Quantitative Proteomics Reveals a Novel Role of Karyopherin Alpha 2

Article pubs.acs.org/jpr

Quantitative Proteomics Reveals a Novel Role of Karyopherin Alpha 2 in Cell Migration through the Regulation of Vimentin−pErk Protein Complex Levels in Lung Cancer Chun-I Wang,† Chih-Liang Wang,∥ Yi-Cheng Wu,⊥ Hsiang-Pu Feng,‡ Pei-Jun Liu,§ Yu-Sun Chang,†,‡ Jau-Song Yu,†,‡,§ and Chia-Jung Yu*,†,‡,§ †

Molecular Medicine Research Center, ‡Department of Cell and Molecular Biology, and §Graduate Institute of Biomedical Sciences College of Medicine, Chang Gung University, Tao-Yuan, Taiwan ∥ Division of Pulmonary Oncology and Interventional Bronchoscopy, Department of Thoracic Medicine, ⊥Division of Thoracic & Cardiovascular Surgery, Chang Gung Memorial Hospital, Linkou, Tao-Yuan, Taiwan S Supporting Information *

ABSTRACT: Karyopherin alpha 2 (KPNA2) is overexpressed in various human cancers and is associated with cancer invasiveness and poor prognosis. Herein, to understand the essential role of KPNA2 protein complexes in cancer progression, we applied stable isotope labeling with amino acids in cell culture (SILAC)-based quantitative proteomic strategy combined with immunoprecipitation (IP) to investigate the differential KPNA2 protein complexes in lung adenocarcinoma cell lines with different invasiveness potentials. We found that 64 KPNA2-interaction proteins displayed a 2-fold difference in abundance between CL1-5 (high invasiveness) and CL1-0 (low invasiveness) cells. Pathway map analysis revealed that the formation of complexes containing KPNA2 and cytoskeleton-remodeling-related proteins, including actin, beta tubulin, tubulin heterodimers, vimentin, keratin 8, keratin 18, and plectin, was associated with cancer invasiveness. IP demonstrated that the levels of KPNA2−vimentin− pErk complexes were significantly higher in CL1-5 cells than in CL1-0 cells. The KPNA2−vimentin−pErk complex was also upregulated in the advanced stage compared with the early-stage lung adenocarcinoma tissues. Importantly, the levels of pErk as well as cell migration ability were significantly reduced in KPNA2-knockdown cells; however, migration was restored by treatment with pErk phosphatase inhibitors. Collectively, our results demonstrate the usefulness of a SILAC-based proteomic strategy for identifying invasiveness-associated KPNA2 protein complexes and provide new insight into the KPNA2-mediated modulation of cell migration. KEYWORDS: KPNA2, Vimentin, pErk, Lung cancer, Invasiveness

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INTRODUCTION The transportation of proteins and RNA into (import) and out of (export) the nucleus occurs through the nuclear pore complex (NPC) and is a vital event in eukaryotic cells. Nucleocytoplasmic shuttling of the large complex (>40 kDa) is mediated by an evolutionarily conserved family of transport factors, designated karyopherins.1 Karyopherin alpha 2 (KPNA2) belongs to the karyopherin family, and delivers numerous cargo proteins to the nucleus, followed by translocation back into cytoplasmic compartments in a RanGTP-dependent manner.2 We previously identified and validated KPNA2 as a potential biomarker for non-small cell lung cancer (NSCLC) by integrating cancer cell secretome and tissue transcriptome data sets.3 We detected KPNA2 overexpression in lung cancer tissues and showed that cancer cells with poor differentiation and high mitosis are independent determinants of nuclear KPNA2 expression in NSCLC. © XXXX American Chemical Society

Aberrant KPNA2 levels have also been observed in various human cancers, including breast cancer, melanoma, cervical cancer, esophageal cancer, lung cancer, ovarian cancer, prostate cancer, liver cancer, bladder cancer, and brain cancer.3−12 The elevated expression of KPNA2 has been correlated with poor prognosis and is associated with tumor invasiveness8−11,13,14 The lung adenocarcinoma cell lines CL1-0 and CL1-5 were derived from a 64-year-old man with poorly differentiated lung adenocarcinoma. A transwell invasion chamber was used to progressively select more invasive cancer cell populations from parental CL1 cells. CL1-5 is a subline with higher metastatic and invasive potential than CL1-0.15 To examine the possible role(s) of KPNA2 in the tumor progression of lung cancer, we previously applied the siRNA approach to suppress the Received: October 23, 2014

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DOI: 10.1021/pr501097a J. Proteome Res. XXXX, XXX, XXX−XXX

Article

Journal of Proteome Research

advanced-stage adenocarcinoma patients carrying wild-type (WT) EGFR were pooled according to the disease stage. Similarly, four earlystage lung adenocarcinoma tissues carrying WT EGFR and four tissues carrying mutated EGFR were pooled according to the EGFR mutation status. The pooled tissue extracts (2 mg) were incubated in 4 μg of an anti-KPNA2 antibody (B-9; Santa Cruz Biotechnology) and 20 μL of Dynabeads protein G (Invitrogen). All the incubations were performed for 2 h at room temperature with rotation, and the samples were then washed twice with Tris buffer A (20 mM Tris-HCl (pH 7.5), 250 mM NaCl, and 0.5 mM DTT) and three times with Tris buffer B (20 mM Tris-HCl (pH 7.5) and 0.5 mM DTT). The resulting protein complexes were eluted with SDS sample buffer, separated via SDS-PAGE, transferred to PVDF membranes, and analyzed by Western blot using primary antibodies against the following proteins: KPNB1 (H-300, Santa Cruz Biotechnology), vimentin (V9, Santa Cruz Biotechnology), Erk (C16, Santa Cruz Biotechnology), pErk (Cell Signaling Technology, Beverly, MA), GAPDH (6C-5, Santa Cruz Biotechnology), and KPNA2 (Proteintech, Chicago, IL). Subsequently, membranes were incubated with the appropriate secondary antibodies, and the signals were visualized by enhanced chemiluminescence according to the manufacturer’s specifications (Millipore Inc., Billerica, MA).

