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Myofibrillar Z-discs Are a Protein Phosphorylation Hot Spot with Protein Kinase C (PKCα) Modulating Protein Dynamics

2016; Elsevier BV; Volume: 16; Issue: 3 Linguagem: Inglês

10.1074/mcp.m116.065425

1535-9484

Lena Reimann, Heike Wiese, Yvonne Leber, Anja N. Schwäble, Anna L. Fricke, Anne Rohland, Bettina L. Knapp, Christian D. Peikert, Friedel Drepper, Peter F. M. van der Ven, Gerald Radziwill, Dieter O. Fürst, Bettina Warscheid,

Calpain Protease Function and Regulation

The Z-disc is a protein-rich structure critically important for the development and integrity of myofibrils, which are the contractile organelles of cross-striated muscle cells. We here used mouse C2C12 myoblast, which were differentiated into myotubes, followed by electrical pulse stimulation (EPS) to generate contracting myotubes comprising mature Z-discs. Using a quantitative proteomics approach, we found significant changes in the relative abundance of 387 proteins in myoblasts versus differentiated myotubes, reflecting the drastic phenotypic conversion of these cells during myogenesis. Interestingly, EPS of differentiated myotubes to induce Z-disc assembly and maturation resulted in increased levels of proteins involved in ATP synthesis, presumably to fulfill the higher energy demand of contracting myotubes. Because an important role of the Z-disc for signal integration and transduction was recently suggested, its precise phosphorylation landscape further warranted in-depth analysis. We therefore established, by global phosphoproteomics of EPS-treated contracting myotubes, a comprehensive site-resolved protein phosphorylation map of the Z-disc and found that it is a phosphorylation hotspot in skeletal myocytes, underscoring its functions in signaling and disease-related processes. In an illustrative fashion, we analyzed the actin-binding multiadaptor protein filamin C (FLNc), which is essential for Z-disc assembly and maintenance, and found that PKCα phosphorylation at distinct serine residues in its hinge 2 region prevents its cleavage at an adjacent tyrosine residue by calpain 1. Fluorescence recovery after photobleaching experiments indicated that this phosphorylation modulates FLNc dynamics. Moreover, FLNc lacking the cleaved Ig-like domain 24 exhibited remarkably fast kinetics and exceedingly high mobility. Our data set provides research community resource for further identification of kinase-mediated changes in myofibrillar protein interactions, kinetics, and mobility that will greatly advance our understanding of Z-disc dynamics and signaling. The Z-disc is a protein-rich structure critically important for the development and integrity of myofibrils, which are the contractile organelles of cross-striated muscle cells. We here used mouse C2C12 myoblast, which were differentiated into myotubes, followed by electrical pulse stimulation (EPS) to generate contracting myotubes comprising mature Z-discs. Using a quantitative proteomics approach, we found significant changes in the relative abundance of 387 proteins in myoblasts versus differentiated myotubes, reflecting the drastic phenotypic conversion of these cells during myogenesis. Interestingly, EPS of differentiated myotubes to induce Z-disc assembly and maturation resulted in increased levels of proteins involved in ATP synthesis, presumably to fulfill the higher energy demand of contracting myotubes. Because an important role of the Z-disc for signal integration and transduction was recently suggested, its precise phosphorylation landscape further warranted in-depth analysis. We therefore established, by global phosphoproteomics of EPS-treated contracting myotubes, a comprehensive site-resolved protein phosphorylation map of the