Proteomic Identification of Common SCF Ubiquitin Ligase FBXO6

Article pubs.acs.org/jpr

Proteomic Identification of Common SCF Ubiquitin Ligase FBXO6-Interacting Glycoproteins in Three kinds of Cells Bin Liu,†,§ Ying Zheng,†,§ Tong-Dan Wang,† Han-Zhang Xu,† Li Xia,† Jian Zhang,† Ying-Li Wu,† Guo-Qiang Chen,*,†,‡ and Li-Shun Wang*,†,‡ †

Shanghai Universities E-Institute for Chemical Biology, Key Laboratory of Cell Differentiation and Apoptosis of Chinese Ministry of Education, Rui-Jin Hospital, Shanghai Jiao Tong University School of Medicine (SJTU-SM), Shanghai 200025, China ‡ Institute of Health Sciences, Shanghai Institutes for Biological Sciences-SJTU-SM, Shanghai 200025, China S Supporting Information *

ABSTRACT: FBOX6 ubiquitin ligase complex is involved in the endoplasmic reticulumassociated degradation pathway by mediating the ubiquitination of glycoproteins. FBXO6 interacts with the chitobiose in unfolded N-glycoprotein, pointing glycoproteins toward E2 for ubiquitination. Although the glycoprotein-recognizing mechanism of FBXO6 is well documented, its bona f ide interacting glycoproteins are largely unknown. Here we utilized a protein purification approach combined with LC−MS to systematically identify the FBXO6-interacting glycoproteins. Following identification of 39 proteins that specifically interact with FBXO6 in all three different cell lines, 293T, HeLa and Jurkat cells, we compared the protein complex organization between wild-type FBXO6 and its mutant, which fails to recognize glycoproteins. Combining these databases, 29 highly confident glycoproteins that interact with FBXO6 in an N-glycan dependent manner are identified. Our data provide valuable information for the discovery of the interacting glycoproteins of FBXO6 and also demonstrate the potential of these approaches as general platforms for the global discovery of interacting glycoproteins of other FBAs (F-box associated regions) containing F-box proteins. KEYWORDS: SCF, FBXO6, glycoprotein, endoplasmic reticulum

1. INTRODUCTION In eukaryotic cells, approximately one-third of proteins are targeted to the secretory pathway and most of these secretory proteins are modified by N-linked oligosaccharides in the endoplasmic reticulum (ER).1,2 The triglucosyl high-mannose oligosaccharide, GlcNAc2-Man9-Glc3 (herein GlcNAc is N-acetylglucosamine, Man is mannose and Glc is glucose), is transferred to the asparagine residue in Asn-X-Ser/Thr motifs using dolichol pyrophosphate as the donor substrate.3,4 Subsequently, the terminal glucose residues can be removed by glucosidases to facilitate folding by the lectin-like chaperones calnexin and calreticulin. Proteins with GlcNAc2-Man9 are competent for ER exit and can transit to their final destinations.5 In the ER quality control system, improperly folded proteins are either subjected to additional folding cycles or selected for a process termed ER-associated degradation (ERAD),2,6 which involves transfer of these proteins from the ER to the cytosol followed by recognization and degradation by the ubiquitin−proteasome system. Recent progress has documented that the ER mannosidase I product GlcNAc2-Man8 seems to serve as a signal for the recognition of ERAD substrates.7 Many proteins have been found to act as E3 ligase in ERAD pathways,2,4−9 and two F box proteins (FBPs), FBXO2 (F-box only protein 2) and FBXO6 (F-box only protein 6), catch more attentions for their glycoproteins binding characters. FBPs are the variable substrate adapters for the Skp1-Cul1-F box protein © 2012 American Chemical Society

(SCF) ubiquitin ligase complexes and dictate the substrate specificity of the ubiquitin ligase.10 FBXO2, FBXO6 and other three FBPs compose a subfamily named F-box associated region (FBA) family for their common FBA domain.11 The FBA domains of these F-box proteins, especially those of FBXO2 and FBXO6, seems to be required for glycoprotein recognizing, because either deletion or mutation of this domain abolished their glycoprotein-recognizing ability.12,13 Thermodynamic analysis of interactions between synthetic N-glycan and FBXO6 has revealed that FBXO6 interacts with the innermost chitobiose in N-glycans of glycoprotein substrates by a small hydrophobic pocket in FBA domain.12,14 Indeed, the introduction of point mutation into the residue in that pocket of FBXO6 impairs the binding activity toward its glycoprotein substrates.15 FBXO2 (Fbs1) is the first identified FBA member and plays critical role in the regulation of neuron function by targeting those vital neuron proteins for ubiquitin mediated degradation.16 As example, FBXO2 involves in the regulation of neurotransmitter receptors in the brain by controlling the stability of NMDA receptors.17 FBXO2 also attenuates Alzheimer’s disease amyloidosis and improves synaptic function by targeting BACE1 for degradation.18 In contrast to the lectin chaperones in the ER such as calnexin and calreticulin, which recognize Received: October 13, 2011 Published: January 23, 2012 1773

dx.doi.org/10.1021/pr2010204 | J. Proteome Res. 2012, 11, 1773−1781

Journal of Proteome Research

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2.4. Construction of Stable Cell Line

