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C during myoblast fusion
Biochimica et Biophysica Acta 1814 (2011) 308–317
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Biochimica et Biophysica Acta j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / b b a p a p
Posttranslational arginine methylation of lamin A/C during myoblast fusion Su-Jin Kim a,1, Byong Chul Yoo b,1, Chang-Sub Uhm c,⁎, Sang-Won Lee a,⁎ a b c
Department of Chemistry, Research Institute for Natural Sciences, Korea University, Seoul 136-701, South Korea Research Institute, National Cancer Center, Gyeonggi 410-769, South Korea Department of Anatomy, College of Medicine, Korea University, Seoul 136-705, South Korea
a r t i c l e
i n f o
Article history: Received 27 June 2010 Received in revised form 31 October 2010 Accepted 18 November 2010 Available online 25 November 2010 Keywords: Skeletal muscle differentiation Lamin A/C Mass spectrometry Protein arginine methylation
a b s t r a c t Protein arginine methylation is a major posttranslational modification that regulates various cellular functions, such as RNA processing and DNA repair. A recent report showed the involvement of protein arginine methyltransferase (PRMT) 4 in chromatin remodeling and gene expression during muscle differentiation in C2C12 cells. Because the fusion of myoblasts is a unique phenomenon observed in skeletal muscle differentiation, the present study focused on the expression and activities of PRMTs during myoblast fusion in primary rat skeletal muscle. NG, NG-asymmetric dimethylarginines (aDMA) and NG, N′G-symmetric dimethylarginines (sDMA) were both found consistently throughout myoblast fusion. However, PRMT1 exhibited the highest activity during myoblast fusion and maintained the elevated activity thereafter, whereas PRMT5 reached its highest activity only after myoblast fusion. To identify the proteins modified by such PRMTs, we conducted 2-dimensional electrophoresis (2-DE) of total proteins before and after myoblast fusion, and protein spots on the 2-DE gel immunoreactive for aDMA and sDMA were identified by mass spectrometric analysis. Among the proteins identified, lamin C2 was in particular observed to be dimethylated. Arginine methylation of lamin may therefore be important for muscle development and maintenance. © 2010 Elsevier B.V. All rights reserved.
1. Introduction The main cellular component of the skeletal muscle is the multinucleated myocyte, which is formed by the fusion of mononucleated myoblasts [1,2]. Usually, 4–25 myoblasts fuse to form a single myotube [3]. Myoblast fusion is associated with cessation of DNA synthesis, production of contractile proteins, and marked alterations in enzymatic activity [1,2,4]. Some of the myoblasts remain as satellite cells in mature skeletal muscle and form new myocytes after muscle injury. Therefore, myoblast fusion is important not only in skeletal muscle development during the embryonic period, but also in the maintenance and repair of adult muscle. However, the mechanisms that control myoblast fusion during myogenesis and muscle regeneration are largely unknown [1,4].
Abbreviations: Asym, asymmetrically dimethylated arginine; aDMA, NG, NG-asymmetric dimethylarginines; sDMA, NG, N′G-symmetric dimethylarginines; EGTA, ethylene glycol-bis (2-aminoethylether)-N,N,N′,N′-tetraacetic acid; GAR, glycine arginine-rich; MMA, N G-monomethyl-L-arginine; PRMT, protein arginine methyltransferase; Sym, symmetrically dimethylated arginine ⁎ Corresponding authors. Chang-Sub Uhm is to be contacted at 1, 5-Ka, Anam-dong, Seongbuk-Ku, Seoul 136-705, South Korea. Tel: + 82 2 920 6150; fax: + 82 2 929 5696. Sang-Won Lee, 1, 5-Ka, Anam-dong, Seongbuk-Ku, Seoul 136-701, South Korea. Tel: + 82 2 3290 3137; fax: + 82 2 3290 3121. E-mail addresses: [email protected] (C.-S. Uhm), [email protected] (S.-W. Lee). 1 These authors contributed equally to this work. 1570-9639/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.bbapap.2010.11.006
Protein arginine methyltransferase (PRMT) methylates guanidino nitrogen atoms of arginyl residues by conversion of S-adenosyl-Lmethionine (AdoMet) to S-adenosyl-L-homocysteine (AdoHcy). PRMTs can be classified into two types: Type I forms NG-monomethyl-Larginine (MMA) and NG, NG-asymmetric dimethyl-L-arginine (aDMA), whereas type II forms MMA and NG, N′G-symmetric dimethyl-L-arginine (sDMA) [5–7] (Diagram 1). Currently, the PRMT family includes at least nine isotypes, designated PRMT1, 2, 3, 4, 5, 6, 7, 8 and 9 [6,8,9]. Protein arginine methylation has been implicated in the pivotal regulation of cellular events such as RNA processing, transcriptional regulation, signal transduction, insulin secretion, and DNA repair [5,7–10]. Furthermore, recent reports have suggested that PRMTs are involved in human diseases such as cancers, cardiovascular disease, HIV infection, multiple sclerosis, and spinal muscular atrophy [8,11]. The presence of methylated arginine in the muscle was first reported by Reporterer and Corbin in 1971 [12]. Subsequently, Chen et al. [10] reported the involvement of PRMT4 in chromatin remodeling and gene expression during muscle differentiation in mouse myoblast C2C12 cells. Because the fusion of myoblasts is a phenomenon unique to skeletal muscle differentiation, we aimed in the present study to investigate the expression and activities of PRMTs during myoblast fusion and the role of arginine methylation in muscle differentiation. We herein report that the activities of PRMTs are significantly changed during myoblast fusion while their expressions remain similar. Of proteins methylated by PRMT, we confirmed that lamin A/C contains an arginine methylation site.
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Diagram 1. Arginine methylation.
2. Materials and methods 2.1. Cell culture Rat myoblasts were cultured as previously described [13–15]. Briefly, primary myoblasts were obtained from embryonic day 20–21 fetal Sprague–Dawley rat hindlimb muscles by mechanical dissociation and trypsinization. The myoblasts were cultured in 80% Dulbecco's modified Eagle's medium (DMEM; Invitrogen, Carlsbad, CA, USA), containing 10% fetal calf serum, 10% horse serum , 1% penicillin streptomycin, and 1% Fungisol® , in an environment of 5–10% CO2 and saturated humidity at 37 °C. Dissociated cells were plated in 100 mm2 gelatin-coated tissue culture dishes. Approximately 48 h after plating, myoblasts were selectively detached from the culture dishes using neutral protease (Dispase, Roche Diagnostic/Boehringer Mannheim Corp., Indianapolis, IN, USA), collected, and replated in 100 mm2 gelatin-coated tissue culture dishes. Some of the cultures were kept from fusing by the addition of 2.0 mM ethylene glycol-bis (2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA; Sigma-Aldrich, St. Louis, MO, USA) to the culture medium. To induce fusion, the medium was replaced with the same medium without EGTA. To remove dividing cells, mainly fibroblasts, 2 mM cytosine arabinoside (Sigma-Aldrich) was added into the culture medium. Proliferating C2C12 myoblasts were grown in 100 mm2 tissue culture dishes in the presence of Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal calf serum , 2 mM glutamine, 1% penicillin streptomycin, and 1% Fungisol® at saturated humidity 37 °C and 5% CO2, until 80% confluence was attained. To induce C2C12 cells to differentiate, confluent myoblast culture medium was changed to differentiation medium (DMEM supplemented with 2% horse serum) and the other steps are the same conditions and methods with primary cell culture preparations [10,16]. 2.2. Microscopy The cultures were observed with a phase-contrast microscope at various time points after replating. For methylation studies, primary
cultured myoblast cells were seeded on 100 mm2 gelatin-coated tissue culture dishes. The muscle cells were cultured similarly to the cultures for morphologic observation. Proteins were extracted from muscle cultures before fusion (attachment and alignment states), during blocked fusion (fusion arrest with EGTA), during early fusion (fusion burst), and after fusion (myofiber). 2.3. Protein extraction and Western blot analysis The medium was removed at appropriate time points, and primary cells and C2C12 cells were washed three times with phosphatebuffered saline (PBS) and harvested by scraping them off the culture dishes with a rubber policeman. The cells were collected by centrifugation (1000 × g, 5 min). Cells were lysed by sonication in RIPA buffer (100 mM Tris–HCl [pH 7.6], 50 mM NaCl, 0.5% NP-40, 1% Triton X-100, 5 mM EDTA (ethylenediaminetetraacetic acid), and protease inhibitors) without SDS. Lysates were clarified by high-speed centrifugation (100 000 × g, 60 min) and quantified by using a Bradford reagent. A 50 μg of protein was used for each SDS-PAGE analysis. SDS-PAGE was performed according to the method of Laemmli [17] with 12.5% acrylamide for running the gels and 5% for stacking the gels in the presence of 0.1% SDS and in the absence of 2-mercaptoethanol [18]. Proteins in the SDS-PAGE gel were electrophoretically transferred onto a PVDF membrane in 25 mM Tris–HCl buffer (pH 8.3) containing 192 mM glycine and 10% methanol at 4 °C at a constant current of first 60 V for 30 min and then 120 V for 40 min. The membrane was dried in air for 30 min, and then incubated with primary antibodies for 16 h at 4 °C. After incubation, the membrane was washed three times with PBS, and the secondary antibodies were applied. The bound antibodies were visualized by an ECL reagent (GE Healthcare Life Sciences, Uppsala, Sweden), and the membrane was then exposed to X-ray film. Western blotting was performed with antibodies against the following proteins: PRMT1 (Upstate Biotechnology, Lake Placid, NY, USA), PRMT4 (CARM1; Upstate Biotechnology), PRMT5 (Upstate Biotechnology), PRMT7 (Upstate Biotechnology), asymmetrically dimethylated arginine
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Fig. 1. Expression and catalytic activities of PRMTs during skeletal muscle differentiation. (A) (Left) Differential expression of PRMTs in cultured skeletal muscle cells. Western blot analyses show that PRMT1 and PRMT5 are continuously expressed before fusion (BF), at early stages of fusion (EF), and after fusion (AF). However, the immunoreactive signal from PRMT4 was very weak and that of PRMT7 was not detected at its expected size. (Right) Expression of PRMT1 and PRMT5 was quantified by densitometry, and actually, myoblast and myotube were little different in protein expression level. (B) Intracellular localization of PRMT1 and PRMT5 in cultured muscle cells. PRMT5 is present in the nuclei and cytoplasm of both myoblasts and myotubes. PRMT1 was observed both in the nuclei and cytoplasm of myoblasts, but mainly in the cytoplasm of myotubes. Insert images are more magnification cy3 images of myoblast and myotube to confirm whether the PRMTs were or were not in the nucleus. Scale bar is a 20 μm. (C) Catalytic activities of PRMT1 and PRMT5. Activity was determined by fluorographic analyses using hnRNP A1 and MBP as the methyl acceptor substrates for PRMT1 and PRMT5, respectively. Both enzyme activities were dramatically upregulated during EF and AF. The highest enzyme activity for PRMT1 was observed during EF and AF, and that for PRMT5 was observed during AF. For fusion blocking (FB), growing cells were kept from fusing by the addition of EGTA. CBB (coomassie brilliant blue) staining is a loading control.