Z-disc and found that it is a phosphorylation hotspot in skeletal myocytes, underscoring its functions in signaling and disease-related processes. In an illustrative fashion, we analyzed the actin-binding multiadaptor protein filamin C (FLNc), which is essential for Z-disc assembly and maintenance, and found that PKCα phosphorylation at distinct serine residues in its hinge 2 region prevents its cleavage at an adjacent tyrosine residue by calpain 1. Fluorescence recovery after photobleaching experiments indicated that this phosphorylation modulates FLNc dynamics. Moreover, FLNc lacking the cleaved Ig-like domain 24 exhibited remarkably fast kinetics and exceedingly high mobility. Our data set provides research community resource for further identification of kinase-mediated changes in myofibrillar protein interactions, kinetics, and mobility that will greatly advance our understanding of Z-disc dynamics and signaling. The highly regular organization of myofibrils, the contractile organelles of cross-striated muscle cells, gives rise to the typical banding pattern of skeletal and cardiac muscle fibers. Myofibrils are mainly composed of an almost crystalline array of thin and thick filaments based on actin and myosin, respectively. The repeating contractile units of myofibrils are the sarcomeres, which are flanked by Z-discs. The latter protein-rich structures provide an essential structural framework by tethering actin filaments at their barbed ends, cross-linking them by antiparallel dimers of α-actinin and linking them to the giant protein titin at its amino terminus. Z-discs not only define the lateral boundaries of adjacent sarcomeres, but also help to connect myofibrils to each other, e.g. via intermediate filaments. In addition, they are involved in linking the contractile apparatus to the sarcolemma and the extracellular matrix via large, membrane-associated protein complexes, the costameres. The function of the Z-disc is not only limited to force transmission, but it is also an important hub for signal transduction events. To fulfil its dual role, Z-discs have to be dynamic and at the same time have to encompass numerous structural proteins. Over the last years, the number of proteins with functions in mechanosensing and signal transduction identified to localize at least temporarily to the Z-disc has steadily increased (reviewed in (1.Luther P.K. The vertebrate muscle Z-disc: sarcomere anchor for structure and signalling.J. Muscle Res. Cell Motil. 2009; 30: 171-185Crossref PubMed Scopus (173) Google Scholar, 2.Frank D. Frey N. Cardiac Z-disc Signaling Network.J. Biol. Chem. 2011; 286: 9897-9904Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, 3.Knöll R. Buyandelger B. Lab M. The sarcomeric Z-disc and Z-discopathies.J. Biomed. Biotechnol. 2011; 2011: 569-628Crossref Scopus (91) Google Scholar)). To date, over 100 gene products are linked to the term "Z-disc" in the human or mouse NCBI gene database (http://www.ncbi.nlm.nih.gov/gene/). However, its precise protein inventory and phosphorylation landscape have not been coherently analyzed. Numerous signaling proteins such as protein kinase C (PKC) 1The abbreviations used are: PKC, protein kinase C; ABD, actin-binding domain; AGC, automatic gain control; BAG, Bcl2-associated athanogene; BP, biological process; CC, cellular component; D, domain; ENAH, enabled homolog; EPS, electrical pulse stimulation; ETD, electron transfer dissociation; FA, formic acid; FDR, false discovery rate; FLNc, filamin C; FRAP, fluorescence recovery after photobleaching; GO, gene ontology; HCD, higher-energy collisional dissociation; HEK293, human embryonic kidney 293; hpH-RP, high pH reversed phase chromatography; HRP, horseradish peroxidase; HSPB1, heat shock protein