nonreducing terminal glucose molecules of the high-mannose oligosaccharides, FBXO2 recognizes and interacts with the innermost portion of the carbohydrate moieties of glycoproteins.12−14 FBXO6 is reported to regulate the stability of Chk1 during DNA damage checkpoint recovery and may play a role in DNA damage response or chemotherapy of tumor cells.19,20 Intriguingly, FBXO6 also involves in the ERAD pathway, because either overexpression of the dominant negative form of FBXO6 or decrease the expression of endogenous FBXO6 resulted in inhibition of ERAD substrates degradation.16 In addition, FBXO6 is relatively ubiquitously expressed in contrast to FBXO2,14 which may endow FBXO6 even more potential on ERAD regulation. However, the interacting glycoproteins, or substrates of FBXO6, are rarely identified and thus its biological implication. So, systematical identification of the FBXO6interacting glycoproteins would be critical for clarifying its substrates as well as biological functions. In this study, we attempt to utilize protein purification approach combined with LC−MS to systematically identify the FBXO6-interacting proteins. We isolated 29 highly confident FBXO6 interacting glycoproteins by comparing the complex components of wild-type FBOX6 (FBXO6WT) and its FBA domain mutant (FBXO6Null). Our data provide a valuable database and information for extensive study of the exact role of FBXO6 and also demonstrate the potential of these approaches as general platforms for the global discovery of interacting glycoproteins of other FBA domain containing F-box proteins and their regulatory networks.

Viral supernatants were produced in HEK293T cells cotransfected with the pBabe-3 × Flag-control or pBabe-3 × Flag-FBXO6 WT/Null constructs and packaging vectors GAGPOL and VSV-G (Clontech). Viral supernatants were collected 48 h after transfection, filter-sterilized, and stored at −80 °C. For 293T and HeLa cells, viral supernatants were added to the cells with 10 μg/mL Ploybrane for 48 h and selected with puromycin (1 μg/mL) for 3 days. For Jurkat cells, 6-well plate was coated with 10 μg/mL Fibronectin for more than 4 h at 37 °C, and the viral supernatants with 10 μg/mL Polybrene were added to Jurkat cells for 48 h and selected with puromycin (1 μg/mL) for 3 days. Positive polyclonal populations were identified based on Western blot for Flag M2 or FBXO6 antibody. 2.5. Protein Purification

For standard purifications, cells from four 15-cm tissue culture dishes at ∼80% confluence were lysed in 6 mL of lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.5% Nonidet P40, Roche complete EDTA-free protease inhibitor cocktail) for 20 min with gentle rocking at 4 °C. Lysates were cleared using centrifugation (13000 rpm, 10 min), the supernatant was filtered through 0.45 μm spin filters (Millipore) to further remove cell debris, and the resulting material subjected to immunoprecipitation (IP) with 50 μL of anti-FLAG M2 affinity resin (Sigma) overnight at 4 °C with gentle inversion. Resin containing immune complexes was washed with 1 mL ice cold lysis buffer 4 times followed by three 1 mL Tris Buffered Saline (TBS) washes. Proteins were eluted with two 50 μL 150 μg/mL 3 × Flag-peptide (Sigma) in TBS for 30 min, and the elutions were pooled for a final volume of 100 μL. Proteins in each elution were precipitated with cold acetone and the resulting pellet washed 2 times with cold acetone.

2. EXPERIMENTAL PROCEDURES 2.1. Cell Culture

2.6. Western Blot

HeLa cells and 293T cells were cultured in Dulbecco’s modified Eagle’s medium (Invitrogen) supplemented with 10% fetal calf serum (Gibco BRL, Gaithersburg, MD). Jurkat cells (from Cell Bank of Shanghai Institutes of Biological Sciences, Shanghai, China) were cultured in RPMI-1640 medium (Sigma, St Louis, MO) supplemented with 10% fetal calf serum. All of these cells were cultured in a 5% CO2/95% air at 37 °C.

Cells were harvested and lysed with ice-cold lysis buffer (62.5 mM Tris-HCl, pH 6.8, 100 mM DTT, 2% w/v SDS, 10% glycerol). After centrifugation at 20000× g for 10 min at 4 °C, proteins in the supernatants were quantified and separated by 10 or 12% SDSPAGE, either stained by Coomassie Brilliant Blue or transferred to NC membrane (Amersham Bioscience, Buckinghamshire, U.K.). After blocking with 5% nonfat milk in PBS, membranes were immunoblotted with antibodies as indicated, followed by HRPlinked secondary antibodies (Cell Signaling). The signals were detected by SuperSignal West Pico Chemiluminescent Substrate kit (Pierce, Rockford, IL) according to manufacturer’s instructions.

2.2. Plasmids

FBXW2, FBXW8, FBXL3, FBXL5, FBXO4, FBXO6, FBXO22 and FBXO34 were amplified from 293T cells by PCR and cloned into the pBabe-3 × Flag retroviral vector. FBXO6Null was generated using QuickChange Site-Directed Mutagenesis Kit (Stratagene). All cDNAs were completely sequenced.