(ASYM24; Upstate Biotechnology), symmetrically dimethylated arginine (SYM10; Upstate Biotechnology), lamin A/C (Sigma-Aldrich), and beta-actin (Sigma-Aldrich). For detection, secondary antibodies (anti-rabbit IgG HRP (Santa Cruz Biotechnology Inc., Santa Cruz, CA), anti-mouse IgG HRP (Santa Cruz Biotechnology Inc.)), and ECL chemiluminescent reagent (GE Healthcare Life Sciences) were used.
2.4. Immunoprecipitation Whole muscle proteins were lysed by sonication in RIPA buffer without SDS. The lamin A/C was monitored by separation with 12.5% SDS-PAGE followed by staining with a Novex® colloidal blue staining kit (Invitrogen). For the immunoprecipitation assay, identical
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2.6. Enzymatic methylation and fluorography
Fig. 2. Alteration of proteins asymmetrically arginine-dimethylated during skeletal muscle differentiation. Antibodies against asymmetric (Asym 24) and symmetric (Sym 10) dimethylated peptides were used for Western blot analysis. The pattern of asymmetric dimethylation changed during muscle differentiation, but the pattern of symmetric dimethylation did not.
amounts of myoblast proteins were incubated with 200 μL of antilamin A/C (Santa Cruz Biotechnology Inc.) for 1 h at 4 °C, while 20 μL of A/G PLUS-Agarose (Santa Cruz Biotechnology Inc.) was incubated with goat anti-mouse IgG for 30 min at 4 °C and rinsed twice with cold phosphate buffer. Then the two samples were mixed for 2 h at 4 °C on a rotator (15 rpm). The beads were sedimented by centrifugation (2000 × g/1 min), the supernatant was removed, and the pellet was rinsed twice with phosphate buffer. The bound proteins were eluted from the beads by boiling the samples in SDS loading buffer (1 M Tris– HCl [pH 6.8], 10% SDS, 50% glycerol, 5% 2-mercaptoethanol, and 1% bromophenol blue). Proteins were separated by SDS-PAGE (12.5%) and stained overnight with GelCode Blue Stain reagent (Thermo Scientific Pierce Protein Research Products, Rockford, IL, USA).
2.5. Immunofluorescence analysis For fluorescent immunohistochemical analysis, myoblast cells were grown on 13 mm cover slips held in 35 mm culture dishes. Cells were washed once with PBS, fixed with 4% paraformaldehyde in PBS (pH 7.4), and blocked with PBS containing 5% normal goat serum and 0.2% Triton X-100 for 30 min. They were then incubated with antibodies against myo-D (Santa Cruz Biotechnology Inc.), myogenin (Santa Cruz Biotechnology Inc.), PRMT1, PRMT5, skeletal actin muscle (Neomarker, Fremont, CA), or lamin A/C antibodies, respectively, for 1 h at room temperature. After washes with PBS, the cells were incubated with Alexa Fluor® 488 goat anti-mouse IgG (1:500, Invitrogen) or Cy3 goat anti-mouse IgG (1:500, Invitrogen) for 30 min at room temperature and washed again. The coverslips were mounted and observed on an Axiovert 200 fluorescent microscope equipped with a 63× oil-immersion objective (Zeiss, Göttingen, Germany).
Protein was extracted as described above. The harvested pellet was resuspended in buffer containing 5 mM sodium phosphate (pH 7.4) containing 5 mM EDTA, 0.27 M sucrose, and protease inhibitor cocktail (Roche Applied Science, Penzberg, Germany), and disrupted by homogenization [19]. The homogenate was then centrifuged at 100,000 × g for 1 h at 4 °C to remove membrane fractions. The supernatant was filtered through VIVASPIN 10,000 mw (Vivaproducts Inc., MA, USA) to remove the endogenous transmethylation inhibitors and the concentrated fraction was used for methylation studies. Protein contents in the supernatant were measured by the Bradford method [20] with an ELISA reader (UniScan®; LabSystem, Helsinki, Finland). Fluorography was performed according to Lim et al. [19,21]. Methylation reactions were carried out with 50 μg cell extract and Ado [methyl-3H]Met (2.5–5.5 μCi, GE Health Care Life Sciences, NJ, USA) in 0.1 M phosphate buffer (pH 7.4) [19]. The reaction mixture without the methyl donor was pre-incubated for 5 min at 37 °C, and then the reaction was initiated by the addition of Ado [methyl-3H]Met and methyltransferase substrates (i.e., hnRNP A1 or MBP) at 37 °C. Recombinant HeLa hnRNP A1 was overexpressed and purified from E. coli and MBP from calf brain was purified. After 30 or 60 min of incubation as indicated, the reactions were stopped by the addition of 5× SDS-PAGE sample buffer, followed by heating at 100 °C for 10 min. Then, the protein was subjected to SDS-PAGE. In order to visualize methylated proteins, the gel was stained with Coomassie Brilliant Blue and developed for fluorography by being soaked in an amplifying solution (NAMP-100, GE Health Care Life Sciences, Uppsala, Sweden), dried, and exposed to diagnostic X-ray film (Hyperfilm MP, GE Health Care Life Sciences) at −70 to −80 °C for 7 days [19,21]. 2.7. Two-dimensional electrophoresis For the isoelectric focusing (IEF), 100 μg of protein was loaded onto 7 cm immobilized pH gradient (IPG) strips (GE Health Care Life Sciences) with a 3–10 nonlinear gradient. The electrophoresis was carried out for about 12 h (200 V 1 h linear, 500 V 1 h linear, 1000 V 1 h linear, 8000 V 1 h linear, 8000 V, for a total of 40 000 Vh rapid). Following IEF, the IPG strips were equilibrated for 10 min in equilibration buffer (50 mM Tris–HCl buffer, pH 8.8, containing 6 M urea, 30% [v/v] glycerol, 2% [w/v] SDS, a few grains of bromophenol blue, 0.2% w/v DTT). The strips were then re-equilibrated for 10 min in the same buffer containing 1.5% (w/v) iodoacetamide. Molecular weight was estimated from the position on a 12.5% SDS polyacrylamide gel (Bio-Rad, Hercules, CA, USA). Coomassie stain solution was applied for 3–12 h, and the gel was washed with distilled water. For 2-dimensional electrophoresis (2-DE) Western blot analysis, a total of six 2-DE gels were prepared by using whole proteome extracted before fusion (BF) and after fusion (AF) (three gels for BF and three gels for AF, respectively). A pair of 2-DE gels (BF and AF) were electrophoretically transferred onto a PVDF membrane and incubated with ASYM 24 primary antibody (Fig. 3B, Asym 24, BF and AF). Another pair of 2-DE gels was Western blotted with SYM 10 primary antibody (Fig. 3B, Sym 10, BF and AF). After incubation, the membranes were washed and the secondary antibody was applied. The immunoreactive protein spots were visualized by an ECL reagent, and matched with same spots on the last pair of 2-DE gels (BF and AF) by comparing its molecular weight and pI point as well as location of neighbor spots. The protein spots that displayed significant difference between BF and AF were picked up for MALDI-TOF analysis. Among 33 spots, 13 spots indicated in Fig. 3B were identified with significant Mascot score (Fig. 3C). Silver staining was done according to the method recommended by the manufacture. Briefly, for silver stain, 100 μg of protein mixed with rehydration buffer (8 M urea, 2% CHAPS, a few grains of
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bromophenol blue) to a final volume of 350 μl was absorbed onto 18-cm immobilized pH gradient (IPG) strips (GE Health Care Life Sciences) with a 3–10 nonlinear gradient. After rehydration, the electrophoresis was carried out for about 20 h, (200 V 1 h linear, 500 V 1 h linear, 1000 V 1 h linear, 8000 V 1 h linear, 8000 V, for a total of 40,000 Vh rapid). Following IEF, the IPG strips were equilibrated for 15 min in equilibration buffer (50 mM Tris–HCl, pH 8.8 buffer, containing 6 M urea, 30% (v/v) glycerol, 2% (w/v) SDS, a few grains of bromophenol blue, 0.2% (w/v) DTT). Strips were then reequilibrated for 10 min in the same buffer containing 1.5% w/v iodoacetamide. Molecular weight separation was achieved on 12.5% SDS polyacrylamide gel using Protean II xi cell gel SDS-PAGE system (Bio-Rad) 2 W per gel for 1 h and 16 W per gel for 3 h. Then, proteins in the gel were fixed with 50% (v/v) methanol, 12% (w/v) acetic acid, and 0.05% (w/v) formaldehyde overnight and washed three times with 50% (v/v) ethyl alcohol for 20 min. The sensitizing solution (0.02% (w/v) sodium thiosulfate) was applied for 1 min to increase sensitivity, and washed three times with distilled water. Proteins were stained with 0.1% silver nitrate staining solution and 0.075% (w/v) formaldehyde for 20–30 min, developed with 6% (w/v) sodium carbonate, 0.05% (v/v) formaldehyde to visualize the proteins. The reaction was stopped by applying stopping solution (50% methanol, 12% (w/v) acetic acid). Reagents used in 2-dimensional electrophoresis (2-DE) included acrylamide, bis-acrylamide, TEMED, Tris base, glycine, SDS, APS, DTT, CHAPS, urea, thiourea, and iodoacetamide, all purchased from Sigma-Aldrich. Immobiline dry strips, IPG buffer, and
IPG cover mineral oil were purchased GE Health Care Life Sciences and colloidal blue staining kit was purchased from Invitrogen. For silver stain, silver nitrate and sodium thiosulfate were purchased from Sigma-Aldrich and methanol, acetic acid, formaldehyde and ethyl alcohol were purchased from Dae Jung Chemical Co., Ltd. (Gyeonggi, Korea). 2.8. MALDI-TOF analysis A total of 33 significant protein spots were excised from the gel and destained with distilled water. Upon reduction of disulfide bonds of the protein with tris (2-carboxyethylphosphine) hydrochloride (Thermo Fisher Scientific Inc.), cysteines were alkylated with iodoacetamide (Sigma-Aldrich). Digestion with modified trypsin (400 ng, Promega, WI, USA) was carried out overnight at 37 °C in a buffer containing 100 mM ammonium bicarbonate buffer, pH 8.3, and 4 mM calcium chloride. α-Cyano 4-hydroxycinnamic acid (20 mg; Bruker Daltonics, Bremen, Germany) was dissolved in 1 ml acetone: ethanol (1:2, v/v), and 0.5 μl of the matrix solution was mixed with an equivalent volume of sample. Analysis was performed using an Ultraflex TOF/TOF system (Bruker Daltonics). The Ultraflex TOF/TOF system was operated in positive ion reflect mode. Each spectrum was the cumulative average of 250–450 laser shots. Mass spectra were first calibrated in the closed external mode using the peptide calibration standard II (Bruker Daltonics), sometimes using the internal statistical mode to achieve maximum calibration mass accuracy, and analyzed
Fig. 3. Identification of proteins asymmetrically arginine-dimethylated during skeletal muscle differentiation. (A) Typical pattern of 2-DE images of whole protein extracted before fusion (BF) and after fusion (AF). The separated protein was stained with a silver nitrate. (B) Western blot analysis of the 2-DE gels. Thirteen protein spots indicated on the 2-DE gels were immunoreactive for dimethylated arginine (Asym 24 and Sym 10) and were selected for mass analysis. (C) Results of MALDI-TOF/TOF analysis of the selected protein spots shown in (B).