beta-1; Ig, immunoglobulin; IMMs, immortalized mouse skeletal myoblasts; IPTG, isopropyl β-d-1-thiogalactopyranoside; LDB3, LIM domain binding 3; LIT, linear ion trap; MF, molecular function; MSA, multi-stage activation; NL, neutral loss; PDZ, post synaptic density protein, Drosophila disc large tumor suppressor, and; zonula occludens-1 protein; PEI, polyethylenimine; PMA, phorbol-12-myristat-13-acetat; PPxY, proline-proline-x-tyrosine; PxxP, proline-rich; ROI, regions of interest; SCX, strong cation exchange; SD, standard deviation; SEM, standard error of the mean; SIM, single ion monitoring; SYNPO, synaptopodin; SYNPO2, myopodin; SYNPO2L, tritopodin (CHAP; synaptopodin 2-like); TiO2, titanium dioxide; VASP, vasodilator-stimulated phosphoprotein; WH1, WASP-homology/EVH1, Ena/VASP homology 1; WT, wildtype; XIRP1, Xin actin-binding repeat containing protein 1. 1The abbreviations used are: PKC, protein kinase C; ABD, actin-binding domain; AGC, automatic gain control; BAG, Bcl2-associated athanogene; BP, biological process; CC, cellular component; D, domain; ENAH, enabled homolog; EPS, electrical pulse stimulation; ETD, electron transfer dissociation; FA, formic acid; FDR, false discovery rate; FLNc, filamin C; FRAP, fluorescence recovery after photobleaching; GO, gene ontology; HCD, higher-energy collisional dissociation; HEK293, human embryonic kidney 293; hpH-RP, high pH reversed phase chromatography; HRP, horseradish peroxidase; HSPB1, heat shock protein beta-1; Ig, immunoglobulin; IMMs, immortalized mouse skeletal myoblasts; IPTG, isopropyl β-d-1-thiogalactopyranoside; LDB3, LIM domain binding 3; LIT, linear ion trap; MF, molecular function; MSA, multi-stage activation; NL, neutral loss; PDZ, post synaptic density protein, Drosophila disc large tumor suppressor, and; zonula occludens-1 protein; PEI, polyethylenimine; PMA, phorbol-12-myristat-13-acetat; PPxY, proline-proline-x-tyrosine; PxxP, proline-rich; ROI, regions of interest; SCX, strong cation exchange; SD, standard deviation; SEM, standard error of the mean; SIM, single ion monitoring; SYNPO, synaptopodin; SYNPO2, myopodin; SYNPO2L, tritopodin (CHAP; synaptopodin 2-like); TiO2, titanium dioxide; VASP, vasodilator-stimulated phosphoprotein; WH1, WASP-homology/EVH1, Ena/VASP homology 1; WT, wildtype; XIRP1, Xin actin-binding repeat containing protein 1. (4.Gu X. Bishop S.P. Increased protein kinase C and isozyme redistribution in pressure-overload cardiac hypertrophy in the rat.Circ. Res. 1994; 75: 926-931Crossref PubMed Scopus (158) Google Scholar) and the protein phosphatase calcineurin (5.Heineke J. Ruetten H. Willenbockel C. Gross S.C. Naguib M. Schaefer A. Kempf T. Hilfiker-Kleiner D. Caroni P. Kraft T. Kaiser R.A. Molkentin J.D. Drexler H. Wollert K.C. Attenuation of cardiac remodeling after myocardial infarction by muscle LIM protein-calcineurin signaling at the sarcomeric Z-disc.Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 1655-1660Crossref PubMed Scopus (123) Google Scholar) were shown to dynamically localize to the Z-disc. Notably, kinase- and phosphatase-mediated phosphorylation and dephosphorylation events may likely control the dynamic shuttling of proteins in and out of the Z-disc as recently revealed for myopodin (6.Faul C. Dhume A. Schecter A.D. Mundel P. Protein kinase A, Ca2+/calmodulin-dependent kinase II, and calcineurin regulate the intracellular trafficking of myopodin between the Z-disc and the nucleus of cardiac myocytes.Mol. Cell. Biol. 2007; 27: 8215-8227Crossref PubMed Scopus (61) Google Scholar), a protein interacting with F-actin, α-actinin, and filamin C (FLNc) (7.Linnemann A. van der Ven P.F. Vakeel P. Albinus B. Simonis D. Bendas G. Schenk J.A. Micheel B. Kley R.A. Fürst D.O. The sarcomeric Z-disc component myopodin is a multiadapter protein that interacts with filamin and alpha-actinin.Eur. J. Cell Biol. 2010; 89: 681-692Crossref PubMed Scopus (52) Google Scholar, 8.Linnemann A. Vakeel P. Bezerra E. Orfanos Z. Djinović-Carugo K. van der Ven P.F.M. Kirfel G. Fürst D.O. Myopodin is an F-actin bundling protein with multiple independent actin-binding regions.J. Muscle Res. Cell Motil. 