2.7. Nano-LC−ESI−MS/MS Analysis

Immunoprecipitation samples were separated by SDS-PAGE, and visualized with colloidal Coomassie blue. The lane from cells was cut into 1 mm slices, and each slice was washed twice with 50 mM NH4HCO3, 50% ACN and dehydrated with ACN. Proteins were reduced and alkylated by treating them with 10 mM DTT and 55 mM iodoacetamide, respectively. After washing with 50 mM NH4HCO3 and ACN, proteins were digested in gel with trypsin (Promega, Madison, WI) and incubated overnight at 37 °C. Tryptic peptides were extracted from the gel pieces with 60% ACN, 0.1% trifluoroacetic acid. The peptide extracts were vacuum centrifuged to dryness. Dried peptides were dissolved to 10 μL of 2% ACN, 0.1% trifluoroacetic acid. Samples were desalted and preconcentrated through a Michrom peptide CapTrap (MW 0.5−50 kDa, 0.5 × 2 mm; Michrom BioResources, Inc., Auburn, CA). The eluant was vacuum centrifuged to dryness then reconstituted in 2% acetontrile (ACN), 0.1% formic acid.

2.3. Antibodies and Reagents

Immunoblotting was performed using antibodies as indicated: monoclonal anti-FBXO6 (7B11, sc-134339, Santa Cruz), monoclonal anti-Cullin1 (D-5, sc-17775, Santa Cruz), monoclonal anti-Skp1 (H-6, sc-5281, Santa Cruz), monoclonal antiRPN1 (E-7, sc-48367, Santa Cruz), monoclonal anti-TFRC (3B82A1, sc-32272, Santa Cruz), monoclonal anti-Ero1-Lα (YW-8, sc-100805, Santa Cruz), monoclonal anti-RPN2 (A-1, sc-166421, Santa Cruz), monoclonal anti-Chk1 (2G11D5, sc56288, Santa Cruz), monoclonal anti-OST48 (E-9, sc-74408, Santa Cruz), polyclonal anti-Erlin-2 (2959, Cell Signaling), polyclonal anti-SMC1 (ab21583, Abcam), polyclonal anti-SMC3 (ab9263, Abcam), monoclonal anti-Flag M2 (F1804, Sigma), peroxidase-conjugated ConA (L6397, Sigma). FLAG M2 Affinity Gel (A2220) was purchased from Sigma. Endo H (P0702L) and PNGase F (P0705L) were purchased from NEB. 1774



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Figure 1. Purification the SCFFBXO6 complex from three different cell lines. (A) Lysates from HeLa cells stably expressing FLAG-CON or FLAGFBXO6 were immunoprecipitated with anti-FLAG M2 affinity gel. Bound proteins were eluted with FLAG peptide and subjected to Western blot with Flag and Cullin1 antibodies. The asterisk indicates IgG heavy chain. (B) Lysates from HeLa cells stably expressing FLAG-CON or FLAGFBXO6 were immunoprecipitated with anti-FLAG M2 affinity gel. Bound proteins were eluted with FLAG peptide, resolved on 10% SDS-PAGE gel, stained by Coomassie Brilliant Blue and analyzed by mass spectrometry. The identified SCFFBXO6 components are listed. (C) Numbers inside the parentheses correspond to proteins unique to FBXO6 immunoprecipitation data set either from HeLa, Jurkat or 293T cells whereas numbers inside the overlaps correspond to proteins common to the three data sets. (D) Immunocomplexes from either 293T-control or 293T-Flag-FBXO6 were probed with the indicated endogenous antibodies. Immunoprecipitation of Cullin1 and Skp1 serve as positive control.

For LC−MS/MS analysis, 10 μL samples were introduced from an autosampler (HTS-PAL, CTC Analytics, Zwingen, Switzerland) at a flow of 1 μL/min for 15 min onto a reverse-phase microcapillary column (0.1 × 150 mm, packed with 5 μm 100 Å Magic C18 resin; Michrom Bioresources) using a HPLC (Paragram MS4, Michrom Bioresources). The reverse phase separation of peptides was performed at a flow of 0.5 μL/min using the following buffers: 2% ACN with 0.1% formic acid (buffer A) and 98% ACN with 0.1% formic acid (buffer B) using a 90 min gradient (0−35% B for 90 min, 80% B for 8 min, 95% B for 12 min, and 0% B for 20 min). The eluate was introduced directly onto a hybrid linear ion trap (LTQ) Orbitrap mass spectrometer (ThermoFinnigan, San Jose, CA, USA) equipped with ADVANCE Spray Source (Michrom Bioresources). A high resolution MS survey scan was obtained for the m/z 350−1800, R = 100000 (at m/z 400) and ion accumulation to a target value of 106. Siloxane (m/z 445.120025) was used as an internal standard. MS/MS spectra were acquired using data-dependent scan from the ten most intense ions with charge states ≥2 in the survey scan (as determined by the Xcalibur mass spectrometer software in real time). Only MS signals exceeding 500 ion counts triggered a MS/MS attempt and 5000 ions were acquired for a MS/MS scan. Dynamic mass exclusion windows of 27 s were used. Singly charged ions and ions with unassigned charge states were excluded from triggering MS/MS scans. The normalized collision energy was set to 35%. Tandem mass spectra were extracted with Xcalibur version 2.0.7. The MS/MS data were