expression of KPNA2 in CL1-0 and CL1-5 cells and assessed the effects on cell migration and survival.16 We found that the migration ability of KPNA2-knockdown CL1-0 and CL1-5 cells were severely impaired compared with that of control cells. KPNA2 overexpression in CL1-0 cells increased migration ability compared with cells transfected with empty vector, confirming that KPNA2 is involved in migration ability. Unexpectedly, cell viability was significantly reduced in KPNA2-knockdown CL1-5 cells but exhibited no changes in CL1-0 cells. This result was partly consistent with our findings that knockdown of KPNA2 led to a significant arrest of the cell cycle at the G2/M phase in CL1-5 and MDA-MB-231cells (high invasiveness), whereas this effect was not apparent in CL1-0 and MCF7 cells (low invasiveness).17 Given the prominent roles of KPNA2 in multiple cellular processes, including cancer progression, the differential protein complexes of KPNA2 between CL1-5 and CL1-0 cells should be compared to determine the molecular mechanisms of KPNA2-mediated cancer progression. We herein applied immunoprecipitation (IP) combined with stable isotope labeling with amino acids in cell culture (SILAC)-based quantitative proteomic technology to identify invasiveness-associated KPNA2 protein complexes and explore the role of KPNA2 complexes in lung cancer tumorigenesis.

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1D SDS-PAGE and In-Gel Digestion of Proteins To obtain equal amounts of KPNA2 protein complexes from CL1-0 and CL1-5, we determined the relative levels of immunoprecipitated KPNA2 from CL1-0 and CL1-5 via Western blot. According to the image quantification of the KPNA2 signals obtained via Western blot, we mixed equal amounts of KPNA2 from these two samples, followed by 1D SDS-PAGE and in-gel digestion. The pooled immunoprecipitated proteins obtained from two cultures of NSCLC cells (5 mg of total cell extracts) were then separated via 10% SDS-PAGE and stained with Coomassie Brilliant Blue G-250. The entire gel lane was cut into 25 pieces and subjected to in-gel tryptic digestion essentially as previously described.19 Briefly, the gel pieces were destained in 10% methanol, reduced with 25 mM NH4HCO3 containing 10 mM dithiothreitol at 60 °C for 30 min, and alkylated with 55 mM iodoacetamide at room temperature for 30 min. The proteins were digested using sequencing-grade modified porcine trypsin (20 μg/mL) (Promega, Madison, WI) overnight at 37 °C. Peptides were extracted with acetonitrile and dried in a SpeedVac.

MATERIALS AND METHODS

Cell Culture and Stable Isotope Labeling by Amino Acids in Cell Culture The human lung adenocarcinoma cancer cell lines CL1-0 and CL1-5 were kindly provided by Professor P.C. Yang (Department of Internal Medicine, National Taiwan University Hospital, Taipei, Taiwan, Republic of China). Cells were maintained in RPMI 1640 with 10% FBS plus antibiotics at 37 °C in a humidified atmosphere of 95% air/ 5% CO2.15 In SILAC experiments, the natural metabolic machinery of the cells is utilized to label all cellular proteins with an isotopic amino acid.18 Briefly, CL1-0 and CL1-5 cells were maintained in arginine- and lysine-depleted RPMI 1640 (Thermo Scientific, Rockford, IL) supplemented with 10% dialyzed FBS (Thermo Scientific) and 0.1 mg/mL light L-lysine and L-arginine or 0.1 mg/mL heavy L[U-13C6]lysine and L-[U-13C6]arginine (Sigma-Aldrich, St. Louis, MO), respectively. The cells were passaged every 3−4 days, and the media were replaced with the corresponding light or heavy labeling medium. The cells achieved almost 100% incorporation of isotopic labeling amino acids in approximately six doubling times and were subjected to IP assays.

Reversed-Phase Liquid Chromatography-Tandem Mass Spectrometry After digestion, each peptide mixture was resuspended in 8 μL of HPLC buffer A (0.1% formic acid, Sigma, St. Louis, MO), and 6.4 μL of the sample was loaded into a trap column (Zorbax 300SB-C18, 0.3 × 5 mm, Agilent Technologies, Wilmington, DE) at a flow rate of 0.2 μL/min in HPLC buffer A. The salts were washed with buffer A at a flow rate of 20 μL/min for 10 min, and the desalted peptides were then separated on a 10 cm analytical C18 column (inner diameter, 75 μm) with a 15 μm tip (New Objective, Woburn, MA). The peptides were eluted by a linear gradient of 0−10% HPLC buffer B (99.9% ACN containing 0.1% formic acid) for 3 min, 10−30% buffer B for 35 min, 30−35% buffer B for 4 min, 35−50% buffer B for 1 min, 50−95% buffer B for 1 min, and 95% buffer B for 8 min at a flow rate of 0.25 μL/min across the analytical column. The LC setup was coupled online to a LTQ-Orbitrap linear ion trap mass spectrometer (Thermo Scientific, San Jose, CA) operated using Xcalibur 2.0.7 software (Thermo Scientific). Full-scan MS was performed using the Orbitrap in a MS range of 350−2000 Da, and the intact peptides were detected at a resolution of 30 000. Internal calibration was performed using the ion signal of (Si(CH3)2O)6H+ at m/z 445.120025 as a lock mass.20 A data-dependent procedure was applied that alternated between one MS survey scan and six MS/MS scans for the six most abundant precursor ions in the MS survey scan with a 2 Da window and fragmented via CID with 35% normalized collision energy. The m/z values selected for MS/MS were dynamically excluded for 180 s. The electrospray voltage applied was 1.8 kV. Both MS and MS/MS spectra were acquired using one microscan with a maximum fill-time of 1000

Immunoprecipitation Assay CL1-0 and CL1-5 cells cultured in the light or heavy medium were extracted in Nonidet P-40 (NP-40) lysis buffer (1% NP-40, 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM Na3VO4, 5 mM EDTA (pH 8.0), 10% glycerol, 10 μg/mL leupeptin, 10 μg/mL aprotinin, 1 mM PMSF) and fractionated by centrifugation (13 000 rpm, 10 min at 4 °C) to obtain cell lysates. For the IP of KPNA2 or vimentin from NSCLC cell lines, cell lysates (2 mg of protein) from CL1-0 and CL15 cells were incubated in 4 μg of an anti-KPNA2 antibody (B-9; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), an antivimentin antibody (V9, Santa Cruz Biotechnology) or control IgG (Santa Cruz Biotechnologies) together with 20 μL of Dynabeads protein G (Invitrogen, Grand Island, NY). For IP of the KPNA2 protein complexes from tissue extracts, lung cancer tissues were lysed in Homo lysis buffer (20 mM Tris-HCl (pH 7.5), 1 mM Na3VO4, 1 mM EDTA (pH 8.0), 1 mM EGTA, 1% Triton X-100, 50 mM NaF, 20 mM sodium pyrophosphate, and 1 mM PMSF) and extracted using a Precellys24 homogenizer (Bertin Technologies, France), followed by centrifugation (13 000 rpm, 10 min at 4 °C) to obtain tissue lysates. Lung tissue extracts obtained from the four early-stage and four B


Article


Gene Knockdown of KPNA2 Using Small Interfering RNA

and 100 ms for MS and MS/MS analysis, respectively. Automatic gain control was used to prevent overfilling of the ion trap, and 5 × 104 ions were accumulated in the ion trap to generate the MS/MS spectra.