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Fig. 3 (continued).
with FlexAnalysis software, version 2.4 (Bruker Daltonics). Peptide mass peaks from each spectrum were submitted to a Mascot peptide mass fingerprinting search (www.matrixscience.com) for analysis with BioTools software, version 3.0 (Bruker Daltonics). The search included peaks with a signal-to-noise ratio greater than 3. The peak list for each sample was used to query the non-redundant Mass Spectrometry Protein Sequence Database for protein identification. Standard settings included the following: enzyme, trypsin; missed cleavage, one; fixed modifications, carbamidomethylation on cysteine; variable modifications, oxidized methionine; protein mass, blank; mass values, MH+ (monoisotopic); mass tolerance, varied between 75 and 100 ppm. 2.9. LC-MS/MS analysis Each immunoprecipitated sample was carried out reduction, alkylation and trypsin digestion. These tryptic peptides separately analyzed using an LTQ-FT mass spectrometer (Thermo Electron, San Jose, CA, USA) and the nanoACQUITY UPLC (NanoA, Waters, Milford,
MA, USA) system. The nanoACQUITY UPLC system was equipped with a capillary column (75 μm ID × 360 μm OD × 75 cm length) built in house and packed with fused-silica capillary with C18-bonded particles (3 μm diameter, 300 Å pore size, Phenomenex, Torrance, CA, USA) [22], also manufactured in house. After the sample injection, each peptide was loaded onto the online SPE column for 6 min in solvent A (0.1% formic acid in distilled water), and peptide separation was achieved by using a linear gradient from 10 to 50% solvent B (0.1% formic acid in acetonitrile) for 60 min at a flow rate of 350 nL/min. The mass spectrometer was operated in a data-dependent tandem MS mode where a full-scan MS experiment (m/z 450–1800) was followed by MS/MS experiments on the three most abundant ions in the precursor MS scan. The normalized energy for peptide dissociation was set to 35% and the dynamic exclusion option was incorporated to prevent reacquisition of MS/MS spectra of the same peptides for 30 s. The resultant MS and MS/MS data from the mass spectrometer were analyzed by using BioworksTM software (v. 3.2, Thermo Electron) against a database that was constructed by combining the IPI rat database (Rat, 3.68, ftp://ftp.ebi.ac.uk) and a common contaminant
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database (http://www.ncbi.nih.gov). All tandem mass spectrometric data (i.e., DTA files) were extracted using ExtractMSn (Version 4.0) of the BioworksTM software. Before a database search was performed, the MS/MS data were filtered and refined through the PE-MMR (postexperiment monoisotopic mass filtering and refinement) [23] program (http://omics.pnl.gov/software/PEMMR.php). The database search was
performed using a m/z tolerance of 10 ppm, and the fragment ion m/z tolerance was ±0.5 Da. The modification options were used for the oxidation of methionine (15.994920 Da), iodoacetamide derivative of alkylation (57.021464 Da), Arg-monomethylation (14.015650 Da), and Arg-dimethylation (28.031300 Da). The search results were filtered using an estimated FP (false positive) rate. The FP rate of peptide
Fig. 4. Methylation of lamin A/C in skeletal muscle. (A) MALDI-TOF/TOF analysis of proteins immunoprecipitated with an anti-lamin A/C antibody. SDS-PAGE was performed using the immunoprecipitate, and the indicated protein bands, which were expected to be lamin A/C, were sliced out of the gel for MALDI-TOF/TOF analysis, and MALDI-TOF/TOF-MS result of lamin A/C identification. (B) Methylation of lamin A/C. Lamin protein expression in the whole cell lysate was checked with an anti-lamin A/C antibody (left), and Western blot analysis of the immunoprecipitate of anti-lamin A/C was performed with antibodies against dimethylated arginine (ASYM 24 and Sym 10; middle and right panels, respectively).
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antibodies against PRMT1 and PRMT4 as type I PRMTs, and PRMT5 and PRMT7 as type II PRMTs. PRMT1 and PRMT5 were continuously expressed in cultured skeletal muscle cells before fusion, at early stages of fusion, and after fusion, but the immunoreactive signal from PRMT4 was very weak and no signal was detected for PRMT7 (Fig. 1A). 3.2. Change of PRMT1 and PRMT5 catalytic activities during myoblast fusion
3.1. Expression of PRMTs during skeletal muscle differentiation
We localized PRMT1 and PRMT5 within cultured muscle cells by using immunofluorescent staining (Fig. 1B). To identify the fusion status, myoblasts and myotubes were identified with antibodies against myo-D and myogenin, respectively [24]. PRMT5 was present in the nuclei and cytoplasm of both myoblasts and myotubes, and PRMT1 was observed in both the nuclei and cytoplasm of myoblasts, but mainly in the cytoplasm of myotubes (Fig. 1B). Because both PRMT1 and PRMT5 were expressed mainly in the cytoplasm, we used whole cytosolic proteins obtained from cultured skeletal muscle cells to determine the enzyme activities. The enzyme activities were measured by fluorographic analyses using heterogeneous nuclear ribonucleoprotein (hnRNP) A1 and MBP as the methyl acceptor substrates for PRMT1 and PRMT5, respectively (Fig. 1C). Both PRMT1 and PRMT5 activities were dramatically upregulated during early fusion and after fusion. The highest enzyme activity for PRMT1 was observed during myoblast fusion (early fusion and after fusion) and that for PRMT5 was during the postfusion stage (Fig. 1C).
To investigate which PRMTs are responsible for arginine methylation in muscle differentiation, we performed Western blotting with
3.3. Alteration of asymmetric arginine-dimethylated proteins during skeletal muscle differentiation
Fig. 4 (continued).
assignment was estimated through a composite target-decoy database search. The values of XCorr and the ΔCn threshold for the 1% FP rate were used to obtain peptide IDs [23]. 3. Results
To clarify whether arginine methylation is involved in muscle differentiation, especially during fusion, we investigated how arginine methylation changed during differentiation using whole proteins extracted from primary cultured rat muscle cells. When antibodies against asymmetric (Asym 24) and symmetric (Sym 10) dimethylated peptides were used for Western blot analysis [29], various proteins with different molecular weight were found to be arginine-methylated (Fig. 2). Interestingly, proteins with asymmetric dimethylation were altered during the muscle fusion, whereas symmetric dimethylation was not affected by different stages of muscle differentiation (Fig. 2). 3.4. Identification of possible substrate proteins of PRMTs after myoblast fusion To identify proteins arginine-methylated by PRMTs whose activity increased during and after myoblast fusion, we used a 2-DE immunoproteomics approach specific for aDMA (Asym 24) and sDMA (Sym 10) to detect dimethylated arginine [29]. Muscle protein extracts before and after fusion were separated by standard 2-DE (Fig. 3A) and transferred to PVDF membranes for Western blotting of arginine-methylated proteins for aDMA (Asym 24) and sDMA (Sym 10) (Fig. 3B). Among the detected protein, significant protein spots with immunoreactive signals for arginine methylation were identified to 13 proteins (Fig. 3B and C) by MALDITOF mass analysis. The candidate proteins asymmetrically dimethylated during after fusion included ATP synthase alpha chain (Q03265), hnRNP A3 (Q8BG05) and A1 (P49312), glyceraldehyde 3-phosphate dehydrogenase (P16858), lamin A (P48678), and lamin C and C2 (P11516) (Fig. 3C). 3.5. Methylated lamin A/C in skeletal muscle Fig. 5. Arginine-methylation site in lamin C2. (A) LC-MS/MS analysis of lamin proteins immunoprecipitated with anti-lamin A/C. The proteins removed from the SDS-PAGE gel (Fig. 4A) were clearly identified as a lamin C2. (B) Dimethylation of arginine 209. Among the peptides matched with lamin C2, dimethylation of arginine 209 was confirmed by peptide sequencing of LRDLEDSLAR.