2013; 34: 61-69Crossref PubMed Scopus (15) Google Scholar). The large cytoskeletal protein FLNc, in turn, constitutes an important hub in the Z-disc interactome with manifold binding partners such as myotilin (9.van der Ven P.F. Wiesner S. Salmikangas P. Auerbach D. Himmel M. Kempa S. Hayess K. Pacholsky D. Taivainen A. Schröder R. Carpén O. Fürst D.O. Indications for a novel muscular dystrophy pathway. gamma-filamin, the muscle-specific filamin isoform, interacts with myotilin.J. Cell Biol. 2000; 151: 235-248Crossref PubMed Scopus (165) Google Scholar), nebulette (10.Eulitz S. Sauer F. Pelissier M.C. Boisguerin P. Molt S. Schuld J. Orfanos Z. Kley R.A. Volkmer R. Wilmanns M. Kirfel G. van der Ven P.F. Fürst D.O. Identification of Xin-repeat proteins as novel ligands of the SH3 domains of nebulin and nebulette and analysis of their interaction during myofibril formation and remodeling.Mol. Biol. Cell. 2013; 24: 3215-3226Crossref PubMed Google Scholar), the Xin actin-binding repeat containing proteins Xin (11.van der Ven P.F. Ehler E. Vakeel P. Eulitz S. Schenk J.A. Milting H. Micheel B. Fürst D.O. Unusual splicing events result in distinct Xin isoforms that associate differentially with filamin c and Mena/VASP.Exp. Cell Res. 2006; 312: 2154-2167Crossref PubMed Scopus (68) Google Scholar) and XIRP2 (12.Kley R.A. Maerkens A. Leber Y. Theis V. Schreiner A. van der Ven P.F. Uszkoreit J. Stephan C. Eulitz S. Euler N. Kirschner J. Müller K. Meyer H.E. Tegenthoff M. Fürst D.O. Vorgerd M. Müller T. Marcus K. A combined laser microdissection and mass spectrometry approach reveals new disease relevant proteins accumulating in aggregates of filaminopathy patients.Mol. Cell. Proteomics. 2013; 12: 215-227Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar), and the calsarcins/myozenins/FATZ proteins (13.Faulkner G. Pallavicini A. Comelli A. Salamon M. Bortoletto G. Ievolella C. Trevisan S. Kojic S. Dalla Vecchia F. Laveder P. Valle G. Lanfranchi G. FATZ, a filamin-, actinin-, and telethonin-binding protein of the Z-disc of skeletal muscle.J. Biol. Chem. 2000; 275: 41234-41242Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar, 14.Frey N. Olson E.N. Calsarcin-3, a novel skeletal muscle-specific member of the calsarcin family, interacts with multiple Z-disc proteins.J. Biol. Chem. 2002; 277: 13998-14004Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, 15.Takada F. Vander Woude D.L. Tong H.Q. Thompson T.G. Watkins S.C. Kunkel L.M. Beggs A.H. Myozenin: An alpha -actinin- and gamma -filamin-binding protein of skeletal muscle Z lines.Proc. Natl. Acad. Sci. U.S.A. 2001; 98: 1595-1600PubMed Google Scholar). Distinct from its two other ubiquitously expressed family members FLNa and FLNb, FLNc is mainly expressed in cross-striated muscles (16.Maestrini E. Patrosso C. Mancini M. Rivella S. Rocchi M. Repetto M. Villa A. Frattini A. Zoppè M. Vezzoni P. Mapping of two genes encoding isoforms of the actin binding protein ABP-280, a dystrophin like protein, to Xq28 and to chromosome 7.Hum. Mol. Genet. 