Gene knockdown of KPNA2 was performed as described previously.3 Briefly, 19 nucleotide RNA duplexes to target human KPNA2 were synthesized and annealed by Dharmacon (Thermo Scientific). The CL1-0 or CL1-5 cells were transfected with control siRNA or KPNA2pooled siRNA (GAAAUGAGGCGUCGCAGAA, GAAGCUACGUGGACAAUGU, AAUCUUACCUGGACACUUU, and GUAAAUUGGUCUGUUGAUG) using Lipofectamine RNAiMAX reagents (Invitrogen) according to the protocol provided by the manufacturer. At 48 h after transfection, cell lysates were prepared for Western blot to determine gene knockdown efficacy.

Database Search and Protein Quantification Pipeline All the MS and MS/MS data were analyzed and processed using Proteome Discoverer (version 1.2, Thermo Scientific). The top 6 fragment ions per 100 Da of each MS/MS spectrum were extracted for a protein database search using the Mascot search engine (version 2.2.03, Matrix Science) against the UniProtKB/Swiss-Prot sequence database (Release 2010_06, containing 517 100 protein sequence entries). The top-six-peaks filter node improves the number of peptides identified with high confidence by reducing the number of peaks in the searched peak lists. This method avoids matching peptide candidates to spurious or noise peaks, thereby avoiding false peptide matches. The search parameters were set as follows: carbamidomethylation (C) as the fixed modification, oxidation (M), N-acetyl (protein), pyro-Glu/Gln (N-term), and the SILAC label (K) and (R) as variable modifications, 6 ppm for MS tolerance, 0.8 Da for MS/MS tolerance, and 2 for missing cleavage. After database searching, the following filter criteria were applied to all the results: six for the minimum peptide length, two for the minimum unique peptides for the assigned protein, and peptide and protein identifications with FDR less than 1% peptide filter were accepted (detailed information is summarized in Supporting Information, Table S1). For precursor ion quantification, Proteome Discoverer was employed using a standard deviation of 2 ppm mass precision to create an extracted ion chromatogram (EIC) for the designated peptide. On the basis of the abundances of light, medium, and heavy isotopic peak patterns, the peptide was quantified. Monoisotopic events that only contained one peak-quantification channel were excluded. The abundances were measured by calculating the area of the EIC of each isotope of a pattern. The quantification value must be comparable to the exact same value of the isotopic pattern peaks for each peptide. To correct for systemic error resulting from sample preparation, we normalized the heavy/light ratio of KPNA2 to 1 and obtained the normalized heavy/light ratio as shown in Supporting Information, Table S1.

Immunofluorescence Assay CL1-5 cells were cultured on glass coverslips using the 12-well culture dish format. Cells were fixed with 4% formaldehyde and treated with permeabilized buffer (0.1% Triton X-100 and 0.05% SDS in phosphate-buffered saline (PBS)). Next, cells were blocked in blocking solution (0.1% saponin and 0.2% bovine serum albumin in PBS) and incubated with primary antibodies. After washing, cells were further incubated with Alexa Fluor 594-conjugated or Alexa Fluor 488conjugated secondary antibodies for 1 h (Molecular Probes, Eugene, OR), and DNA was stained with Hoechst 33258. Following a second wash with PBS, cells were mounted in 90% glycerol in PBS containing 1 mg/mL ρ-phenylenediamine and observed under a Zeiss Axio Imager Z1 microscope (Carl Zeiss, Gottingen, Germany).

Preparation of the Cytosolic and Nuclear Extracts A protocol developed by Grenklo et al. was modified and used to extract the vimentin from soluble cytosolic, insoluble filamentous cytomatrix, and nuclear fractions for IP.21 Briefly, cells grown on Petri dishes were incubated in NP-40 lysis buffer for 1 min, and the supernatant was collected as the soluble cytosolic fraction. The remaining nuclear proteins and insoluble filamentous cytomatrix proteins were harvested by scraping on ice in nuclear extraction reagent (Thermo Scientific) and continuously vortexing for 15 s every 10 min for a total of 40 min. After centrifugation (13 000 rpm) for 10 min at 4 °C, the supernatant (nuclear extract) was immediately transferred to a clean prechilled tube. The insoluble filamentouscytomatrix-containing pellet was resuspended in NP-40 lysis buffer followed by sonication using a Bioruptor ultrasonicator (Diagenode, Denville, NJ) and centrifugation (13 000 rpm) for 10 min at 4 °C. The supernatants were combined with the soluble cytosolic fraction to obtain the total cytosolic extract.

Patient Population and Clinical Specimens Written informed consent was received from all the patients involved in this study before data collection, and the study was approved by the Institutional Review Board of Chang Gung Memorial Hospital, TaoYuan, Taiwan. The nine surgically resected lung adenocarcinoma cancer tissue samples with WT EGFR (3 males and 6 females; age range = 50−81 years; 5 early-stage and 4 advanced-stage disease patients) and 4 surgically resected lung adenocarcinoma cancer tissue samples with L858R mutated EGFR (1 male and 3 females; age range = 52−78 years; all with early-stage disease) were obtained from patients subjected to surgery at Chang Gung Memorial Hospital. The patients’ medical records were reviewed, and identities were diligently protected.

Transwell Migration Assay Cells were transfected with control or KPNA2 siRNA for 48 h. After transfection, the cells were treated with dual-specificity phosphatase (DUSP) inhibitor (10 μM) for 15 min, harvested by trypsinization, and suspended in OPTI-MEM (Invitrogen). Cell migration was assayed using a 24-well format transwell chamber (8.0 μm pore size filter, Corning, Canton, NY). The cell suspension (300 μL, 3 × 104 cells) was added to each insert of the upper chamber, and each lower chamber was filled with 600 μL of OPTI-MEM containing 10 μL/mL fibronectin. After 6 h of incubation at 37 °C, the chambers were gently washed twice with PBS and fixed with methanol, followed by Giemsa staining. Cells that had traversed the filter to the lower chamber were counted microscopically (200×) in 9 different fields per filter.

