance of DNA integrity and genomic stability. Nuclear NBN Briefly, 19-nucleotide RNA duplexes for targeting human KPNA2 were generally acts as a tumor suppressor protein (17–20). Inhibi- synthesized and annealed by Dharmacon (Thermo Fisher Scientific, tion or blockage of the interactions between KPNA2 and NBN Lafayette, CO). In brief, CL1-5 cells cultured in stable isotope labeling results in reduction of DSB repair, cell cycle checkpoint sig- medium were transfected with control siRNA (Heavy) and KPNA2 naling and radiation-induced nuclear focus accumulation (21), pooled siRNA (Light) (GAAAUGAGGCGUCGCAGAA, GAAGC- signifying inhibition of the tumor suppressor function of nu- UACGUGGACAAUGU, AAUCUUACCUGGACACUUU, GUAAAUUG- GUCUGUUGAUG)), respectively using Lipofectamine RNAiMAX re- clear NBN, as it loses interactions with KPNA2. In addition, a agents (Invitrogen) according to the protocol provided by the recent study showed that cytoplasmic NBN plays an onco- manufacturer. At 48 h after transfection, cell lysates were prepared for genic role via binding and activation of the PI3-kinase/AKT Western blotting to determine gene knockdown efficacy. pathway and promotes tumorigenesis (22). Therefore, KPNA2 Subcellular Fractionation—CL1-5 cells transfected with control appears to be a major determinant of the subcellular localiza- siRNA or KPNA2 siRNA were mixed at an equal ratio (1:1) and subjected to a subcellular fractionation using the ProteoJETTM Cyto- tion and the biological functions of its cargo proteins, such as plasmic and Nuclear Protein Extraction Kit K0311 (Fermentas, Can- NBN. ada), according to the manufacturer’s instructions. The efficacy of We previously identified and validated KPNA2 as a potential fractionation was determined via Western blotting using GAPDH as biomarker for nonsmall cell lung cancer (NSCLC) by integra- the cytosolic control and Lamin B as the nuclear control protein. tion of cancer cell secretome and tissue transcriptome data In-solution Digestion of Proteins and Micro-column Desalting—In- solution digestion was performed with the protocols described below. sets (23). We detected KPNA2 overexpression in lung cancer Briefly, the protein mixture (100 ␮g) was dissolved in 50 mM tissues and showed that cancer cells with poor differentiation NH4HCO3, reduced with 10 mM dithiothreitol (DTT) for 30 min at 56 °C and high mitosis are independent determinants for nuclear and alkylated using 30 mM iodoacetamide in the dark for 45 min at KPNA2 expression in NSCLC. Data obtained from exogenous room temperature. Next, 30 mM DTT was added to quench iodoac- expression and KPNA2 knockdown experiments supported etamide
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