searched against The International Protein Index (IPI) human database (ipi.HUMAN.v3.73 downloaded from ftp.ebi.ac.uk/pub/ databases/IPI) containing 179304 entries using SORCERERSEQUEST (Bioworks version 3.5, ThermoFinnigan), which was run on the Sage-N Sorcerer2 (Thermo Electron, San Jose, CA). Peptide (parent ion) tolerance of 10 ppm, fragment ion tolerance of 1 Da and 3 missed cleavages were allowed, and fixed modification of carbamidomethylation on Cys (157 Da) and differential modification of oxidation on Met (16 Da) were used. The XCorr scores cutoffs for each charge state were as follows: 2.5 (+2), 3.0 (+3), 3.5 (+4). The results were further analyzed by Scaffold 3 proteome software (Portland, OR), which integrates both Protein Prophet and Peptide Prophet. Two unique peptides must be identified independently for each protein, the peptide probability must be 95% or higher, and the protein probability must be 95% or higher.

3. RESULTS 3.1. Identification of the FBXO6-Interacting Proteins in Three Cell Lines

Interaction proteomics provides the most direct approach available for identifying physiologically relevant protein complexes and networks.21 To identify proteins interacting with FBXO6, the lysate of human epithelial carcinoma HeLa cells with stable retrovirustransduced expression of Flag-FBXO6 or Flag was precipitated by anti-Flag M2 resin, followed by Western blot. As depicted in Figure 1A and Supplementary Figure S4 (Supporting Information), 1775



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Table 1. FBOX6 Core Interactor Lista HeLa cells

Jurkat cells

AC

gene

protein name

peptides

coverage (%)

IPI00440493 IPI00303476 IPI00027235 IPI00553185 IPI00743825 IPI00793277 IPI00014310 IPI00297084 IPI00026942 IPI00386755 IPI00025869 IPI00796919 IPI00168262 IPI00908404 IPI00296713 IPI00219516 IPI00607861 IPI00007765 IPI00903213 IPI00043598 IPI00296922 IPI00163381 IPI00217975 IPI00299904 IPI00008787 IPI00333985 IPI00922838 IPI00465308 IPI00943008 IPI00030255 IPI00290945 IPI00399307 IPI00025874 IPI00383680 IPI00301364 IPI00297492 IPI00152377 IPI00022462 IPI00305383

ATP5A1 ATP5B ATRN CCT3 COPS2 CRTAP CUL1 DDOST ERLIN2 ERO1L GLA GLB1 GLT25D1 GNS GRN GUSB H6PD HSPA9 IGF2R IKBIP LAMB2 LEPRE1 LMNB1 MCM7 NAGLU NOMO2 ORP150 PIGS PLOD1 PLOD3 PON2 PRCP RPN1 RPN2 SKP1 STT3A STT3B TFRC UQCRC2

ATP synthase subunit alpha, mitochondrial ATP synthase subunit beta, mitochondrial Attractin, Inflammatory response T-complex protein 1 subunit gamma COP9 signalosome complex subunit 2 Cartilage-associated protein Cullin1 Oligosaccharyl transferase subunit DDOST SPFH domain-containing protein 2 Endoplasmic oxidoreductin-1-like protein Alpha-galactosidase A Beta-galactosidase, Hydrolase Procollagen galactosyltransferase 1 N-Acetylglucosamine-6-sulfatase Cytokine granulin Beta-glucuronidase GDH/6PGL endoplasmic bifunctional protein Heat shock protein 70 family A9 Insulin-like growth factor 2 receptor Inhibitor of NF kappa-B interacting protein Laminin subunit beta-2 Prolyl 3-hydroxylase 1, Oxidoreductase Lamin-B1 DNA replication licensing factor MCM7 Alpha-N-acetylglucosaminidase, Hydrolase Nodal modulator 2, Transmembrane helix Hypoxia up-regulated protein 1, Chaperone GPI transamidase component PIG-S Procollagen-lysine,2-oxoglutarate 5-dioxygenase 1 Procollagen-lysine,2-oxoglutarate 5-dioxygenase 3 Serum paraoxonase/arylesterase 2 Lysosomal Pro-X carboxypeptidase Oligosaccharyl transferase subunit RPN1 Oligosaccharyl transferase subunit RPN2 S-phase kinase-associated protein 1 Oligosaccharyl transferase subunit STT3A Oligosaccharyl transferase subunit STT3B Transferrin receptor protein 1 Cytochrome b-c1 complex subunit 2, mitochondrial

4 7 3 5 11 3 53 10 9 2 2 13 8 5 4 5 16 7 2 2 25 2 2 5 7 2 37 2 28 16 2 3 18 6 5 2 5 22 2

9.80 19.70 2.73 10.60 28.40 8.38 54.50 31.80 29.80 5.56 6.99 24.40 13.50 10.10 8.77 9.17 26.00 12.00 0.80 5.84 16.80 2.72 5.94 8.34 13.60 2.29 37.60 5.95 41.00 24.80 6.67 6.58 31.30 17.80 31.30 3.12 7.75 37.80 6.62

293T cells

peptides

coverage (%)

peptides

coverage (%)