Pathway Map Analysis After quantification analysis, differentially expressed proteins of interest were converted into gene symbols and uploaded into MetaCore Version 6.5 build 27009 (GeneGo, St. Joseph, MI) for pathway map analysis. MetaCore consists of curated protein interaction networks based on manually annotated and regularly updated databases. The databases describe millions of relationships between proteins based on publications on proteins and small molecules. These relationships include direct protein interactions, transcriptional regulation, binding, enzyme−substrate interactions, and other structural or functional relationships. The pathways module contains 350 interactive maps for >2000 established pathways in human signaling, regulation, and metabolism. The pathway maps were used to map the canonical pathway of uploaded proteins. The relevant pathway maps were then prioritized on the basis of their significance with respect to the uploaded data sets.

Cell Viability Assay Cells were transfected with control or KPNA2 siRNA for 24 h. After transfection, the cells (5 × 103 cells/well) plated in 24-well plates were treated with DUSP inhibitor (0.25 μM) for 15 min. After incubation at 37 °C for the indicated time intervals, cell viability was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenytetrazolium bromide (MTT) colorimetric growth assay. Briefly, MTT solution (5 mg/ mL) was added, and the cells were incubated at 37 °C for 1 h. The supernatant was aspirated, cells were treated with dimethyl sulfoxide (500 μL), and the absorbance was measured at 540 nm using an ELISA reader (SpectraMax M2; Molecular Devices). C


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Figure 1. Quantitative proteomic analysis of differentially abundant KPNA2 protein complexes between two lung adenocarcinoma cell lines with different invasiveness potentials. (A) Schematic diagrams show the workflow designed for profiling KPNA2-interaction proteins via proteomics-based analysis. The processes included SILAC labeling, IP, GeLC−MS/MS analysis, protein identification, and quantification using Proteome Discoverer software. (B) CL1-0 and CL1-5 cells were cultured in light and heavy labeling media, respectively, followed by IP with anti-KPNA2 antibodies. Equal amounts of immunoprecipitated KPNA2 protein complexes obtained from these two lines (500 μg of total cell lysates) were mixed and subjected to SDS-PAGE analysis and silver staining. (C) Comparison of KPNA2-interaction proteins identified in the current study with well-known KPNA2interaction proteins in GeneCards. (D) Verification of differentially abundant KPNA2-interaction proteins between CL1-0 and CL1-5 cells. The KPNA2 protein complexes prepared from CL1-0 or CL1-5 cell lysates (2 mg) by IP were detected by Western blot. The normalized ratios (CL1-5 heavy/CL1-0 light) of KPNA2, vimentin, and γ-actin derived from SILAC-based proteomics analysis in experiments 1 and 2 (Exp. 1 and 2) are presented. GAPDH was used as an internal control.

Statistical Analysis

respectively. After six doubling times, the KPNA2 protein complex was obtained by IP using the KPNA2-specific antibody. The silver-stained SDS-PAGE of the immunoprecipitated products demonstrated the similar patterns of these complexes from experiments 1 and 2 (Exp. 1 and 2, Figure 1B). Equal amounts of KPNA2-immunoprecipitated products obtained from CL1-0 and CL1-5 cells were mixed and separated via SDS-PAGE, followed by in-gel protein digestion and LC−MS/MS analysis. Following the experimental workflow, 808 and 965 nonredundant proteins in experiments 1 and 2 were identified, respectively. Among these proteins, 751 and 883 nonredundant proteins in experiments 1 and 2 were quantified, respectively (Supporting Information, Table S1). A total of 646 nonredundant proteins were quantified in both experiments (Supporting Information, Table S2). The ratio distributions of quantified proteins in both experiments are presented in the Supporting Information, Figure S1A. The correlation of these 646 quantified proteins between the two independent experiments was 0.71 (p < 0.001, Spearman’s correlation) and is presented in the Supporting Information, Figure S1B. To globally verify the KPNA2-interaction proteins,

All data were processed using GraphPad Prism software (GraphPad Software, Inc., La Jolla, CA). All continuous variables were expressed as the means ± standard deviation (SD). The unpaired t test was employed to analyze the variations observed on the transwell migration assay. For quantitative analysis of the MTT assay, twoway ANOVA was used. A p value less than 0.05 was considered to be statistically significant.

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RESULTS

Identification and Quantification of KPNA2 Protein Complexes from Two Lung Adenocarcinoma Cell Lines with Different Invasiveness Potentials Using a SILAC-Based Proteomic Approach

To better understand the essential role of KPNA2 in cancer progression, we used a SILAC-based quantitative proteomic method combined with IP to systematically analyze the differentially abundant KPNA2 protein complexes between CL1-0 and CL1-5 cell lines. A schematic diagram of the experimental workflow is shown in Figure 1A. Briefly, CL1-0 and CL1-5 cells were cultured in light or heavy labeling media, D


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Table 1. Protein List of 64 KPNA2-Interaction Proteins Displaying a Two-Fold Difference in Abundance between the CL1-5 and CL1-0 Cell Lines Exp. 1 gene name

peptidesa

H/L countb

KRT18 ACTG1 MYH9 SPTAN1 CAPZA1 VIM CAPZB TIMP3 KRT8 SPATS2L ACTL6A MYO1C HIST1H1B PLEC1 LMNA SMARCA2 ACTR2 SQRDL ACSL3 PBRM1 ERLIN1 CDYL BAG2 ARHGEF2 LUZP1 CSRP1 RAB10 SEC22B DBN1 ABCE1 TRIP13 BAT2L1 RRBP1 TMOD3

18 24 123 15 8 32 9 2 26 15 5 8 5 19 10 11 3 7 4 16 3 7 2 14 4 7 4 2 8 5 3 28 8 5