Among the asymmetrically methylated proteins we identified (Fig. 3C), we further validated the methylation of lamin A/C because it is involved in muscle function [25–27]. To confirm that lamin A/C is
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arginine-methylated during myoblast fusion, we extracted total protein from cells at each stage of myogenesis and immunoprecipitated them using an anti-lamin A/C antibody. The immunoprecipitated proteins were identified by MALDI-TOF mass analysis (Fig. 4A, indicated in the enlarged image), and the result was confirmed by Western blot analysis employing anti-lamin A/C, Asy 24, and Sym 10 antibodies (Fig. 4B). To identify the arginine methylation site in lamin, we analyzed the lamin proteins immunoprecipitated with anti-lamin A/C by using LCMS/MS. Proteins extracted from the immunoprecipitate were clearly identified as a lamin C2 (Fig. 5A). Among the peptides that matched to lamin C2, we confirmed the dimethylation of Arg 209 by peptide sequencing of LRDLEDSLAR (Fig. 5B). 4. Discussion Alterations in the amount, posttranslational modifications, or activities of certain proteins during muscle differentiation implicate these proteins as having key roles in muscle fusion [10]. Therefore, the identification of proteins differentially modified by PRMTs is a critical step for understanding the molecular mechanism(s) of muscle fusion. PRMTs are known to be highly expressed in the cells wrapping up protein synthesis [9]. Because myoblasts are actively growing cells and need a great amount of proteins for fusion and after-fusion growth, we expected to find high expression of major PRMTs. Among the PRMTs we evaluated during muscle fusion, PRMT1 and PRMT5 were dominantly expressed without regard to the muscle fusion process (Fig. 1A). Though a role for PRMT4 in terminal differentiation of C2C12 muscle cells has been suggested [10], we detected very little PRMT4 in primary rat muscle culture (Fig. 1A). We also note that the expression of PRMT1 and PRMT5 was explicitly different in C2C12 cells and primary muscle cells (Supplementary Fig. 1). Both PRMT5 and PRMT7 belong to type II and symmetrically dimethylate Sm proteins in vitro [28,29]. They are involved in the biogenesis of small nuclear ribonucleoproteins (snRNPs). Nuclear PRMT5 is well known as a regulator of transcriptional elongation [28], but the role of PRMT7, a recent addition to the PRMT family, is not yet known [29]. Our present results indicate that both type I (PRMT1) and II (PRMT5) PRMTs are expressed during fusion. It is not, however, clear why PRMT1 and PRMT5 were selectively expressed. Kwak et al. [30] reported that either PRMT1 or PRMT5 can methylate SPT5, a component of important transcriptional elongational factors, to decrease association of SPT5 with RNA polymerase II; however, the role of SPT5 in muscle fusion is not known. Interestingly, we found dramatic upregulation of enzyme activity of PRMT1 during early fusion and after fusion, and of PRMT5 after fusion (Fig. 1C), suggesting that relative expression of the enzymes may be important for muscle fusion, and PRMTs may have different roles in myoblast fusion and maintenance. Methylation of HnRNP A1 and MBP had previously been employed to check the activity of PRMT1 and PRMT5, respectively [8,19,31], as hnRNP A1 and MBP are ones of the well known substrates for PRMT1 and PRMT5, respectively. From fluorography experiment (Fig. 1C), we were able to confirm that the activities of PRMTs were indeed increased. Our objective then was to find novel substrates for the PRMTs in primary culture under the increased activities. Our finding that PRMT1 is localized to both the nuclei and cytoplasm of myoblasts (Fig. 1B) is in good agreement with a previous report, in which a photobleaching experiment showed that PRMT1 is highly mobile both in the cytoplasm and the nucleus [32]. This fact may be evidence that PRMT1 moves from the nucleus to the cytoplasm before and after fusion. Though the best-known substrates for PRMT1 are present in the nucleus, the enzyme itself is not preferentially located in the nucleus [33]; in fact, PRMT1 is distributed quite variably in both the cytoplasm and nucleus [34]. Considering that treatment with a PRMT1 inhibitor leads to a significant nuclear accumulation of PRMT1 [32], the preferential localization of PRMT1 in
the cytoplasm may be necessary for increasing methylation during myoblast fusion. PRMT5 was observed in the nuclei and cytoplasm of both myoblasts and myotubes (Fig. 1B), but the significance of this subcellular localization is not clear. We identified 13 proteins with immunoreactive signals against aDMA and sDMA during muscle fusion (Figs. 2 and 3). Thirteen proteins, including hnRNPs, the best-known substrates for asymmetrical arginine dimethylation [35] and lamin A/C, were identified by MALDI-TOF analysis. Lamins are intermediate filaments that form the essential structural meshwork of the nuclear lamina of all mammalian cells. The nuclear lamina is located beneath the inner nuclear membrane and maintains the shape and mechanical properties of the nuclei. The nuclear lamina had been suggested as a molecular docking site for peripheral heterochromatin. Lamin A and C are the major products of the LMNA gene, and mutations in LMNA have been linked to certain human genetic disorders, broadly termed laminopathies [25]. Laminopathies affect the development of striated muscle, adipose tissue, peripheral nerve, and skeletal muscle [27]. For example, recent studies have demonstrated the important role of lamin A/C in Emery–Dreifuss muscular dystrophy and limb-girdle muscular dystrophy [25,26]. The cultured cells of lamin A/C knockout mice show impaired differentiation kinetics and reduced differentiation potential [25]. Furthermore, differentiation of muscle in Emdnull mice is delayed, and this delay is associated with disordered transcriptional pathways regulated by the retinoblastoma (Rb1) and myo-D genes [25,26]. Our observation that PRMT1 is localized to myoblast nuclei suggests that lamins are dimethylated before the completion of myoblast fusion. This finding indicates possible involvement of lamin methylation in myoblast fusion, and the data shown in Figs. 4 and 5 clearly suggest that lamin is a substrate for PRMTs. Arginine methylation occurs at arginines within glycine and arginine-rich (GAR) motifs [8,35]. In the lamin C2 sequences, there are 4 GAR motifs. Unfortunately, we did not identify methylated arginine at these 4 sites (Arg 7, 287, 289 and 327; Fig. 5). Among the peptides matched with lamin C2, we did confirm dimethylation of Arg 209 by peptide sequencing of LRDLEDSLAR (Fig. 5B). Ultimately, this study revealed the relationship of lamin, PRMT, and muscle differentiation, but the specific roles of lamin and arginine methylation were not actually disclosed; however, the role of this specific modification in muscle differentiation has to be studied further. By extension, we will have to study whether modifying PRMT could be a treatment for Emery–Dreifuss muscular dystrophy and limb-girdle muscular dystrophy. In summary, PRMTs showed changes in their catalytic activities during myoblast fusion of primary muscle cells. The highest activities of PRMT1 and PRMT5 were observed during and after myoblast fusion, respectively, suggesting that each PRMT has a different role in myoblast fusion. Among the possible substrates, mass spectrometric evidences confirmed that lamin C2 was arginine-methylated during muscle fusion, but its functional significance in muscle differentiation is still not clear. Supplementary materials related to this article can be found online at doi:10.1016/j.bbapap.2010.11.006. Acknowledgments We thank Steffen Pahlich, Dr. Heinz Gehring, and Dr. Peter Gehrig (Department of Biochemistry University of Zurich and Functional Genomic Center Zurich (FGCZ) Switzerland) for allowing us to use the LC-MALDI-TOF/TOF-MS, and Woon Ki Paik, Sangduk Kim, Yong-Chul Lim, and Young-Ho Kwon (Graduate School of Biomedical Sciences, Korea University College of Medicine) for helping with the interpretation of the data and experiment. This work was supported in part by 21C Frontier Functional Proteomics Project (grant no. FPR08A1-010), Converging Research Center for Mass Spectrometric Diagnosis (2010K001300) and Priority Research Centers Program through the
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National Research Foundation (NRF20100020209) from Korean Ministry of Education Science & Technology. S. K. also acknowledges a Korea University Grant. Y.B. acknowledges the Korean National Cancer Center Research Grant 1010050. References [1] V. Horsley, K.M. Jansen, S.T. Mills, G.K. Pavlath, IL-4 acts as a myoblast recruitment factor during mammalian muscle growth, Cell 113 (2003) 483–494. [2] S. Charge, M.A. Rudnicki, Fusion with the fused: a new role for interleukin-4 in the building of muscle, Cell 113 (2003) 422–423. [3] S.K. Doberstein, R.D. Fetter, A.Y. Mehta, C.S. Goodman, Genetic analysis of myoblast fusion: blown fuse is required for progression beyond the prefusion complex, J. Cell Biol. 136 (1997) 1249–1261. [4] D.L. Allen, R.R. Roy, V.R. Edgerton, Myonuclear domains in muscle adaptation and disease, Muscle Nerve 22 (1999) 1350–1360. [5] J.D. Gary, S. Clarke, RNA and protein interactions modulated by protein arginine methylation, Prog. Nucleic Acid Res. Mol. Biol. 61 (1998) 65–131. [6] S. Pahlich, R.P. Zakaryan, H. Gehring, Protein arginine methylation: cellular functions and methods of analysis, Biochim. Biophys. Acta (2006). [7] M.R. Stallcup, Role of protein methylation in chromatin remodeling and transcriptional regulation, Oncogene 20 (2001) 3014–3020. [8] W.K. Paik, D.C. Paik, S. Kim, Historical review: the field of protein methylation, Trends Biochem. Sci. 32 (2007) 146–152. [9] Y.H. Lee, M.R. Stallcup, Minireview: protein arginine methylation of nonhistone proteins in transcriptional regulation, Mol. Endocrinol. 23 (2009) 425–433. [10] S.L. Chen, K.A. Loffler, D. Chen, M.R. Stallcup, G.E. Muscat, The coactivator-associated arginine methyltransferase is necessary for muscle differentiation: CARM1 coactivates myocyte enhancer factor-2, J. Biol. Chem. 277 (2002) 4324–4333. [11] M.T. Bedford, S. Richard, Arginine methylation an emerging regulator of protein function, Mol. Cell 18 (2005) 263–272. [12] M. Reporterer, J.L. Corbin, NG, NG,-dimethylarginine in myosin during muscle development, Biochem. Biophys. Res. Commun. 43 (1971) 644–650. [13] M.P. Daniels, Localization of actin, beta-spectrin, 43 × 10(3) Mr and 58 × 10(3) Mr proteins to receptor-enriched domains of newly formed acetylcholine receptor aggregates in isolated myotube membranes, J. Cell Sci. 97 (Pt 4) (1990) 615–626. [14] E.K. Dutton, C.S. Uhm, S.J. Samuelsson, A.E. Schaffner, S.C. Fitzgerald, M.P. Daniels, Acetylcholine receptor aggregation at nerve-muscle contacts in mammalian cultures: induction by ventral spinal cord neurons is specific to axons, J. Neurosci. 15 (1995) 7401–7416. [15] S.J. Kim, S. Kim, H. Shin, C.S. Uhm, Intercellular interaction observed by atomic force microscopy, Ultramicroscopy 108 (2008) 1148–1151. [16] B.W. Kim, J.W. Lee, H.J. Choo, C.S. Lee, S.Y. Jung, J.S. Yi, Y.M. Ham, J.H. Lee, J. Hong, M.J. Kang, S.G. Chi, S.W. Hyung, S.W. Lee, H.M. Kim, B.R. Cho, D.S. Min, G. Yoon, Y.G. Ko, Mitochondrial oxidative phosphorylation system is recruited to detergentresistant lipid rafts during myogenesis, Proteomics 10 (2010) 2498–2515. [17] U.K. Laemmli, Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature 227 (1970) 680–685. [18] W.F. Glass 2nd, R.C. Briggs, L.S. Hnilica, Identification of tissue-specific nuclear antigens transferred to nitrocellulose from polyacrylamide gels, Science 211 (1981) 70–72.