1993; 2: 761-766Crossref PubMed Scopus (69) Google Scholar). In healthy muscle, it predominantly localizes at Z-discs, whereas a minor portion is found beneath the sarcolemma in association with the dystrophin-associated glycoprotein complex (17.Thompson T.G. Chan Y.M. Hack A.A. Brosius M. Rajala M. Lidov H.G. McNally E.M. Watkins S. Kunkel L.M. Filamin 2 (FLN2): A muscle-specific sarcoglycan interacting protein.J. Cell Biol. 2000; 148: 115-126Crossref PubMed Scopus (235) Google Scholar). During myofibril development, FLNc assists in Z-disc assembly by acting as a molecular scaffold (18.van der Ven P.F. Obermann W.M. Lemke B. Gautel M. Weber K. Fürst D.O. Characterization of muscle filamin isoforms suggests a possible role of gamma-filamin/ABP-L in sarcomeric Z-disc formation.Cell Motil. Cytoskelet. 2000; 45: 149-162Crossref PubMed Scopus (123) Google Scholar). Mutations in its gene cause severe myopathies and cardiomyopathies (reviewed in (19.Fürst D.O. Goldfarb L.G. Kley R.A. Vorgerd M. Olivé M. van der Ven P.F. Filamin C-related myopathies: pathology and mechanisms.Acta Neuropathol. 2013; 125: 33-46Crossref PubMed Scopus (81) Google Scholar)). All filamin isoforms feature an aminoterminal actin-binding domain (ABD) and a rod of 24 immunoglobulin-like (Ig-like) domains. Flexibility is mainly provided by hinge regions between Ig-like domains 15 and 16 (hinge 1) and 23 and 24 (hinge 2). Depending on cell type and differentiation stage, alternative splicing may remove hinge 1 in FLNc and FLNb (20.Xie Z. Xu W. Davie E.W. Chung D.W. Molecular cloning of human ABPL, an actin-binding protein homologue.Biochem. Biophys. Res. Commun. 1998; 251: 914-919Crossref PubMed Scopus (63) Google Scholar, 21.van der Flier A. Kuikman I. Kramer D. Geerts D. Kreft M. Takafuta T. Shapiro S.S. Sonnenberg A. Different splice variants of filamin-B affect myogenesis, subcellular distribution, and determine binding to integrin [beta] subunits.J. Cell Biol. 2002; 156: 361-376Crossref PubMed Scopus (91) Google Scholar). The carboxyterminal Ig-like domain 24 mediates homodimerization, resulting in filamin dimers capable of cross-linking actin filaments (22.Himmel M. Van Der Ven P.F. Stöcklein W. Fürst D.O. The limits of promiscuity: isoform-specific dimerization of filamins.Biochemistry. 2003; 42: 430-439Crossref PubMed Scopus (54) Google Scholar, 23.Pudas R. Kiema T.-R. Butler P.J.G. Stewart M. Ylänne J. Structural basis for vertebrate filamin dimerization.Structure. 2005; 13: 111-119Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 24.Sjekloća L. Pudas R. Sjöblom B. Konarev P. Carugo O. Rybin V. Kiema T.R. Svergun D. Ylänne J. Djinović Carugo K. Crystal structure of human filamin C domain 23 and small angle scattering model for filamin C 23–24 dimer.J. Mol. Biol. 2007; 368: 1011-1023Crossref PubMed Scopus (23) Google Scholar), whereas hinge 2 was suggested to fulfil a regulatory role in dimerization (22.Himmel M. Van Der Ven P.F. Stöcklein W. Fürst D.O. The limits of promiscuity: isoform-specific dimerization of filamins.Biochemistry. 2003; 42: 430-439Crossref PubMed Scopus (54) Google Scholar). FLNc features a unique insertion of 82 amino acids in Ig-like domain 20, which is sufficient for Z-disc targeting (18.van der Ven P.F. Obermann W.M. Lemke B. Gautel M. Weber K. Fürst D.O. Characterization of muscle filamin isoforms suggests a possible role of gamma-filamin/ABP-L in sarcomeric Z-disc formation.Cell Motil. Cytoskelet. 2000; 45: 149-162Crossref PubMed Scopus (123) Google Scholar). This insert is also likely important for establishing diverse protein interaction and scaffolding functions for cytoplasmic signaling processes. Compatible with its role in intracellular signaling events, FLNc was proposed to shuttle between the Z-disc and the sarcolemma or other compartments, particularly in disease states (17.Thompson T.G. Chan Y.M. Hack A.A. Brosius M. Rajala M. Lidov H.G. McNally E.M. Watkins S. Kunkel L.M. Filamin 2 (FLN2): A muscle-specific sarcoglycan interacting protein.J. Cell Biol. 2000; 148: 115-126Crossref PubMed Scopus (235) Google Scholar, 18.van der Ven P.F. Obermann W.M. Lemke B. Gautel M. Weber K. Fürst D.O. Characterization of muscle filamin isoforms suggests a possible role of gamma-filamin/ABP-L in sarcomeric Z-disc formation.Cell Motil. Cytoskelet. 