3 1 72 1 3 54 5 2 1 14 2 6 9 4 7 1 3 6 5 17 4 10 2 14 4 7 2 2 3 4 3 71 11 1

Exp. 1

Exp. 2 H/L ratio 84.99 67.01 54.09 45.35 33.73 32.49 24.12 15.16 14.62 14.51 13.04 11.08 10.28 10.16 7.71 6.33 5.52 4.99 4.79 3.54 3.53 3.36 3.36 3.19 3.14 2.92 2.81 2.74 2.42 2.42 2.41 2.39 2.36 2.20

peptidesa

H/L countb

H/L ratio

28 29 135 126 12 42 12 2 37 15 8 11 2 174 24 9 7 15 13 25 8 6 2 17 21 9 4 2 23 5 11 29 8 13

16 6 174 35 12 193 9 1 12 12 6 12 2 104 33 1 7 16 12 25 13 4 2 13 17 12 2 2 10 5 11 79 9 1

94.32 8.09 55.00 69.16 39.49 29.24 27.76 11.08 107.58 17.09 25.05 6.35 12.38 23.88 12.20 6.68 8.30 10.73 7.01 5.50 5.05 6.89 3.53 4.82 14.00 5.13 2.28 3.02 101.04 4.11 2.56 4.42 3.07 47.36

gene name

peptidesa

H/L countb

PURA PSME3 POLR1B CBX8 KRT4 NOLC1 HSPB1 DDX41 AIFM1 MORF4L2 SDAD1 KRT6A NKRF LYAR JUP DHX33 KRT77 USP36 NQO1 KRT14 PHF6 A2M THRAP3 KRT2 KRT1 KRT5 KRT10 KRT9 ALB HRNR

6 7 34 2 17 25 10 28 4 6 16 42 18 18 21 23 9 28 2 38 17 5 15 58 53 41 40 45 12 26

7 12 48 2 2 125 11 32 3 2 17 3 18 39 3 39 1 31 3 6 33 2 16 19 3 28 33 46 5 2

Exp. 2 H/L ratio

peptidesa

H/L countb

2.08 2.03 0.49 0.48 0.44 0.42 0.40 0.38 0.38 0.37 0.36 0.35 0.32 0.31 0.30 0.27 0.26 0.26 0.23 0.16 0.15 0.13 0.12 0.11 0.07 0.07 0.05 0.05 0.04 0.03

6 5 33 3 28 25 13 25 5 3 8 51 15 21 29 10 23 22 2 40 14 5 9 57 54 46 41 45 35 31

8 8 40 2 4 112 25 21 3 1 8 3 15 46 4 17 2 22 2 9 28 1 11 17 6 37 80 88 7 2

H/L ratio 3.42 2.62 0.47 0.41 0.18 0.36 0.49 0.49 0.48 0.18 0.41 0.06 0.41 0.41 0.43 0.45 0.49 0.47 0.23 0.06 0.16 0.29 0.22 0.06 0.06 0.10 0.06 0.05 0.25 0.04

a

Number of identified peptides. bNumber of peptide ratios that were actually used to calculate the protein ratio.

Table 2. Top Three Significant Pathway Maps of 64 Differentially Abundant KPNA2-Interaction Proteins Displaying a TwoFold Change no.

pathway map

total nodes

root nodes

p value

FDR

1 2 3

cytoskeleton remodeling_keratin filaments cytoskeleton remodeling_neurofilaments wtCFTR and delta508 traffic/clathrin coated vesicles formation (norm and CF)

36 25 19

7 4 3

1.475 × 10−12 3.379 × 10−07 1.231 × 10−05

1.357 × 10−10 1.554 × 10−05 3.775 × 10−04

abundant KPNA2-interaction proteins, vimentin and γ-actin, via Western blot. The consistency between Western blot and MSbased quantitative results, as depicted in Figure 1D, supports the theory that the SILAC strategy is a reliable and feasible method to analyze differentially abundant KPNA2 protein complexes between the two cell lines.

we examined the overlap between our proteomic data set and 397 well-known KPNA2-interacion proteins identified at least twice in the UniProtKB, MINT, STRING, and I2D protein− protein interaction databases. As shown in Figure 1C, 121 proteins of our 646 quantified proteins were reported in the previous study (Supporting Information, Table S2). Using a two-fold change and reproducibility (experiments 1 and 2) as the criteria for the selection of differentially abundant KPNA2interaction proteins between the two cell lines, 36 and 28 more abundant proteins were identified in the KPNA2 protein complexes in CL1-5 cells and CL1-0 cells, respectively (Table 1). To confirm that the quantitative information obtained from MS-based proteomic analysis reflects the protein interaction levels in vivo, we examined two of these 64 differentially

Pathway Map Analysis of Invasiveness-Associated KPNA2 Protein Complexes

To clarify the potential pathways or processes related to the differentially abundant KPNA2 protein complexes between CL1-0 and CL1-5 cells, the 64 proteins displaying a 2-fold difference between these two cell lines were further examined using the MetaCore bioinformatics tool. The differentially abundant KPNA2-interaction proteins were highly correlated E


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Figure 2. Pathway map analysis of invasiveness-associated KPNA2 protein complexes. The 64 differentially abundant KPNA2-interaction proteins were uploaded to the MetaCore mapping tools, and the most significant pathway involved in cytoskeleton remodeling of keratin filaments is shown. Bars labeled in red denote differentially abundant KPNA2-interaction proteins. The interaction mechanism is labeled with a symbol in the hexagon in the middle of the interaction arrow: B, binding; +P, phosphorylation; CS, complex subunit.

with the cytoskeleton-remodeling keratin filaments pathway (Table 2). Figure 2 shows the most significant pathway in which the seven cytoskeleton-remodeling-related proteins (actin, tubulin beta, tubulin heterodimers, vimentin, keratin 8, keratin 18, and plectin) were up-regulated in CL1-5 cells compared to CL1-0 cells. Our results suggest that complex formation of KPNA2 and cytoskeleton-remodeling-related proteins is associated with cancer invasiveness.

links pErk to dynein retrograde motor protein via direct binding of vimentin to importin β in injured nerves.23 In the present study, we proposed that the interaction between vimentin and KPNA2 may participate in cell signaling and contribute to cell invasiveness. To test this hypothesis, we treated cells with epidermal growth factor (EGF) to trigger pErk signaling, followed by coimmunoprecipitation (co-IP), to examine the interaction between KPNA2 and vimentin and/or pErk in lung cancer cells. As shown in Figure 3A, the levels of pErk−vimentin−KPNA2 complexes were significantly higher in CL1-5 cells than in CL1-0 cells, and the level of pErk was similar between these two cell lines. Next, we used an antivimentin antibody in the IP assay to confirm the presence of differentially abundant complexes in these two lung cell lines. Figure 3B shows that vimentin indeed interacted with KPNA2 and pErk and that the levels of KPNA2−vimentin−pErk complexes in CL1-5 cells were higher than those in CL1-0 cells. To examine whether this event was specific to lung cancer cells, we selected two breast cancer cell lines (MCF7 and MDA-MB231) with differential invasiveness and performed the same