24 2 9 20 23 11 109 23 31 15 9 21 13 12 4 16 4 16 28 11 3 5 7 16 2 71 80 14 35 3 3 13 49 21 27 5 11 3 13

52.30 6.24 8.10 47.70 52.80 33.70 82.70 49.60 79.10 42.70 26.60 33.40 23.60 25.90 7.59 42.20 5.94 32.70 14.20 36.30 1.78 7.47 22.00 28.20 3.63 62.30 58.90 38.90 56.30 43.20 9.87 28.80 64.60 42.90 85.00 7.23 15.30 42.60 41.10

3 6 2 28 9 4 74 1 24 15 18 11 5 4 2 8 19 2 12 11 10 9 7 12 13 45 37 3 21 21 6 4 2 10 31 8 16 6 10

59.00 20.00 1.92 51.20 29.80 13.70 71.60 43.20 77.90 42.70 50.30 24.40 9.97 7.19 4.36 24.50 33.90 4.86 5.26 36.30 6.45 14.10 22.00 21.10 27.30 57.90 44.00 10.30 45.30 42.30 37.60 14.50 63.30 28.60 84.70 11.30 23.10 9.72 30.00

a

AC, the accession number in the International Protein Index (IPI) human database; Gene, the gene name in IPI database; peptides, the number of unique peptides; coverage, percentage sequence coverage.

Flag-FBXO6, Chk119 and cullin1, a molecular scaffold for SCF complex,22 could be effectively pulled down in Flag-FBXO6 but not Flag-expressing cells, suggesting the effectiveness of the coIP assay to isolate the FBOX6-interacting proteins. Thus, the immunoprecipitates were run on SDS-PAGE, followed by Coomassie Brilliant Blue staining (Figure 1B). The gel was continuously cut and subjected to LC−MS/MS analysis. Totally, 120 FBXO6-interacting unique proteins (Figure 1C) were identified in Flag-FBXO6-expressing HeLa cells. As reported,14 FBXO6 is ubiquitously expressed in various tissues. To identify the common FBXO6-interacting proteins in the different cells, we further applied the same strategy to other two human cell lines, human embryonic kidney 293T cells and human T cell lymphoblast-like Jurkat cells, to create two more FBXO6 interactomes. Totally 485 and 245 FBOX6-interacting proteins were identified respectively in Jurkat and 293T cells

(Figure 1C). With the comparison of these data sets from three cells (Tables S1−3, Supporting Information), we identified 39 common putative FBOX6-interacting proteins, including 37 novel ones besides the known SCF complex components Cullin1 and Skp1 (Figure 1C, Table 1). Here we termed these proteins as the core binding proteins of FBXO6. Furthermore, we randomly selected six proteins besides cullin-1 and Skp1 to be confirmed by Western blot. In line with the mass spectrometry analysis, all six proteins including ErolL, TFRC, RPN1, Erlin 2, SMC1, SMC3 could be specifically detected in Flag-FBXO6 immunoprecipitate (Figure 1D), supporting the confidence of these FBOX6-interacting proteins. 3.2. Ontological Analysis for the Core FBXO6-Interacting Proteins

Ontological analysis showed that 66.7% of the previously identified 39 core binding proteins of FBXO6 belongs to 1776



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3.3. Comparison of Protein Complex Organization between FBXO6WT and FBXO6Null

binding proteins, and 12.8 and 5.1% present oligosaccharyl transferase and procollagen galactosyltransferase activities respectively (Figure 2A). They mainly involved in biological

In line with the previous reports that FBXO6 mainly recognized glycoproteins,14 the glycoproteins were accumulated in FBXO6 immunocomplex, as assessed by probe with Con A (Supplementary Figure S1, Supporting Information), a lectin that binds high mannose glycans.15 By gene ontology analysis, 29 (76%) of the 39 core binding proteins of FBXO6 are glycoproteins (Supplementary Figure S2, Supporting Information). According to the previous report,12 FBA domain of FBOX6 is required for its glycoprotein recognized activity (Figure 3A). To systematically identify the bona f ide FBXO6-interacting glycoproteins, Flag-FBXO6WT and Flag-FBXO6 Null, a FBA domain mutant of FBXO6 which losts its glycoprotein recognized activity, were infected into 293T cells, respectively. Their lysates were subjected to immunoprecipitation with anti-FLAG M2 resin. Immunoprecipitates from FBXO6WT and FBXO6Null cells were subjected to Western blot with Cullin1 and Flag antibody or probed with ConA (Figure S3, Supporting Information). Consistent with the previous report,12 less glycoproteins were immunoprecipitated by FBXO6Null, although FBXO6Null still formed complex with Cullin1 (Figure S3, lower panel) and Skp1 (data not shown). Next, the immunoprecipitates from FBXO6WT and FBXO6Null cells were subjected to SDS-PAGE and stained by Coomassie Brilliant Blue (Figure 3B). Proteins were trypsinized and subjected to LC−MS/MS analysis. Identified proteins that interacted with FBXO6WT and FBXO6Null were shown in Tables S4 and S5, respectively (Supporting Information). We then focused on the proteins that exclusively interacted with FBXO6WT. One hundred and forty proteins were identified (Figure 3C) and shown in Table S6 (Supporting Information). Interestingly, similar to the core interactors, 66% of these proteins are glycoproteins (Figure 3D). These 140 proteins were further overlapped with the core binding proteins of FBXO6 to find the common interacting glycoproteins (Figure 4A). According to these criteria and gene ontology analysis, around 30 proteins were identified (Table 2) and only one (lamin B) of these 30 proteins was not glycoproteins (Figure 4B). Using available antibodies, we validated the interactions of several proteins identified in the list above. Indeed, those proteins we tested only did bind to FBXO6WT, but not to FBXO6Null (Figure 4C). These observations further validated our MS data and revealed a novel list of interacting glycoproteins of FBXO6.