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Biochimica et Biophysica Acta j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / b b a p a p
Posttranslational arginine methylation of lamin A/C during myoblast fusion Su-Jin Kim a,1, Byong Chul Yoo b,1, Chang-Sub Uhm c,⁎, Sang-Won Lee a,⁎ a b c
Department of Chemistry, Research Institute for Natural Sciences, Korea University, Seoul 136-701, South Korea Research Institute, National Cancer Center, Gyeonggi 410-769, South Korea Department of Anatomy, College of Medicine, Korea University, Seoul 136-705, South Korea
a r t i c l e
i n f o
Article history: Received 27 June 2010 Received in revised form 31 October 2010 Accepted 18 November 2010 Available online 25 November 2010 Keywords: Skeletal muscle differentiation Lamin A/C Mass spectrometry Protein arginine methylation
a b s t r a c t Protein arginine methylation is a major posttranslational modification that regulates various cellular functions, such as RNA processing and DNA repair. A recent report showed the involvement of protein arginine methyltransferase (PRMT) 4 in chromatin remodeling and gene expression during muscle differentiation in C2C12 cells. Because the fusion of myoblasts is a unique phenomenon observed in skeletal muscle differentiation, the present study focused on the expression and activities of PRMTs during myoblast fusion in primary rat skeletal muscle. NG, NG-asymmetric dimethylarginines (aDMA) and NG, N′G-symmetric dimethylarginines (sDMA) were both found consistently throughout myoblast fusion. However, PRMT1 exhibited the highest activity during myoblast fusion and maintained the elevated activity thereafter, whereas PRMT5 reached its highest activity only after myoblast fusion. To identify the proteins modified by such PRMTs, we conducted 2-dimensional electrophoresis (2-DE) of total proteins before and after myoblast fusion, and protein spots on the 2-DE gel immunoreactive for aDMA and sDMA were identified by mass spectrometric analysis. Among the proteins identified, lamin C2 was in particular observed to be dimethylated. Arginine methylation of lamin may therefore be important for muscle development and maintenance. © 2010 Elsevier B.V. All rights reserved.
1. Introduction The main cellular component of the skeletal muscle is the multinucleated myocyte, which is formed by the fusion of mononucleated myoblasts [1,2]. Usually, 4–25 myoblasts fuse to form a single myotube [3]. Myoblast fusion is associated with cessation of DNA synthesis, production of contractile proteins, and marked alterations in enzymatic activity [1,2,4]. Some of the myoblasts remain as satellite cells in mature skeletal muscle and form new myocytes after muscle injury. Therefore, myoblast fusion is important not only in skeletal muscle development during the embryonic period, but also in the maintenance and repair of adult muscle. However, the mechanisms that control myoblast fusion during myogenesis and muscle regeneration are largely unknown [1,4].
Abbreviations: Asym, asymmetrically dimethylated arginine; aDMA, NG, NG-asymmetric dimethylarginines; sDMA, NG, N′G-symmetric dimethylarginines; EGTA, ethylene glycol-bis (2-aminoethylether)-N,N,N′,N′-tetraacetic acid; GAR, glycine arginine-rich; MMA, N G-monomethyl-L-arginine; PRMT, protein arginine methyltransferase; Sym, symmetrically dimethylated arginine ⁎ Corresponding authors. Chang-Sub Uhm is to be contacted at 1, 5-Ka, Anam-dong, Seongbuk-Ku, Seoul 136-705, South Korea. Tel: + 82 2 920 6150; fax: + 82 2 929 5696. Sang-Won Lee, 1, 5-Ka, Anam-dong, Seongbuk-Ku, Seoul 136-701, South Korea. Tel: + 82 2 3290 3137; fax: + 82 2 3290 3121. E-mail addresses: [email protected] (C.-S. Uhm), [email protected] (S.-W. Lee). 1 These authors contributed equally to this work. 1570-9639/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.bbapap.2010.11.006
Protein arginine methyltransferase (PRMT) methylates guanidino nitrogen atoms of arginyl residues by conversion of S-adenosyl-Lmethionine (AdoMet) to S-adenosyl-L-homocysteine (AdoHcy). PRMTs can be classified into two types: Type I forms NG-monomethyl-Larginine (MMA) and NG, NG-asymmetric dimethyl-L-arginine (aDMA), whereas type II forms MMA and NG, N′G-symmetric dimethyl-L-arginine (sDMA) [5–7] (Diagram 1). Currently, the PRMT family includes at least nine isotypes, designated PRMT1, 2, 3, 4, 5, 6, 7, 8 and 9 [6,8,9]. Protein arginine methylation has been implicated in the pivotal regulation of cellular events such as RNA processing, transcriptional regulation, signal transduction, insulin secretion, and DNA repair [5,7–10]. Furthermore, recent reports have suggested that PRMTs are involved in human diseases such as cancers, cardiovascular disease, HIV infection, multiple sclerosis, and spinal muscular atrophy [8,11]. The presence of methylated arginine in the muscle was first reported by Reporterer and Corbin in 1971 [12]. Subsequently, Chen et al. [10] reported the involvement of PRMT4 in chromatin remodeling and gene expression during muscle differentiation in mouse myoblast C2C12 cells. Because the fusion of myoblasts is a phenomenon unique to skeletal muscle differentiation, we aimed in the present study to investigate the expression and activities of PRMTs during myoblast fusion and the role of arginine methylation in muscle differentiation. We herein report that the activities of PRMTs are significantly changed during myoblast fusion while their expressions remain similar. Of proteins methylated by PRMT, we confirmed that lamin A/C contains an arginine methylation site.
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Diagram 1. Arginine methylation.