KPNA2−Vimentin−pErk Complex Is Associated with Invasiveness in NSCLC and Breast Cancer Cells

On the basis of MetaCore analysis, we were interested in why many cytoskeleton-remodeling-related proteins differentially interacted with KPNA2 between CL1-5 and CL1-0 cells. As shown in Figure 1d, we found that the levels of KPNA2−γ-actin complex but not KPNA2 or γ-actin in CL1-5 cells were much higher than those in CL1-0 cells. A similar pattern was observed for vimentin. Notably, the intermediate filament cytoskeleton may be affected by extracellular stimulation and may participate in cell signaling.22 Perlson et al. showed that vimentin binds to phosphorylated extracellular signal regulated kinase (pErk) and F


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Figure 3. Level of KPNA2−vimentin−pErk complex is associated with invasiveness in NSCLC and breast cancer cells. CL1-0 and CL1-5 cells were treated with EGF (25 ng/mL) for 15 min, followed by co-IP using (A) anti-KPNA2 antibodies or (B) antivimentin antibodies, as described in Materials and Methods. The precipitated protein complexes were analyzed by Western blot using antibodies against KPNB1, vimentin, pErk, Erk, GAPDH, and KPNA2 as indicated. The values represent the quantified signals of Western blot obtained from CL1-5 cells and normalized to those of CL1-0 cells. (C) MCF7 and MDA-MB-231 cells were treated with EGF (25 ng/mL) for 15 min, followed by co-IP using anti-KPNA2 antibodies or antivimentin antibodies. (D) Precipitated protein complexes were analyzed by Western blot as described in Figure 3A,B.

examinations. As shown in Figure 3C,D, MDA-MB-231 cells contained more KPNA2−vimentin−pErk complexes than MCF7 cells, suggesting that the KPNA2−vimentin−pErk complex levels were positively associated with cancer invasiveness and that this characteristic was not specific to lung cancer cells.

stage cancer) were higher than those in WT EGFR cancer tissues (four pooled samples with early-stage cancer). These results collectively suggest that KPNA2−vimentin−pErk complex was associated with invasiveness in vivo and that this functional complex may be regulated by the EGFRmediated signaling pathway.

KPNA2−Vimentin−pErk Complex Is Up-Regulated in Advanced Stage Lung Adenocarcinoma Compared with That in Early-Stage Cancer Tissues

KPNA2 Forms a Complex with Vimentin and pErk in the Cytoplasm

Considering that Erks are phosphorylated upon stimulation, followed by nuclear translocation via interaction with KPNA7,24 we determined the subcellular localization of KPNA2−vimentin−pErk complex formation. We treated CL1-5 cells with EGF at the indicated times (0−60 min) and then examined the subcellular distribution of KPNA2, vimentin, and pErk using immunofluorescence. As shown in Figure 5A, in control cells, pErk and vimentin were primarily distributed in the cytoplasm, and KPNA2 was ubiquitously distributed throughout the nucleus and cytoplasm. After EGF was treated for 15 min, we found that the proportion of KPNA2 distributed in the nucleus was increased; however, pErk and vimentin remained distributed in the cytoplasm. Importantly, the distribution of pErk but not vimentin in the nucleus was

To verify whether KPNA2−vimentin−pErk complexes exist in vivo and are associated with invasiveness, we performed IP using lung cancer tissues from eight adenocarcinoma patients (WT EGFR), including four pooled tissues with early-stage (I and II) disease and four pooled tissues with advanced-stage (III and IV) disease. Consistent with the results obtained from lung cancer cell lines, the levels of KPNA2−vimentin−pErk complexes in advanced-stage cancer tissues were higher than those in early-stage cancer tissues (Figure 4A). Interestingly, we found that the dysregulated EGFR pathway may enhance KPNA2−vimentin−pErk complex formation. Figure 4B shows that the levels of KPNA2−vimentin−pErk complex in mutated EGFR (L858R) cancer tissues (four pooled samples with earlyG


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Figure 4. KPNA2−vimentin−pErk complex is up-regulated in advanced-stage lung adenocarcinoma tissues compared with early-stage lung adenocarcinoma tissues. (A) Lung tissue extracts obtained from four early-stage (I and II) and four advanced-stage (III and IV) adenocarcinoma patients carrying WT EGFR were pooled according to the disease stage. IP was performed using anti-KPNA2 antibodies, and the immunoprecipitated protein complexes were analyzed via Western blot using antibodies against KPNB1, vimentin, pErk, Erk, and KPNA2 as indicated. γ-actin was used as the internal control. The values represent the quantified signals of Western blot obtained from advanced-stage tissue samples and normalized to those in early-stage tissue samples. (B) KPNA2−vimentin−pErk complex may be regulated by the EGFR signaling pathway. Early-stage lung adenocarcinoma tissues carrying WT EGFR (4 individuals) or mutated EGFR (4 individuals) were pooled according to the EGFR mutation status. IP was performed using anti-KPNA2 antibodies, and the immunoprecipitated protein complexes were analyzed via Western blot as described in Figure 4A. The values represent the quantified Western blot signals obtained from the mutated EGFR tissue samples and normalized to those in WT EGFR tissue samples.

complex form of pErk by preventing pErk from phosphatasemediated dephosphorylation.

increased following EGF treatment for 60 min (Figure 5A,B). We performed immunofluorescence by fixing cells with methanol to preserve the cytoskeleton structure, and we consistently observed weak staining for vimentin in the nucleus of CL1-5 cells (Supporting Information, Figure S2). We further confirmed the assembly of the cytosolic complexes via subcellular fractionation followed by co-IP. After EGF treatment for 15 min, we observed that the levels of pErk were increased in the cytosolic vimentin-IP products (Figure 5D). These results support that KPNA2 was not responsible for the nuclear entry of pErk and suggest that KPNA2−vimentin−pErk formed a complex in the cytoplasm upon EGF treatment.