Figure 2. Ontological analysis for the core FBXO6-interacting proteins. (A) Gene Ontology classification of the FBXO6 core interactors by molecular function, biological process and cellular compartment. See text for details. (B) Interaction network for proteins identified in association with FBXO6. Black lines, this study; red lines, HPRD.

3.4. Interaction between FBXO6 and Glycosylated Ero1L

There are several dozen of FBPs reported in human tissues. To investigate the specificity of binding between those proteins and FBXO6, we test 7 other human FBPs using Ero1L as a probe. Flag-tagged versions of these FBPs were retrovirally expressed in 293T cells and then immunoprecipitated to evaluate their interaction with endogenous Ero1L proteins. We found that each FBPs could pull down cullin1, the common partner of this family, while the only F-box protein able to immunoprecipitate Ero1L was FBXO6 (Figure 5A). FBXO6 was documented to interact with glycoproteins via chitobiose.7 Interestingly, 29 of 39 the core binding proteins are glycoproteins. To test the dependence of glycosylation for the interaction of these proteins with FBXO6, We utilized endoglycosidase Endo H and amidase PNGase F. Removing N-glycans by either Endo H or PNGase generated a faster shift form of Ero1L in both lysate and immunoprecipitates of 293T FlagFBXO6 cells (Figure 5B), indicating FBXO6 interacted with

processes such as cellular protein metabolism, protein modification and protein N-linked glycosylation. Furthermore, about half (43.6%) are localized to ER compartment, and about 10−15% are localized to lysosome, nucleus, mitochondria and membrane, respectively (Figure 2A). Furthermore, by mining the known protein−protein interactions between these proteins through the public Human Protein Reference Database (HPRD, www.hprd.org), we built a map of the FBXO6 interactome (Figure 2B). In addition to the known SCF complex components cullin 1 and Skp 1, more intriguingly, five out of eight subunits of the oligosaccharyltransferase (OST) complex and three subunits of the respiratory chain protein presented in the novel interactome of FBXO6. 1777



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Figure 3. Comparison of protein complex organization between FBXO6WT and FBXO6Null. (A) The diagram of FBXO6WT and FBXO6Null. (B) Lysates from 293T cells stably expressing either FBXO6WT or FBXO6Null were immunoprecipitated with anti-FLAG M2 resin. Bound proteins were eluted with FLAG peptide, resolved on 10% SDS-PAGE gel, stained by Coomassie Brilliant Blue and analyzed by mass spectrometry. The identified SCF FBXO6 components from FBXO6WT and FBXO6Null are listed. (C) Numbers outside the overlaps correspond to proteins unique to either FBXO6WT or FBXO6Null immunoprecipitation data set whereas numbers inside the overlaps correspond to proteins common to the two data sets. (D) Ratio of glycoproteins in FBXO6WT unique interacted proteins.

Figure 4. Validation of glycoproteins interacting with FBXO6WT. (A) Numbers outside the overlaps correspond to proteins unique to either FBXO6WT core interactors or proteins exclusively interact with FBXO6, whereas numbers inside the overlaps correspond to proteins common to the two data sets. (B) Ratio of glycoprotein in proteins which are common to the two data sets. Twenty-nine out of the common 30 proteins are glycoproteins. (C) Immunocomplexes from either 293T FBXO6WT or 293T FBXO6Null were immunoblotted with the indicated antibodies. Both FBXO6WT and FBXO6Null interacted with Cullin1, only FBXO6WT interacted with the indicated glycoproteins.

(Figure 5D, left panel) and significantly decreased the glycosylation of Ero1L (Figure 5E). Tunicamycin also decreased the ConA recognized glycoproteins among the FBXO6 interaction proteins (Figure 5D, right panel) and only glycosylated Ero1L was shown to interact with FBXO6 (Figure 5E). These results indicated that FBXO6 interacted with the proteins in the list in an N-glycosylation dependent manner.