2. Materials and methods 2.1. Cell culture Rat myoblasts were cultured as previously described [13–15]. Briefly, primary myoblasts were obtained from embryonic day 20–21 fetal Sprague–Dawley rat hindlimb muscles by mechanical dissociation and trypsinization. The myoblasts were cultured in 80% Dulbecco's modified Eagle's medium (DMEM; Invitrogen, Carlsbad, CA, USA), containing 10% fetal calf serum, 10% horse serum , 1% penicillin streptomycin, and 1% Fungisol® , in an environment of 5–10% CO2 and saturated humidity at 37 °C. Dissociated cells were plated in 100 mm2 gelatin-coated tissue culture dishes. Approximately 48 h after plating, myoblasts were selectively detached from the culture dishes using neutral protease (Dispase, Roche Diagnostic/Boehringer Mannheim Corp., Indianapolis, IN, USA), collected, and replated in 100 mm2 gelatin-coated tissue culture dishes. Some of the cultures were kept from fusing by the addition of 2.0 mM ethylene glycol-bis (2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA; Sigma-Aldrich, St. Louis, MO, USA) to the culture medium. To induce fusion, the medium was replaced with the same medium without EGTA. To remove dividing cells, mainly fibroblasts, 2 mM cytosine arabinoside (Sigma-Aldrich) was added into the culture medium. Proliferating C2C12 myoblasts were grown in 100 mm2 tissue culture dishes in the presence of Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal calf serum , 2 mM glutamine, 1% penicillin streptomycin, and 1% Fungisol® at saturated humidity 37 °C and 5% CO2, until 80% confluence was attained. To induce C2C12 cells to differentiate, confluent myoblast culture medium was changed to differentiation medium (DMEM supplemented with 2% horse serum) and the other steps are the same conditions and methods with primary cell culture preparations [10,16]. 2.2. Microscopy The cultures were observed with a phase-contrast microscope at various time points after replating. For methylation studies, primary
cultured myoblast cells were seeded on 100 mm2 gelatin-coated tissue culture dishes. The muscle cells were cultured similarly to the cultures for morphologic observation. Proteins were extracted from muscle cultures before fusion (attachment and alignment states), during blocked fusion (fusion arrest with EGTA), during early fusion (fusion burst), and after fusion (myofiber). 2.3. Protein extraction and Western blot analysis The medium was removed at appropriate time points, and primary cells and C2C12 cells were washed three times with phosphatebuffered saline (PBS) and harvested by scraping them off the culture dishes with a rubber policeman. The cells were collected by centrifugation (1000 × g, 5 min). Cells were lysed by sonication in RIPA buffer (100 mM Tris–HCl [pH 7.6], 50 mM NaCl, 0.5% NP-40, 1% Triton X-100, 5 mM EDTA (ethylenediaminetetraacetic acid), and protease inhibitors) without SDS. Lysates were clarified by high-speed centrifugation (100 000 × g, 60 min) and quantified by using a Bradford reagent. A 50 μg of protein was used for each SDS-PAGE analysis. SDS-PAGE was performed according to the method of Laemmli [17] with 12.5% acrylamide for running the gels and 5% for stacking the gels in the presence of 0.1% SDS and in the absence of 2-mercaptoethanol [18]. Proteins in the SDS-PAGE gel were electrophoretically transferred onto a PVDF membrane in 25 mM Tris–HCl buffer (pH 8.3) containing 192 mM glycine and 10% methanol at 4 °C at a constant current of first 60 V for 30 min and then 120 V for 40 min. The membrane was dried in air for 30 min, and then incubated with primary antibodies for 16 h at 4 °C. After incubation, the membrane was washed three times with PBS, and the secondary antibodies were applied. The bound antibodies were visualized by an ECL reagent (GE Healthcare Life Sciences, Uppsala, Sweden), and the membrane was then exposed to X-ray film. Western blotting was performed with antibodies against the following proteins: PRMT1 (Upstate Biotechnology, Lake Placid, NY, USA), PRMT4 (CARM1; Upstate Biotechnology), PRMT5 (Upstate Biotechnology), PRMT7 (Upstate Biotechnology), asymmetrically dimethylated arginine
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Fig. 1. Expression and catalytic activities of PRMTs during skeletal muscle differentiation. (A) (Left) Differential expression of PRMTs in cultured skeletal muscle cells. Western blot analyses show that PRMT1 and PRMT5 are continuously expressed before fusion (BF), at early stages of fusion (EF), and after fusion (AF). However, the immunoreactive signal from PRMT4 was very weak and that of PRMT7 was not detected at its expected size. (Right) Expression of PRMT1 and PRMT5 was quantified by densitometry, and actually, myoblast and myotube were little different in protein expression level. (B) Intracellular localization of PRMT1 and PRMT5 in cultured muscle cells. PRMT5 is present in the nuclei and cytoplasm of both myoblasts and myotubes. PRMT1 was observed both in the nuclei and cytoplasm of myoblasts, but mainly in the cytoplasm of myotubes. Insert images are more magnification cy3 images of myoblast and myotube to confirm whether the PRMTs were or were not in the nucleus. Scale bar is a 20 μm. (C) Catalytic activities of PRMT1 and PRMT5. Activity was determined by fluorographic analyses using hnRNP A1 and MBP as the methyl acceptor substrates for PRMT1 and PRMT5, respectively. Both enzyme activities were dramatically upregulated during EF and AF. The highest enzyme activity for PRMT1 was observed during EF and AF, and that for PRMT5 was observed during AF. For fusion blocking (FB), growing cells were kept from fusing by the addition of EGTA. CBB (coomassie brilliant blue) staining is a loading control.
(ASYM24; Upstate Biotechnology), symmetrically dimethylated arginine (SYM10; Upstate Biotechnology), lamin A/C (Sigma-Aldrich), and beta-actin (Sigma-Aldrich). For detection, secondary antibodies (anti-rabbit IgG HRP (Santa Cruz Biotechnology Inc., Santa Cruz, CA), anti-mouse IgG HRP (Santa Cruz Biotechnology Inc.)), and ECL chemiluminescent reagent (GE Healthcare Life Sciences) were used.
2.4. Immunoprecipitation Whole muscle proteins were lysed by sonication in RIPA buffer without SDS. The lamin A/C was monitored by separation with 12.5% SDS-PAGE followed by staining with a Novex® colloidal blue staining kit (Invitrogen). For the immunoprecipitation assay, identical
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2.6. Enzymatic methylation and fluorography
Fig. 2. Alteration of proteins asymmetrically arginine-dimethylated during skeletal muscle differentiation. Antibodies against asymmetric (Asym 24) and symmetric (Sym 10) dimethylated peptides were used for Western blot analysis. The pattern of asymmetric dimethylation changed during muscle differentiation, but the pattern of symmetric dimethylation did not.
amounts of myoblast proteins were incubated with 200 μL of antilamin A/C (Santa Cruz Biotechnology Inc.) for 1 h at 4 °C, while 20 μL of A/G PLUS-Agarose (Santa Cruz Biotechnology Inc.) was incubated with goat anti-mouse IgG for 30 min at 4 °C and rinsed twice with cold phosphate buffer. Then the two samples were mixed for 2 h at 4 °C on a rotator (15 rpm). The beads were sedimented by centrifugation (2000 × g/1 min), the supernatant was removed, and the pellet was rinsed twice with phosphate buffer. The bound proteins were eluted from the beads by boiling the samples in SDS loading buffer (1 M Tris– HCl [pH 6.8], 10% SDS, 50% glycerol, 5% 2-mercaptoethanol, and 1% bromophenol blue). Proteins were separated by SDS-PAGE (12.5%) and stained overnight with GelCode Blue Stain reagent (Thermo Scientific Pierce Protein Research Products, Rockford, IL, USA).
2.5. Immunofluorescence analysis For fluorescent immunohistochemical analysis, myoblast cells were grown on 13 mm cover slips held in 35 mm culture dishes. Cells were washed once with PBS, fixed with 4% paraformaldehyde in PBS (pH 7.4), and blocked with PBS containing 5% normal goat serum and 0.2% Triton X-100 for 30 min. They were then incubated with antibodies against myo-D (Santa Cruz Biotechnology Inc.), myogenin (Santa Cruz Biotechnology Inc.), PRMT1, PRMT5, skeletal actin muscle (Neomarker, Fremont, CA), or lamin A/C antibodies, respectively, for 1 h at room temperature. After washes with PBS, the cells were incubated with Alexa Fluor® 488 goat anti-mouse IgG (1:500, Invitrogen) or Cy3 goat anti-mouse IgG (1:500, Invitrogen) for 30 min at room temperature and washed again. The coverslips were mounted and observed on an Axiovert 200 fluorescent microscope equipped with a 63× oil-immersion objective (Zeiss, Göttingen, Germany).
Protein was extracted as described above. The harvested pellet was resuspended in buffer containing 5 mM sodium phosphate (pH 7.4) containing 5 mM EDTA, 0.27 M sucrose, and protease inhibitor cocktail (Roche Applied Science, Penzberg, Germany), and disrupted by homogenization [19]. The homogenate was then centrifuged at 100,000 × g for 1 h at 4 °C to remove membrane fractions. The supernatant was filtered through VIVASPIN 10,000 mw (Vivaproducts Inc., MA, USA) to remove the endogenous transmethylation inhibitors and the concentrated fraction was used for methylation studies. Protein contents in the supernatant were measured by the Bradford method [20] with an ELISA reader (UniScan®; LabSystem, Helsinki, Finland). Fluorography was performed according to Lim et al. [19,21]. Methylation reactions were carried out with 50 μg cell extract and Ado [methyl-3H]Met (2.5–5.5 μCi, GE Health Care Life Sciences, NJ, USA) in 0.1 M phosphate buffer (pH 7.4) [19]. The reaction mixture without the methyl donor was pre-incubated for 5 min at 37 °C, and then the reaction was initiated by the addition of Ado [methyl-3H]Met and methyltransferase substrates (i.e., hnRNP A1 or MBP) at 37 °C. Recombinant HeLa hnRNP A1 was overexpressed and purified from E. coli and MBP from calf brain was purified. After 30 or 60 min of incubation as indicated, the reactions were stopped by the addition of 5× SDS-PAGE sample buffer, followed by heating at 100 °C for 10 min. Then, the protein was subjected to SDS-PAGE. In order to visualize methylated proteins, the gel was stained with Coomassie Brilliant Blue and developed for fluorography by being soaked in an amplifying solution (NAMP-100, GE Health Care Life Sciences, Uppsala, Sweden), dried, and exposed to diagnostic X-ray film (Hyperfilm MP, GE Health Care Life Sciences) at −70 to −80 °C for 7 days [19,21]. 2.7. Two-dimensional electrophoresis For the isoelectric focusing (IEF), 100 μg of protein was loaded onto 7 cm immobilized pH gradient (IPG) strips (GE Health Care Life Sciences) with a 3–10 nonlinear gradient. The electrophoresis was carried out for about 12 h (200 V 1 h linear, 500 V 1 h linear, 1000 V 1 h linear, 8000 V 1 h linear, 8000 V, for a total of 40 000 Vh rapid). Following IEF, the IPG strips were equilibrated for 10 min in equilibration buffer (50 mM Tris–HCl buffer, pH 8.8, containing 6 M urea, 30% [v/v] glycerol, 2% [w/v] SDS, a few grains of bromophenol blue, 0.2% w/v DTT). The strips were then re-equilibrated for 10 min in the same buffer containing 1.5% (w/v) iodoacetamide. Molecular weight was estimated from the position on a 12.5% SDS polyacrylamide gel (Bio-Rad, Hercules, CA, USA). Coomassie stain solution was applied for 3–12 h, and the gel was washed with distilled water. For 2-dimensional electrophoresis (2-DE) Western blot analysis, a total of six 2-DE gels were prepared by using whole proteome extracted before fusion (BF) and after fusion (AF) (three gels for BF and three gels for AF, respectively). A pair of 2-DE gels (BF and AF) were electrophoretically transferred onto a PVDF membrane and incubated with ASYM 24 primary antibody (Fig. 3B, Asym 24, BF and AF). Another pair of 2-DE gels was Western blotted with SYM 10 primary antibody (Fig. 3B, Sym 10, BF and AF). After incubation, the membranes were washed and the secondary antibody was applied. The immunoreactive protein spots were visualized by an ECL reagent, and matched with same spots on the last pair of 2-DE gels (BF and AF) by comparing its molecular weight and pI point as well as location of neighbor spots. The protein spots that displayed significant difference between BF and AF were picked up for MALDI-TOF analysis. Among 33 spots, 13 spots indicated in Fig. 3B were identified with significant Mascot score (Fig. 3C). Silver staining was done according to the method recommended by the manufacture. Briefly, for silver stain, 100 μg of protein mixed with rehydration buffer (8 M urea, 2% CHAPS, a few grains of