DUSP Inhibitor Treatment Can Restore Migration Ability in KPNA2-Knockdown Cells

To examine whether the restored levels of pErk can rescue the decreasing migration ability shown in KPNA2-knockdown cells. We performed gene knockdown of KPNA2, followed by treatment with DUSP inhibitor, and assessed the effects on cell migration. Figure 7A shows that the pErk levels were decreased in KPNA2-knockdown CL1-5 cells and were restored upon DUSP inhibitor treatment. Data obtained from the transwell migration assay (6 h incubation) indicated that the migration ability of KPNA2-knockdown cells was severely impaired compared with that in control cells (100 versus 51.5%). Notably, the migration ability was restored to 71.8% in KPNA2-knockdown cells, whereas it displayed no significant change in control cells (Figure 7B). In addition, we performed a MTT assay to examine whether the restored levels of pErk rescued the decreased viability shown in KPNA2-knockdown cells. The treatment of a low dose of the DUSP inhibitor (0.25 μM), which displays low cell toxicity, indeed restored the pErk level in KPNA2-knockdown cells (Supporting Information, Figure S4). However, the MTT assay revealed that DUSP inhibitor treatment did not significantly alter the control- or KPNA2-siRNA-transfected cells (Figure 7C). These results collectively suggest that the KPNA2−vimentin−pErk complex level contributes to KPNA2-mediated modulation of cell migration in NSCLC.

KPNA2 Prevents pErk from Phosphatase-Mediated Dephosphorylation

To examine the role of KPNA2 in complex integrity and in maintaining the phosphorylation levels of Erk in complexes, we applied gene knockdown of KPNA2. As shown in Figure 6A, the phosphorylation level of Erk was decreased in KPNA2knockdown CL1-5 cells but not CL1-0 cells compared with the control cells. The decreased pErk level can be restored upon DUSP inhibitor treatment in CL1-5 cells. We also examined the levels of Erk kinase (pMEK) in KPNA2-knockdown CL1-5 cells. We found that the level of pMEK was not changed in KPNA2-knockdown CL1-5 cells (Supporting Information, Figure S3), suggesting that the reduction of pErk was not caused by down-regulated pMEK kinase activity. Knockdown of KPNA2 indeed reduced the level of pErk but not KPNB1 in the vimentin complex. Importantly, DUSP inhibitor treatment can restore pErk levels in the vimentin complex in KPNA2knockdown CL1-5 cells (Figure 6B). These results suggest that KPNA2 plays a vital role in maintaining the levels of the

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DISCUSSION In the current study, we employed IP combined with a SILAC approach to systematically analyze invasiveness-associated H


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Figure 5. KPNA2 forms a complex with vimentin and pErk in the cytoplasm. CL1-5 cells were treated with EGF (25 ng/mL) for the indicated time, followed by dual labeling with antivimentin and (A) anti-pErk antibodies, (B) anti-pErk and anti-KPNA2 antibodies, or (C) anti-KPNA2 and antivimentin antibodies to detect the expressions of KPNA2, pErk, and vimentin as indicated. (D) CL1-5 cells were treated with EGF (25 ng/mL) for the indicated time, followed by subcellular fractionation and co-IP using antivimentin antibodies. The immunoprecipitated protein complexes obtained from the cytosolic extracts (2 mg, upper panel) or nuclear extracts (600 μg, lower panel) were analyzed via Western blot using antibodies against vimentin, pErk, and KPNA2 as indicated. GAPDH and transcription factor NRF (NKRF) were used as the control for cytosolic and nuclear fraction, respectively.

to be potential KPNA2 interaction partners. Among these proteins, 121 were detected in our data set (Figure 1C and Supporting Information, Table S2). However, Erk or phosphorylated Erk was not successfully identified or quantified in this study, likely because the phosphorylated protein levels are generally relatively low and because the phosphorylation sites on the proteins may vary, implying that any given phosphoprotein is heterogeneous, which may account for the

KPNA2 protein complexes in lung cancer cells. Using this approach, immunoprecipitated proteins from different cell populations can be processed in a single experiment after equal mixing of immunoprecipitated bait, thus minimizing experimental variations, contamination, and artifacts that originate from separate sample processing.25−27 On the basis of UniProtKB, MINT, STRING, and I2D protein−protein interaction databases, numerous proteins have been reported I


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Figure 6. KPNA2 prevents pErk from phosphatase-mediated dephosphorylation. (A) CL1-0 and CL1-5 cells transfected with control or KPNA2 siRNA, followed by treatment with DUSP inhibitor (10 μM) for 15 min. After treatment, the cells were lysed, and KPNA2, pErk, and Erk were detected via Western blot. β-actin was used as an internal control. (B) CL1-5 cells transfected with control or KPNA2 siRNA for 48 h. The cells were then treated with EGF (25 ng/mL) or cotreated with DUSP inhibitor (10 μM) for 15 min, followed by co-IP using antivimentin antibodies. The immunoprecipitated protein complexes were analyzed by Western blot using antibodies against KPNB1, vimentin, pErk, GAPDH, and KPNA2 as indicated.

Figure 7. DUSP inhibitor treatment restores the migration ability in KPNA2-knockdown cells. (A) CL1-5 cells were transfected with control or KPNA2 siRNA, followed by treatment with DUSP inhibitor for 15 min. After treatment, the cells were lysed and detected using KPNA2 and pErk antibodies by Western blot. β-actin was used as an internal control. (B) Quantitative analysis of the migration assay. Data are presented as the mean obtained from three independent experiments. Error bars indicate the standard error. A p value less than 0.05 using unpaired t tests was considered statistically significant. (C) Quantitative analysis of the MTT assay. Data are presented as the mean obtained from three independent experiments. Error bars indicate standard deviation.