Ero1L containing N-glycosylation. To test whether the Nglycosylation of these proteins, for example Ero1L, was required for FBXO6 binding, we removed protein N-glycans of 293T Flag-FBXO6 cells lysate with Endo H (Figure 5B, left panel). We found that the treated lysate with Endo H significantly abolished the binding of FBXO6 to Ero1L (Figure 5C). Tunicamycin blocked the synthesis N-linked glycoproteins 1778



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Table 2. Core Proteins Specifically Interact with FBXO6WT but Not with FBXO6Null gene/location Endoplasmic reticulum DDOST RPN1 RPN2 STT3A STT3B ERLIN2 ERO1L GLA GLT25D1 H6PD IKBIP LEPRE1 NOMO2 ORP150 PIGS PLOD1 PLOD3 Membrane ATRN IGF2R PON2 TFRC Secreted CRTAP GRN LAMB2 Lysosome GLB1 GNS GUSB NAGLU PRCP Nucleus LMNB1

glycoproteins. The data sets from LC−MS analysis demonstrated that the interacting proteins of FBXO6Null were significantly less than FBXO6WT. Subtractive analysis of them indicated that there were 140 FBXO6WT specific interacting proteins, of which 67% were glycoproteins. Interestingly, when we further compared these proteins with the core interactors of FBXO6, 30 proteins were isolated and 29 of them were glycoproteins. Similar to core binding proteins, nearly half of these glycoproteins are localized to ER, and the rest are mostly lysosome, membrane and secreted glycoproteins, whereas the mitochondrial proteins are not included and indeed these proteins are not glycoproteins as far as we know. Among these ER resident glycoproteins, we found lots of regulators of protein glycosylation. In those glycosylation regulators, five subunits of OST were copurified with FBXO6. Mammalian OST is an oligomeric membrane protein consisting of seven to eight nonidentical subunits.23 OST with different catalytic subunits and distinct enzymatic properties are coexpressed in mammalian cells. These subunits cooperate and act sequentially to mediate protein N-glycosylation.24 We further confirmed the interaction and these subunits may interact with FBXO6 in an N-glycan-dependent manner, as FBXO6Null failed to interact with these glycoproteins. The interaction between FBXO6 and OST subunits suggests a role of FBXO6 in the regulation of global glycosylation. However, this potential FBXO6 regulatory function may not only go through the ERAD pathway. These ER resident glycoproteins may also act as a scaffold to recruit FBXO6. The biological significance of those interactions needs further investigations. Ero1L, a thiol oxidase, is a heavy glycosylated protein.25 In our unbiased screen, the only F-box protein that interacted with Ero1L was FBXO6. Interestingly, Ero1L interacted with FBXO6 depended on its N-glycan, as abolishing the glycan chain in vitro inhibited the interaction between Ero1L and FBXO6. Tunicamycin treated cells posed both glycosylated and nonglycosylated forms of Ero1L, only glycosylated Ero1L interacts with FBXO6. Ero1L exists as a collection of oxidized and reduced forms and covalently binds PDI (protein disulfide isomerase),25 which catalyzes disulfide formation in newly synthesized proteins entering the mammalian endoplasmic reticulum.26 A dynamic equilibrium between Ero1L and glutathione disulfide-mediated oxidation of PDI constitutes an important element of ER redox homeostasis.27 The interaction between FBXO6 and Ero1L suggests a role of FBXO6 in the regulation of ER redox homeostasis and further researches are needed to clarify the exact role of FBXO6 in this important biological process. Usually, it is much difficult to expect a certain protein could be identified for one hundred percent in such proteomic analysis. As one example, one previously identified FBXO6 substrate Chk119 was not identified in our purification by mass spectrometry. However, using Western blotting, we are able to detect the presence of Chk1 in the FBXO6- immunoprecipitates (Supplementary Figure S4, Supporting Information). In addition, the cell types, the reagent in the sample preparation, or protein abundance may interfere with the identification of an individual protein. Further research is encouraged to identify the bona f ide substrates of FBXO6 under more specific circumstances or with more specific strategies. Our research highlights the purification of the interaction glycoproteins of FBXO6, which may lead to uncover the exact biological role of FBXO6 in ERAD pathway as well as ER redox

protein name Oligosaccharyl transferase subunit DDOST Oligosaccharyl transferase subunit RPN1 Oligosaccharyl transferase subunit RPN2 Oligosaccharyl transferase subunit STT3A Oligosaccharyl transferase subunit STT3B SPFH domain-containing protein 2 Endoplasmic oxidoreductin-1-like protein Alpha-galactosidase A Procollagen galactosyltransferase 1, Glycosyltransferase GDH/6PGL endoplasmic bifunctional protein Inhibitor of NF kappa-B kinase-interacting protein Prolyl 3-hydroxylase 1, Oxidoreductase Nodal modulator 2, Transmembrane helix Hypoxia up-regulated protein 1, Chaperone GPI transamidase component PIG-S Procollagen-lysine,2-oxoglutarate 5-dioxygenase 1 Procollagen-lysine,2-oxoglutarate 5-dioxygenase 3 Attractin, Inflammatory response Insulin-like growth factor 2 receptor Serum paraoxonase/arylesterase 2 Transferrin receptor protein 1 Cartilage-associated protein Cytokine granulin Laminin subunit beta-2 Beta-galactosidase, Hydrolase N-Acetylglucosamine-6-sulfatase Beta-glucuronidase Alpha-N-acetylglucosaminidase, Hydrolase Lysosomal Pro-X carboxypeptidase Lamin-B1