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bromophenol blue) to a final volume of 350 μl was absorbed onto 18-cm immobilized pH gradient (IPG) strips (GE Health Care Life Sciences) with a 3–10 nonlinear gradient. After rehydration, the electrophoresis was carried out for about 20 h, (200 V 1 h linear, 500 V 1 h linear, 1000 V 1 h linear, 8000 V 1 h linear, 8000 V, for a total of 40,000 Vh rapid). Following IEF, the IPG strips were equilibrated for 15 min in equilibration buffer (50 mM Tris–HCl, pH 8.8 buffer, containing 6 M urea, 30% (v/v) glycerol, 2% (w/v) SDS, a few grains of bromophenol blue, 0.2% (w/v) DTT). Strips were then reequilibrated for 10 min in the same buffer containing 1.5% w/v iodoacetamide. Molecular weight separation was achieved on 12.5% SDS polyacrylamide gel using Protean II xi cell gel SDS-PAGE system (Bio-Rad) 2 W per gel for 1 h and 16 W per gel for 3 h. Then, proteins in the gel were fixed with 50% (v/v) methanol, 12% (w/v) acetic acid, and 0.05% (w/v) formaldehyde overnight and washed three times with 50% (v/v) ethyl alcohol for 20 min. The sensitizing solution (0.02% (w/v) sodium thiosulfate) was applied for 1 min to increase sensitivity, and washed three times with distilled water. Proteins were stained with 0.1% silver nitrate staining solution and 0.075% (w/v) formaldehyde for 20–30 min, developed with 6% (w/v) sodium carbonate, 0.05% (v/v) formaldehyde to visualize the proteins. The reaction was stopped by applying stopping solution (50% methanol, 12% (w/v) acetic acid). Reagents used in 2-dimensional electrophoresis (2-DE) included acrylamide, bis-acrylamide, TEMED, Tris base, glycine, SDS, APS, DTT, CHAPS, urea, thiourea, and iodoacetamide, all purchased from Sigma-Aldrich. Immobiline dry strips, IPG buffer, and
IPG cover mineral oil were purchased GE Health Care Life Sciences and colloidal blue staining kit was purchased from Invitrogen. For silver stain, silver nitrate and sodium thiosulfate were purchased from Sigma-Aldrich and methanol, acetic acid, formaldehyde and ethyl alcohol were purchased from Dae Jung Chemical Co., Ltd. (Gyeonggi, Korea). 2.8. MALDI-TOF analysis A total of 33 significant protein spots were excised from the gel and destained with distilled water. Upon reduction of disulfide bonds of the protein with tris (2-carboxyethylphosphine) hydrochloride (Thermo Fisher Scientific Inc.), cysteines were alkylated with iodoacetamide (Sigma-Aldrich). Digestion with modified trypsin (400 ng, Promega, WI, USA) was carried out overnight at 37 °C in a buffer containing 100 mM ammonium bicarbonate buffer, pH 8.3, and 4 mM calcium chloride. α-Cyano 4-hydroxycinnamic acid (20 mg; Bruker Daltonics, Bremen, Germany) was dissolved in 1 ml acetone: ethanol (1:2, v/v), and 0.5 μl of the matrix solution was mixed with an equivalent volume of sample. Analysis was performed using an Ultraflex TOF/TOF system (Bruker Daltonics). The Ultraflex TOF/TOF system was operated in positive ion reflect mode. Each spectrum was the cumulative average of 250–450 laser shots. Mass spectra were first calibrated in the closed external mode using the peptide calibration standard II (Bruker Daltonics), sometimes using the internal statistical mode to achieve maximum calibration mass accuracy, and analyzed
Fig. 3. Identification of proteins asymmetrically arginine-dimethylated during skeletal muscle differentiation. (A) Typical pattern of 2-DE images of whole protein extracted before fusion (BF) and after fusion (AF). The separated protein was stained with a silver nitrate. (B) Western blot analysis of the 2-DE gels. Thirteen protein spots indicated on the 2-DE gels were immunoreactive for dimethylated arginine (Asym 24 and Sym 10) and were selected for mass analysis. (C) Results of MALDI-TOF/TOF analysis of the selected protein spots shown in (B).
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Fig. 3 (continued).
with FlexAnalysis software, version 2.4 (Bruker Daltonics). Peptide mass peaks from each spectrum were submitted to a Mascot peptide mass fingerprinting search (www.matrixscience.com) for analysis with BioTools software, version 3.0 (Bruker Daltonics). The search included peaks with a signal-to-noise ratio greater than 3. The peak list for each sample was used to query the non-redundant Mass Spectrometry Protein Sequence Database for protein identification. Standard settings included the following: enzyme, trypsin; missed cleavage, one; fixed modifications, carbamidomethylation on cysteine; variable modifications, oxidized methionine; protein mass, blank; mass values, MH+ (monoisotopic); mass tolerance, varied between 75 and 100 ppm. 2.9. LC-MS/MS analysis Each immunoprecipitated sample was carried out reduction, alkylation and trypsin digestion. These tryptic peptides separately analyzed using an LTQ-FT mass spectrometer (Thermo Electron, San Jose, CA, USA) and the nanoACQUITY UPLC (NanoA, Waters, Milford,
MA, USA) system. The nanoACQUITY UPLC system was equipped with a capillary column (75 μm ID × 360 μm OD × 75 cm length) built in house and packed with fused-silica capillary with C18-bonded particles (3 μm diameter, 300 Å pore size, Phenomenex, Torrance, CA, USA) [22], also manufactured in house. After the sample injection, each peptide was loaded onto the online SPE column for 6 min in solvent A (0.1% formic acid in distilled water), and peptide separation was achieved by using a linear gradient from 10 to 50% solvent B (0.1% formic acid in acetonitrile) for 60 min at a flow rate of 350 nL/min. The mass spectrometer was operated in a data-dependent tandem MS mode where a full-scan MS experiment (m/z 450–1800) was followed by MS/MS experiments on the three most abundant ions in the precursor MS scan. The normalized energy for peptide dissociation was set to 35% and the dynamic exclusion option was incorporated to prevent reacquisition of MS/MS spectra of the same peptides for 30 s. The resultant MS and MS/MS data from the mass spectrometer were analyzed by using BioworksTM software (v. 3.2, Thermo Electron) against a database that was constructed by combining the IPI rat database (Rat, 3.68, ftp://ftp.ebi.ac.uk) and a common contaminant
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database (http://www.ncbi.nih.gov). All tandem mass spectrometric data (i.e., DTA files) were extracted using ExtractMSn (Version 4.0) of the BioworksTM software. Before a database search was performed, the MS/MS data were filtered and refined through the PE-MMR (postexperiment monoisotopic mass filtering and refinement) [23] program (http://omics.pnl.gov/software/PEMMR.php). The database search was
performed using a m/z tolerance of 10 ppm, and the fragment ion m/z tolerance was ±0.5 Da. The modification options were used for the oxidation of methionine (15.994920 Da), iodoacetamide derivative of alkylation (57.021464 Da), Arg-monomethylation (14.015650 Da), and Arg-dimethylation (28.031300 Da). The search results were filtered using an estimated FP (false positive) rate. The FP rate of peptide
Fig. 4. Methylation of lamin A/C in skeletal muscle. (A) MALDI-TOF/TOF analysis of proteins immunoprecipitated with an anti-lamin A/C antibody. SDS-PAGE was performed using the immunoprecipitate, and the indicated protein bands, which were expected to be lamin A/C, were sliced out of the gel for MALDI-TOF/TOF analysis, and MALDI-TOF/TOF-MS result of lamin A/C identification. (B) Methylation of lamin A/C. Lamin protein expression in the whole cell lysate was checked with an anti-lamin A/C antibody (left), and Western blot analysis of the immunoprecipitate of anti-lamin A/C was performed with antibodies against dimethylated arginine (ASYM 24 and Sym 10; middle and right panels, respectively).
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antibodies against PRMT1 and PRMT4 as type I PRMTs, and PRMT5 and PRMT7 as type II PRMTs. PRMT1 and PRMT5 were continuously expressed in cultured skeletal muscle cells before fusion, at early stages of fusion, and after fusion, but the immunoreactive signal from PRMT4 was very weak and no signal was detected for PRMT7 (Fig. 1A). 3.2. Change of PRMT1 and PRMT5 catalytic activities during myoblast fusion
3.1. Expression of PRMTs during skeletal muscle differentiation
We localized PRMT1 and PRMT5 within cultured muscle cells by using immunofluorescent staining (Fig. 1B). To identify the fusion status, myoblasts and myotubes were identified with antibodies against myo-D and myogenin, respectively [24]. PRMT5 was present in the nuclei and cytoplasm of both myoblasts and myotubes, and PRMT1 was observed in both the nuclei and cytoplasm of myoblasts, but mainly in the cytoplasm of myotubes (Fig. 1B). Because both PRMT1 and PRMT5 were expressed mainly in the cytoplasm, we used whole cytosolic proteins obtained from cultured skeletal muscle cells to determine the enzyme activities. The enzyme activities were measured by fluorographic analyses using heterogeneous nuclear ribonucleoprotein (hnRNP) A1 and MBP as the methyl acceptor substrates for PRMT1 and PRMT5, respectively (Fig. 1C). Both PRMT1 and PRMT5 activities were dramatically upregulated during early fusion and after fusion. The highest enzyme activity for PRMT1 was observed during myoblast fusion (early fusion and after fusion) and that for PRMT5 was during the postfusion stage (Fig. 1C).