KPNA2 protein complexes in the pathway maps and provided a basis for identifying KPNA2-interaction proteins. Erk1/2, also called mitogen-activated protein kinase (MAPK), is activated by dual phosphorylation at both the Thr202 and Tyr204 residues. This activation is mediated by activated MEK1, which is in turn phosphorylated by RAF-1, and RAF-1 is activated by RAS.31,32 Because the phosphorylation of both Tyr and Thr residues is required to induce full Erk1/2 activity, the removal of phosphate from just one is sufficient for full inactivation. Thus, protein Ser/Thr phosphatases, protein Tyr phosphatases, and DUSPs act to inactivate Erk1/2 under various conditions.33 Notably, Erk activation is positively correlated with tumor stage and lymph node metastasis and is associated with advanced and aggressive NSCLC.34 In addition, the expression of DUSP6, the cytoplasmic DUSP with high specificity for Erk, is downregulated in lung cancer and negatively associated with tumor

underdetection of pErk in our study. Notably, 7 and 4 differentially KPNA2 interaction proteins that we identified in the most and second-most significant pathways, respectively, belong to the family of cytoskeleton proteins (Table 2 and Figure 2). Among these cytoskeleton-remodeling-related proteins, vimentin and other cytoskeleton proteins have been reported to be involved in intracellular signaling pathways that regulate cell responses to injuries, cell growth, death, and migration. For example, the loss of keratin 8/18 expression during epithelial−mesenchymal transition is associated with metastasis and chemoresistance,28 actin dynamics control serum response factor activity by regulating its coactivator MAL,29 and keratin 10 function as a negative modulator of cell cycle progression involving changes in the phosphoinositide 3kinase signal transduction pathway.30 Immunoprecipitated quantitative proteome analysis combined with bioinformatics tools effectively facilitated our search for invasiveness-associated J


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molecules, intermediate filaments, and growth stimuli, contribute to the differential formation of KPNA2-protein complexes and are associated with cancer invasiveness. In conclusion, we applied an IP−MS strategy to identify invasiveness-associated KPNA2 protein complexes in NSCLC cells and showed for the first time that KPNA2 plays a role in the maintenance of vimentin−pErk complex levels in vitro and in vivo. Although future work is needed to determine the detailed mechanisms, including the upstream signaling and PTM status of KPNA2 complexes functioning in cancer progression, our study provides new insight into how KPNA2 modulates cancer invasiveness.

histological grade.35 These results are consistent with our finding that the KPNA2−vimentin−pErk complex was upregulated in advanced-stage lung adenocarcinoma tissues (Figure 4A) and that DUSP inhibitor treatment restored migration ability in KPNA2-knockdown cells (Figure 7A,B). Forced expression of DUSP6 has been shown to markedly suppress the growth of A549, a lung cancer cell line bearing a KRAS mutation, and to attenuate cell proliferation significantly.35 In this study, we found that KPNA2 prevents pErk from phosphatase-mediated dephosphorylation and that a DUSP inhibitor restores the pErk levels in the vimentin complex (Figure 6B). However, we did not observe the expected result in its effect on the restoration of cell viability in KPNA2-knockdown cells (Figure 7C). We speculate that restored pErk in KPNA2-knockdown cells was not sufficient to rescue the decreasing viability caused by KPNA2 depletion in CL1-5 cells, and this assumption should be further investigated. Vimentin is an intermediate filament protein characteristic of mesenchymal cells, such as fibroblasts and endothelial cells.36 Vimentin has a three-domain structure comprised of aminoand carboxy-terminal head and tail domains flanking a central α-helical rod domain.37−39 The rod domain contains an element responsible for the cytoplasmic retention of vimentin.40,41 Notably, the main pErk-binding domain in vimentin is within the second coiled-coil region in the rod domain.42 This result is consistent with our finding that KPNA2 forms a complex with vimentin and pErk in the cytoplasm (Figure 5). Highly dynamic and complex posttranslational modifications (PTMs), such as phosphorylation of vimentin, appear to be likely regulator mechanisms for cell attachment, migration, and cell signaling.43 Interestingly, vimentin bound to pErk in CL1-5 cells, whereas the complexes were significantly decreased in CL1-0 cells (Figure 3B). Previously, Wang showed that CL1-0 and CL1-5 cells contain differentially phosphorylated vimentin, in which the phosphorylation of vimentin in CL1-5 cells was up-regulated compared with that in CL1-0 cells.44 Considering that KPNA2 is a phosphoprotein (S61, S62, and S490) with N-acetylserine at the second residue,45−47 we speculate that the differential PTM status of vimentin, KPNA2, and/or other KPNA2-interaction partners may contribute to complex formation as well as cancer invasiveness. It is known that CL1-5 cells display higher metastatic and invasive potential than CL1-0 cells.15 Interestingly, the protein level of KPNA2 in CL1-5 cells is lower than that in CL1-0 cells. The KPNA2 gene status is WT in both of these cell lines. We used a SILAC-based quantitative proteomic approach combined with network analysis to identify the differentially abundant proteome between CL1-5 and CL1-0 cells. A total of 5757 and 5244 nonredundant proteins were identified and quantified, respectively (unpublished data). To clarify the pathways related to the differential malignancy of these two cell lines, 875 proteins displaying a 2-fold change, including 500 upregulated and 375 down-regulated proteins in CL1-5 compared to CL1-0, were further examined using MetaCore software. We found that the GeneGo Pathway Maps derived from the 500 differentially up-regulated proteins (CL1-5 > CL1-0) are predominantly involved in cytoskeleton remodeling, cell adhesion, and VEGF signaling. This finding is consistent with the present result that the levels of pErk−vimentin−KPNA2 complexes are positively associated with cancer invasiveness. Therefore, we propose that multiple proteins/oncogenic factors, such as the microenvironment, cell signaling, adhesion

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ASSOCIATED CONTENT

* Supporting Information S

Lists of proteins identified and quantified in two independent KPNA2-immunoprecipitated protein complexes and of 646 nonredundant proteins identified and quantified in both independent KPNA2-immunoprecipitated protein complexes as well as figures showing ratio distributions and correlation of the quantified protein levels between experiments 1 and 2, detection of vimentin by immunofluorescence staining with a methanol-fixed method, KPNA2 knockdown effect on levels of pMEK, and DUSP inhibitor treatment effect on pErk level in KPNA2-knockdown cells. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel.: 886-3-2118800 ext. 3424. Fax: 886-3-2118042. E-mail: [email protected]. Present Address

C.-J.Y.: Department of Cell and Molecular Biology, College of Medicine, Chang Gung University, 259 Wen-Hwa First Road, Kwei-Shan, Tao-Yuan, Taiwan. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by grants from the Chang Gung Medical Research Fund (CMRPD1C0091-2), the Ministry of Science and Technology, Taiwan, R.O.C. (101-2320-B-182035-MY3), and the Ministry of Education, Taiwan, R.O.C. (EMRPD1D0721).

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NOTE ADDED AFTER ASAP PUBLICATION This paper was published ASAP on March 13, 2015, with an error to Table S1 in the Supporting Information. The corrected version reposted March 19, 2015.

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Quantitative Proteomics Reveals a Novel Role of Karyopherin Alpha 2

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