2000; 45: 149-162Crossref PubMed Scopus (123) Google Scholar, 25.Bönnemann C.G. Thompson T.G. van der Ven P.F. Goebel H.H. Warlo I. Vollmers B. Reimann J. Herms J. Gautel M. Takada F. Beggs A.H. Fürst D.O. Kunkel L.M. Hanefeld F. Schröder R. Filamin C accumulation is a strong but nonspecific immunohistochemical marker of core formation in muscle.J. Neurol. Sci. 2003; 206: 71-78Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 26.Nilsson M.I. Nissar A.A. Al-Sajee D. Tarnopolsky M.A. Parise G. Lach B. Fürst D.O. van der Ven P.F. Kley R.A. Hawke T.J. Xin is a marker of skeletal muscle damage severity in myopathies.Am. J. Pathol. 2013; 183: 1703-1709Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). Phosphorylation events and calpain-dependent cleavage of FLNc may likely be involved in regulating functions and localization of the protein (27.Kawamoto S. Hidaka H. 1-(5-Isoquinolinesulfonyl)-2-methylpiperazine (H-7) is a selective inhibitor of protein kinase C in rabbit platelets.Biochem. Biophys. Res. Commun. 1984; 125: 258-264Crossref PubMed Scopus (358) Google Scholar, 28.Tigges U. Koch B. Wissing J. Jockusch B.M. Ziegler W.H. The F-actin Cross-linking and Focal Adhesion Protein Filamin A Is a Ligand and in Vivo Substrate for Protein Kinase C.J. Biol. Chem. 2003; 278: 23561-23569Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 29.Raynaud F. Jond-Necand C. Marcilhac A. Fürst D. Benyamin Y. Calpain 1-gamma filamin interaction in muscle cells: a possible in situ regulation by PKC-alpha.Int. J. Biochem. Cell Biol. 2006; 38: 404-413Crossref PubMed Scopus (2) Google Scholar), however precise information concerning phosphorylation and cleavage sites and the physiological effects thereof have remained obscure. In this work, we differentiated mouse C2C12 myoblasts into multinucleated myotubes by serum reduction followed by electrical pulse stimulation (EPS) to generate contracting myotubes comprising fully assembled sarcomeres with mature Z-discs. By quantitative proteomics, we determined changes in the abundance of proteins in EPS-treated contracting myotubes versus myoblasts and differentiated myotubes. We found that contracting C2C12 myotubes exhibited increased levels of proteins needed for ATP synthesis, reflecting their higher energy consumption because of contractile activity. Through phosphoproteomics analysis of contracting C2C12 myotubes, we further identified the myofibrillar Z-disc as major site of protein phosphorylation. Based on these data, we established a detailed site-specific phosphorylation map of the Z-disc proteome, highlighting the large number of highly phosphorylated Z-disc and Z-disc-associated proteins including FLNc. We focused on a phosphosite cluster in the hinge 2 region of FLNc for a detailed analysis. Through in vitro kinase assays coupled to high resolution MS we precisely mapped serine residues serving as specific PKCα substrate sites. These data were further confirmed by a cellular approach. In a newly established top-down and a targeted MS-based approach, we determined a distinct tyrosine residue positioned directly carboxyterminal to these phosphorylation sites as the major calpain 1 cleavage site in FLNc. PKCα-mediated phosphorylation not only controls cleavage by calpain 1, but also modulates FLNc dynamics. Interestingly, an FLNc variant lacking Ig-like domain 24, thus mimicking calpain 1 cleaved FLNc, exhibited remarkably fast kinetics and exceedingly high mobility. C2C12 and C2 myoblasts were cultured in high glucose DMEM medium (Life Technologies, Darmstadt, Germany) supplemented with 15% FCS (PAA, GE Healthcare Life Sciences, Freiburg, Germany), 1% nonessential amino acids, 1% penicillin/streptomycin and 1% sodium pyruvate (all Life Technologies) in six-well plates (Techno Plastic Products AG, Trasadingen, Switzerland) to a confluency of ∼90%. Differentiation was induced by reduction of the FCS content to 2% (30.Ong S.E. Blagoev B. Kratchmarova I. Kristensen D.B. Steen H. Pandey A. Mann M. Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics.Mol. Cell. Proteomics. 2002; 1: 376-386Abstract Full Text Full Text PDF PubMed Scopus (4569) Google Scholar) in the absence of sodium pyruvate. Differentiation medium was changed every 48 h until complete myotube formation was observed (day 5–6). After myotube development, sarcomere formation was improved by EPS (0.5 Hz, 4 ms, 10–12 V) with a C-Pace EP Culture Pacer (IonOptix, Milton, MA) for 16–24 h. SILAC labeling of C2C12 myoblasts was performed with high glucose SILAC-DMEM medium (GE Healthcare Life Sciences) supplemented with dialyzed 15% FCS (PAA), 1% nonessential amino acids,1% sodium pyruvate, 1% proline (all Life Technologies), 84 mg/l arginine and 146 mg/l lysine (Cambridge Isotope Laboratories Inc., Tewksbury, MA) for at least nine cell doublings. Light, medium, and heavy stable isotope labeling by amino acids in cell culture (SILAC) was performed with 13C6 l-arginine and 12C6 l-lysine, 12C6 l-arginine and D4 l-lysine, and 13C615N4 l-arginine and 13C615N2 l-lysine, respectively. Labeled myoblasts were seeded into six-well plates and differentiation was induced by reduction of the dialyzed FCS content to 2% in the absence of sodium pyruvate (30.Ong S.E. Blagoev B. Kratchmarova I. Kristensen D.B. Steen H. Pandey A. Mann M. Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics.Mol. Cell. Proteomics. 2002; 1: 376-386Abstract Full Text Full Text PDF PubMed Scopus (4569) Google Scholar). Immortalized mouse skeletal myoblasts (IMMs) were cultivated and transfected as described before ((31.Winter L. Staszewska I. Mihailovska E. Fischer I. Goldmann W.H. Schröder R. Wiche G. Chemical chaperone ameliorates pathological protein aggregation in plectin-deficient muscle.J. Clin. Investig. 2014; 124: 1144-1157Crossref PubMed Scopus (66) Google Scholar); (32.Molt S. Buhrdel J.B. Yakovlev S. Schein P. Orfanos Z. Kirfel G. Winter L. Wiche G. van der Ven P.F. Rottbauer W. Just S. Belkin A.M. Furst D.O. Aciculin interacts with filamin C and Xin and is essential for myofibril assembly, remodeling and maintenance.J. Cell Sci. 2014; 127: 3578-3592Crossref PubMed Scopus (44) Google Scholar)). IMMs with passage numbers of up to 40 were used for experiments. Human embryonic kidney cells (HEK293) were cultured in DMEM supplemented with 10% FCS and 1% sodium pyruvate. Transient transfections of HEK293 and C2 cells were performed using polyethylenimine (PEI, 1 μg/μl Polysciences Europe GmbH) and Lipofectamine2000 (Invitrogen, Darmstadt, Germany), respectively. To this end, cells were grown to a confluence of 70% in 6 cm dishes and for each transfection, a total of 4.5 μg DNA was used. PEI was mixed in a 3:1 ratio with DNA in a 6-fold volume of Opti-MEM (Life Technologies) and incubated for 15 min. C2 cells were transfected in solution according to the manufacture's protocol. Transfected cells were grown for 24 h before cell lysis. All cell lines were regularly tested to be mycoplasma-negative. For lysis of C2C12 myotubes, plates were placed on ice and cells were washed twice with ice-cold PBS (CM-PBS; 0.75 mm CaCl2, 0.75 mm MgCl2, 155 mm NaCl2, 2.7 mm KCl, 2 mm KH2PO4, 10 mm Na2HPO4, pH 7.4). 