4. DISCUSSION In the present study, we employed an efficient in vivo Flagtagging approach to identify a number of new interacting partners of FBXO6. As FBXO6 is ubiquitously expressed, the interaction proteins of FBXO6 maybe varied among different cell lines. To isolate the interacting proteins of FBXO6, we utilized 3 different types of cells with stable expressed FlagFBXO6 for purification. Indeed, the interacting proteins of FBXO6 are greatly varied among these cell lines. For example, the interacting proteins were more abundant in Jurkat cells than the other two cells and some hemopoietic system specifically expressed proteins were copurified, such as leukemia inhibitory factor receptor (LIFR) and leukocyte elastase inhibitor, (SERPINB1) (Table S2, Supporting Information). By comparing these data sets from different cell lines, we also got a comment list of the interacting proteins of FBXO6, which we termed the core interactors of FBXO6 that might provide clues to investigate the conserved function of it. As is reported, FBXO6-recognized glycoproteins depended on its FBA domain. To identify the glycroproteins of FBXO6 interaction proteins, we compared the interacting proteins between FBXO6WT and FBXO6Null which cannot recognize 1779



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Figure 5. Interaction between FBXO6 and glycosylated Ero1L. (A) 293T cells were infected with retroviruses encoding the indicated FLAG-tagged F-box proteins (FBPs). Exogenous proteins were immunoprecipitated (IP) from cell extracts with an anti-FLAG M2 resin, and immunocomplexes were probed with Ero1L, CUL1 and Flag antibodies. Lane 1 shows a whole-cell extract (WCE) from cells infected with an empty virus (EV). (B) Lysates and immunocomplex from 293T cells stably expressing FLAG-FBXO6 were treated with or without either Endo H or PNGase F for 1 h at 37 °C. Loading buffer was added to stop the reaction. Samples were analyzed by immunoblotting with the indicated antibodies. CHO, carbohydrate oligosaccharide. (C) Lysates from 293T cells stably expressing FLAG-FBXO6 were treated with (+) or without (−) Endo H at 37 °C for 1 h, and then they were immunoprecipitated with anti-FLAG antibody and probed with Ero1L antibody. Only glycosylated Ero1L can be immunoprecipitated by FBXO6. (D) 293T cells stably expressing FLAG-FBXO6 were treated with (+) or without (−) 10 μg/mL tunicamycin for 36 h, lysates were immunoprecipitated with anti-FLAG antibody, eluted with FLAG peptide, separated by SDS-PAGE and probed by ConA-HRP. (E) 293T cells stably expressing FLAG-FBXO6 were treated with (+) or without (−) 10 μg/mL tunicamycin for 36 h, lysates were immunoprecipitated with anti-FLAG antibody, eluted with FLAG peptide, separated by SDS-PAGE and immunoblotted with the indicated antibodies.

FBXO6 core interactors. Figure S3. FBXO6 interacting glycoproteins in 293T cells stably expressing wild type FBXO6 or mutant one. Figure S4. Detecting the presence of Chk1 in the FBXO6- immunoprecipitates by Western blotting. Table S1. List of FBXO6 interacting proteins in HeLa cells. Table S2. List of FBXO6 interacting proteins in Jurkat cells. Table S3. List of FBXO6 interacting proteins in 293T cells. Table S4. List of FBXO6WT interacting proteins in 293T cells. Table S5. List of FBXO6Null interacting proteins in 293T cells. Table S6. List of proteins exclusively interacted with FBXO6WT but not with FBXO6Null in 293T cells. This material is available free of charge via the Internet at http://pubs.acs.org.

homeostasis. Moreover, the purification strategy we provided here may further be utilized to other FBA domain containing F-box proteins. For example, FBXO2 interacting glycoproteins can be purified by this strategy in neuron cells.

5. CONCLUSION We utilized a protein purification approach combined with LC−MS to systematically identify the interacting glycoproteins of FBXO6. Thirty-nine proteins are identified that specifically interact with FBXO6 in all three different cell lines, 293T, HeLa and Jurkat cells. Then, we compared the protein complex organization between wild-type FBXO6 and its mutant, which fails to recognize glycoproteins. Combining these databases, 29 highly confident glycoproteins which interact with FBXO6 in an N-glycan dependent manner are identified. Our data provide valuable information for the discovery of the interacting glycoproteins of FBXO6 and also demonstrate the potential of these approaches as general platforms for the global discovery of interacting glycoproteins of other F-box proteins containing FBA domain.

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AUTHOR INFORMATION

Corresponding Author

* Li-Shun Wang, Tel: 86-21-63846590-776529. Fax: 86-2164154900. E-mail: [email protected]. Guo-Qiang Chen, Tel: 86-21-63846590-776529. Fax: 86-21-64154900. E-mail: [email protected]. Author Contributions

ASSOCIATED CONTENT

§

These authors contributed equally to this work.

S Supporting Information *

Notes

Figure S1. Isolation of glycoproteins from 293T cells stably expressing FBXO6. Figure S2. The ratios of glycoprotein in

The authors declare no competing financial interest. 1780



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ACKNOWLEDGMENTS This work is supported in part by grants from National Basic Research Program of China (973 Program) (NO2009CB918404), National Natural Science Foundation of China (NSFC, 90813034, 30971488, 81071668, 31170783, 31100980), Science and Technology Commission of Shanghai (08JC1413700, 11QH1401700) as well as Leading Academic Discipline Project of Shanghai Municipal Education Commission (J50201).

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Proteomic Identification of Common SCF Ubiquitin Ligase FBXO6

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