To investigate which PRMTs are responsible for arginine methylation in muscle differentiation, we performed Western blotting with
3.3. Alteration of asymmetric arginine-dimethylated proteins during skeletal muscle differentiation
Fig. 4 (continued).
assignment was estimated through a composite target-decoy database search. The values of XCorr and the ΔCn threshold for the 1% FP rate were used to obtain peptide IDs [23]. 3. Results
To clarify whether arginine methylation is involved in muscle differentiation, especially during fusion, we investigated how arginine methylation changed during differentiation using whole proteins extracted from primary cultured rat muscle cells. When antibodies against asymmetric (Asym 24) and symmetric (Sym 10) dimethylated peptides were used for Western blot analysis [29], various proteins with different molecular weight were found to be arginine-methylated (Fig. 2). Interestingly, proteins with asymmetric dimethylation were altered during the muscle fusion, whereas symmetric dimethylation was not affected by different stages of muscle differentiation (Fig. 2). 3.4. Identification of possible substrate proteins of PRMTs after myoblast fusion To identify proteins arginine-methylated by PRMTs whose activity increased during and after myoblast fusion, we used a 2-DE immunoproteomics approach specific for aDMA (Asym 24) and sDMA (Sym 10) to detect dimethylated arginine [29]. Muscle protein extracts before and after fusion were separated by standard 2-DE (Fig. 3A) and transferred to PVDF membranes for Western blotting of arginine-methylated proteins for aDMA (Asym 24) and sDMA (Sym 10) (Fig. 3B). Among the detected protein, significant protein spots with immunoreactive signals for arginine methylation were identified to 13 proteins (Fig. 3B and C) by MALDITOF mass analysis. The candidate proteins asymmetrically dimethylated during after fusion included ATP synthase alpha chain (Q03265), hnRNP A3 (Q8BG05) and A1 (P49312), glyceraldehyde 3-phosphate dehydrogenase (P16858), lamin A (P48678), and lamin C and C2 (P11516) (Fig. 3C). 3.5. Methylated lamin A/C in skeletal muscle Fig. 5. Arginine-methylation site in lamin C2. (A) LC-MS/MS analysis of lamin proteins immunoprecipitated with anti-lamin A/C. The proteins removed from the SDS-PAGE gel (Fig. 4A) were clearly identified as a lamin C2. (B) Dimethylation of arginine 209. Among the peptides matched with lamin C2, dimethylation of arginine 209 was confirmed by peptide sequencing of LRDLEDSLAR.
Among the asymmetrically methylated proteins we identified (Fig. 3C), we further validated the methylation of lamin A/C because it is involved in muscle function [25–27]. To confirm that lamin A/C is
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arginine-methylated during myoblast fusion, we extracted total protein from cells at each stage of myogenesis and immunoprecipitated them using an anti-lamin A/C antibody. The immunoprecipitated proteins were identified by MALDI-TOF mass analysis (Fig. 4A, indicated in the enlarged image), and the result was confirmed by Western blot analysis employing anti-lamin A/C, Asy 24, and Sym 10 antibodies (Fig. 4B). To identify the arginine methylation site in lamin, we analyzed the lamin proteins immunoprecipitated with anti-lamin A/C by using LCMS/MS. Proteins extracted from the immunoprecipitate were clearly identified as a lamin C2 (Fig. 5A). Among the peptides that matched to lamin C2, we confirmed the dimethylation of Arg 209 by peptide sequencing of LRDLEDSLAR (Fig. 5B). 4. Discussion Alterations in the amount, posttranslational modifications, or activities of certain proteins during muscle differentiation implicate these proteins as having key roles in muscle fusion [10]. Therefore, the identification of proteins differentially modified by PRMTs is a critical step for understanding the molecular mechanism(s) of muscle fusion. PRMTs are known to be highly expressed in the cells wrapping up protein synthesis [9]. Because myoblasts are actively growing cells and need a great amount of proteins for fusion and after-fusion growth, we expected to find high expression of major PRMTs. Among the PRMTs we evaluated during muscle fusion, PRMT1 and PRMT5 were dominantly expressed without regard to the muscle fusion process (Fig. 1A). Though a role for PRMT4 in terminal differentiation of C2C12 muscle cells has been suggested [10], we detected very little PRMT4 in primary rat muscle culture (Fig. 1A). We also note that the expression of PRMT1 and PRMT5 was explicitly different in C2C12 cells and primary muscle cells (Supplementary Fig. 1). Both PRMT5 and PRMT7 belong to type II and symmetrically dimethylate Sm proteins in vitro [28,29]. They are involved in the biogenesis of small nuclear ribonucleoproteins (snRNPs). Nuclear PRMT5 is well known as a regulator of transcriptional elongation [28], but the role of PRMT7, a recent addition to the PRMT family, is not yet known [29]. Our present results indicate that both type I (PRMT1) and II (PRMT5) PRMTs are expressed during fusion. It is not, however, clear why PRMT1 and PRMT5 were selectively expressed. Kwak et al. [30] reported that either PRMT1 or PRMT5 can methylate SPT5, a component of important transcriptional elongational factors, to decrease association of SPT5 with RNA polymerase II; however, the role of SPT5 in muscle fusion is not known. Interestingly, we found dramatic upregulation of enzyme activity of PRMT1 during early fusion and after fusion, and of PRMT5 after fusion (Fig. 1C), suggesting that relative expression of the enzymes may be important for muscle fusion, and PRMTs may have different roles in myoblast fusion and maintenance. Methylation of HnRNP A1 and MBP had previously been employed to check the activity of PRMT1 and PRMT5, respectively [8,19,31], as hnRNP A1 and MBP are ones of the well known substrates for PRMT1 and PRMT5, respectively. From fluorography experiment (Fig. 1C), we were able to confirm that the activities of PRMTs were indeed increased. Our objective then was to find novel substrates for the PRMTs in primary culture under the increased activities. Our finding that PRMT1 is localized to both the nuclei and cytoplasm of myoblasts (Fig. 1B) is in good agreement with a previous report, in which a photobleaching experiment showed that PRMT1 is highly mobile both in the cytoplasm and the nucleus [32]. This fact may be evidence that PRMT1 moves from the nucleus to the cytoplasm before and after fusion. Though the best-known substrates for PRMT1 are present in the nucleus, the enzyme itself is not preferentially located in the nucleus [33]; in fact, PRMT1 is distributed quite variably in both the cytoplasm and nucleus [34]. Considering that treatment with a PRMT1 inhibitor leads to a significant nuclear accumulation of PRMT1 [32], the preferential localization of PRMT1 in
the cytoplasm may be necessary for increasing methylation during myoblast fusion. PRMT5 was observed in the nuclei and cytoplasm of both myoblasts and myotubes (Fig. 1B), but the significance of this subcellular localization is not clear. We identified 13 proteins with immunoreactive signals against aDMA and sDMA during muscle fusion (Figs. 2 and 3). Thirteen proteins, including hnRNPs, the best-known substrates for asymmetrical arginine dimethylation [35] and lamin A/C, were identified by MALDI-TOF analysis. Lamins are intermediate filaments that form the essential structural meshwork of the nuclear lamina of all mammalian cells. The nuclear lamina is located beneath the inner nuclear membrane and maintains the shape and mechanical properties of the nuclei. The nuclear lamina had been suggested as a molecular docking site for peripheral heterochromatin. Lamin A and C are the major products of the LMNA gene, and mutations in LMNA have been linked to certain human genetic disorders, broadly termed laminopathies [25]. Laminopathies affect the development of striated muscle, adipose tissue, peripheral nerve, and skeletal muscle [27]. For example, recent studies have demonstrated the important role of lamin A/C in Emery–Dreifuss muscular dystrophy and limb-girdle muscular dystrophy [25,26]. The cultured cells of lamin A/C knockout mice show impaired differentiation kinetics and reduced differentiation potential [25]. Furthermore, differentiation of muscle in Emdnull mice is delayed, and this delay is associated with disordered transcriptional pathways regulated by the retinoblastoma (Rb1) and myo-D genes [25,26]. Our observation that PRMT1 is localized to myoblast nuclei suggests that lamins are dimethylated before the completion of myoblast fusion. This finding indicates possible involvement of lamin methylation in myoblast fusion, and the data shown in Figs. 4 and 5 clearly suggest that lamin is a substrate for PRMTs. Arginine methylation occurs at arginines within glycine and arginine-rich (GAR) motifs [8,35]. In the lamin C2 sequences, there are 4 GAR motifs. Unfortunately, we did not identify methylated arginine at these 4 sites (Arg 7, 287, 289 and 327; Fig. 5). Among the peptides matched with lamin C2, we did confirm dimethylation of Arg 209 by peptide sequencing of LRDLEDSLAR (Fig. 5B). Ultimately, this study revealed the relationship of lamin, PRMT, and muscle differentiation, but the specific roles of lamin and arginine methylation were not actually disclosed; however, the role of this specific modification in muscle differentiation has to be studied further. By extension, we will have to study whether modifying PRMT could be a treatment for Emery–Dreifuss muscular dystrophy and limb-girdle muscular dystrophy. In summary, PRMTs showed changes in their catalytic activities during myoblast fusion of primary muscle cells. The highest activities of PRMT1 and PRMT5 were observed during and after myoblast fusion, respectively, suggesting that each PRMT has a different role in myoblast fusion. Among the possible substrates, mass spectrometric evidences confirmed that lamin C2 was arginine-methylated during muscle fusion, but its functional significance in muscle differentiation is still not clear. Supplementary materials related to this article can be found online at doi:10.1016/j.bbapap.2010.11.006. Acknowledgments We thank Steffen Pahlich, Dr. Heinz Gehring, and Dr. Peter Gehrig (Department of Biochemistry University of Zurich and Functional Genomic Center Zurich (FGCZ) Switzerland) for allowing us to use the LC-MALDI-TOF/TOF-MS, and Woon Ki Paik, Sangduk Kim, Yong-Chul Lim, and Young-Ho Kwon (Graduate School of Biomedical Sciences, Korea University College of Medicine) for helping with the interpretation of the data and experiment. This work was supported in part by 21C Frontier Functional Proteomics Project (grant no. FPR08A1-010), Converging Research Center for Mass Spectrometric Diagnosis (2010K001300) and Priority Research Centers Program through the
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