150 μl precooled lysis buffer (7 m urea, 2 m thiourea, 1 mm sodium orthovanadate, 10 mm β-glycerophosphate, 9.5 mm sodium fluoride, 10 mm sodium pyrophosphate) were added per well, cells were scraped from the dish and sonified 2× for 10 s on ice for complete cell lysis. Insoluble material was removed by centrifugation for 20 min at 21,000 × g and 4 °C and the protein concentration was estimated using the Bradford assay (BioRad, München, Germany). Reduction and alkylation of proteins from phosphoproteome replicate 2 and dimethyl- and SILAC-labeled samples was performed as described (33.Francavilla C. Hekmat O. Blagoev B. Olsen J.V. SILAC-based temporal phosphoproteomics.Methods Mol. Biol. 2014; 1188: 125-148Crossref PubMed Scopus (9) Google Scholar) with slight modifications. Each reaction was carried out for 30 min using a final concentration of 1 mm dithiothreitol (DTT) and 55 mm 2-chloroacetamide before alkylation was quenched with a final concentration of 5 mm DTT. A lysate volume equal to a total protein amount of 100 μg (for each sample used for stable isotope dimethyl labeling), 7 mg (replicate 1), 8.1 mg (replicate 2), and 2 mg (for each SILAC replicate) was diluted 1:4 with 50 mm ammonium bicarbonate solution and digested with sequencing grade trypsin (1:50) (Promega, Mannheim, Germany) for 3.5 h at 200 rpm and 42 °C. Peptides were desalted using an Oasis HLB cartridge (Waters Corporation, Milford, MA) according to the manufacturer's protocol. Eluates were aliquoted, lyophilized and stored at −80 °C. Triple stable isotope dimethyl labeling was performed as previously described (34.Boersema P.J. Raijmakers R. Lemeer S. Mohammed S. Heck A.J. Multiplex peptide stable isotope dimethyl labeling for quantitative proteomics.Nat. Protocols. 2009; 4: 484-494Crossref PubMed Scopus (1055) Google Scholar). To this end, 100 μg tryptic digests of myoblasts (day 0), myotubes (day 5), and EPS-stimulated myotubes at day 5 were dissolved in 100 mm TEAB buffer and incubated for 1 h with triple dimethyl labeling reagents (all Sigma Aldrich, St. Louis, MO). A label switch was performed for each replicate (n = 3) and the completeness of the peptide labeling reaction was confirmed using an aliquot of 10 μg before mixing. Stable isotope-labeled peptide samples were mixed in a 1:1:1 ratio, desalted as described above, and dried in vacuo. For each dimethyl and SILAC replicate, 300 μg and 2 mg of total peptide were dissolved in 200 μl buffer A (10 mm ammonium hydroxide, pH 10.5), sonicated for 3 min, centrifuged at 20,000 × g for 4 min and filtrated into a sample vial using a 0.2 μm syringe filter (Phenomenex, Aschaffenburg, Germany). Peptides samples were separated by high-pH reversed phase (hpH-RP) chromatography using a Dionex Ultimate 3000 system equipped with a RP Gemini C18 column ( 4.6 mm × 25 mm, 3 μ, 110 Å, Phenomenex) and operated at a flow rate of 200 μl/min and 750 μl/min, respectively. All separations were carried out using a binary solvent system (A: 10 mm ammonium hydroxide, pH 10.5; B: 10 mm ammonium hydroxide, pH 10.5, 90% acetonitrile) at a temperature of 40 °C. Peptide loading and binding was performed at 1% B for 5 min, followed by peptide elution by increasing buffer B to 20% in 35 min and further to 45% in 20 min. Ninety fractions were collected in a 96 well plate at 50 s intervals from minute 2 to 77. Every 30th fraction was pooled and acidified with TFA to a final concentration of 1%. From SILAC replicates, the collected fractions 19 and 20 were used for enrichment of phosphopeptides by TiO2 beads. Tryptic digests were dissolved in 200 μl SCX buffer A (5 mm potassium dihydrogen phosphate, 20% acetonitrile (ACN, v/v), pH 2.8). The supernatant was loaded onto a Polysulfoethyl-A column (Ø 4.6 mm, 20 cm, 5 μm, 200 Å, PolyLC, Columbia, MD) equilibrated with SCX buffer A using a Dionex Ultimate 3000 UHPLC system. Peptides were separated at a flow rate of 700 μl/min applying a linear gradient of 0–30% SCX buffer B (5 mm potassium dihydrogen phosphate, 20% ACN