Mitsugumin 53 (MG53) as an e3 ligase in skeletal muscle

Thesis for the Degree of Doctor of Philosophy Mitsugumin 53 (MG53) as an E3 ligase in skeletal muscle by Nguyen Thi Huynh Nga Major in Cell Biology Department of Biotechnology School of Life Sciences and Biotechnology Korea University August 2014 Under guidance of Professor Young-Gyu Ko Mitsugumin 53 (MG53) as an E3 ligase in skeletal muscle A thesis Submitted to School of Life Sciences and Biotechnology Korea University For the Degree of Doctor of Philosophy by Nguyen Thi Huynh Nga August 2014 Nguyen Thi Huynh Nga 의 理學 博士學位 論文 審査를 完了함 2014 年 月 Table of Contents Table of Contents I List of Tables IV List of Figures V List of Abbreviations VIII Abstract Chapter I: General introduction & Literature review Lipid rafts Mitsugumin 53 10 Skeletal myogenesis 18 The ubiquitin proteasome system (UPS) 25 Focal adhesion kinase (FAK) 29 Objectives 33 References 35 Chapter II: Materials and methods 44 Cell culture and differentiation 45 Adenoviral preparation and infection 45 Plasmids for transient transfection and luciferase assay 46 Immunoblotting, immunoprecipitation and immunofluorescence 46 I Measurement of the myogenic index 51 RNA interference 52 In vitro binding assay 52 Pulse-chase analysis 53 RT-PCR 53 10 Quantitative real-time PCR 54 11 IRS-1 ubiquitination and FAK ubiquitination 55 12 Statistical analysis 56 References 57 Chapter III: Results and discussions 59 Part 1: MG53-induced IRS-1 ubiquitination negatively regulates skeletal myogenesis 60 1.1 RING domain of MG53 is required for the negative regulation of skeletal myogenesis 61 1.2 MG53 is a ubiquitin E3 ligase targeting IRS-1 68 1.3 UBE2H is involved in MG53-mediated IRS-1 ubiquitination 79 1.4 Discussion 88 Part 2: MG53 ligase ubiquitinates focal adhesion kinase during skeletal myogenesis 94 2.1 FAK protein is down-regulated during skeletal myogenesis 95 II 2.2 FAK interacts with MG53 and UBE2H 105 2.3 MG53 induces FAK degradation 111 2.4 The RING domain of MG53 is required for FAK ubiquitination118 2.5 The E2 enzyme UBE2H is essential for MG53-induced FAK ubiquitination 124 2.6 Discussion 128 References 134 III List of Tables Chapter I: General introduction & Literature review Table Diseases for which rafts and raft proteins are targets Chapter II: Materials and methods Table List of primary antibodies for immunoblotting (IB), immunoprecipitation (IP) and immunofluorescence (IFA) 48 Table List of secondary antibodies for immunoblotting (IB) and immunofluorescence (IFA) 51 Table List of primer sequences 54 IV List of Figures Chapter I: General introduction & Literature review Figure The structure of lipid rafts Figure Identification of TRIM72 in the lipid rafts of skeletal muscle 10 Figure A schematic representation of the proposed function of MG53 in muscle membrane repair 16 Figure Hierarchy of transcription factors regulating progression 19 Figure Morphology of C2C12 cells in different phases 21 Figure A signaling pathway mediated by IRS-1 in which skeletal muscle differentiation is inhibited by MG53 (TRIM72) 22 Figure The ubiquitin system 26 Figure Molecular architecture of focal contacts 30 Chapter III: Results and discussions Part 1: MG53-induced IRS-1 ubiquitination negatively regulates skeletal myogenesis 60 Figure RING domain-disrupting MG53 mutants 62 Figure The RING domain of MG53 is required to negatively regulate myogenesis 64 V Figure IRS-1 signaling and the IRS-1 expression level in MG53overexpressed C2C12 myoblasts and MG53-knockdowned C2C12 myotubes 66 Figure MG53 induces the degradation of IRS-1 but not of IRS-2 69 Figure MG53 is an E3-ligase enzyme inducing IRS-1 ubiquitination 72 Figure UBE2H is an E2 enzyme for MG53 80 Figure UBE2H is an E2 enzyme for MG53-mediated IRS-1 ubiquitination 83 Figure MG53-mediated negative feedback regulation of skeletal myogenesis 90 Part 2: MG53 ligase ubiquitinates focal adhesion kinase during skeletal myogenesis 94 Figure I Expression of FAK during myogenic differentiation in primary myoblast cultures 96 Figure II Focal adhesion kinase (FAK) phosphorylation at Tyr-397 98 Figure The protein expression level of FAK decreases 101 Figure FAK interacts with MG53 and UBE2H 107 Figure The B-box of MG53 is a binding domain for FAK 112 VI Figure The RING domain is required for MG53-induced FAK degradation 115 Figure FAK ubiquitination requires the RING domain of MG53 119 Figure UBE2H is required for MG53-induced FAK ubiquitination 125 VII vice versa Indeed, MG53 is another common E3 ligase for the ubiquitination of IRS-1 and FAK It is also of interest to reinforce that both of these two E3 ligases, Cbl and SOCS proteins take part in skeletal myogenesis as well (59, 60) Being an essential mediator of the cell membrane repair process, MG53 nucleates assembly of the membrane repair patch at the membrane injury site, thus, providing therapeutic benefits for muscular dystrophy (61, 62) The involvement of FAK in dystrophinopathy and its regulation of caveolin 3, which interacts with MG53 provide a link between FAK and MG53 in the disease (24, 62, 63) Although FAK and IRS-1 share a common E2 ligase (UBE2H) and E3 ligase (MG53) during skeletal myogenesis, their MG53-interacting domains are different For example, among the different domains of MG53, the coiled-coil domain is utilized for IRS-1 interaction, and the B-box domain is utilized for FAK interaction Although the FAK expression level decreased during myogenesis of C2C12 cells and MyoD-overexpressing MEFs, phosphorylation of FAK at Tyr576/577 was increased gradually (Figures 1a, c) These findings suggest that FAK ubiquitination might require its phosphorylation because many proteins are ubiquitinated and degraded in a phosphorylation-dependent process (64) However, the molecular interaction between MG53 and FAK was not prevented in the presence of λ phosphatase (Figures 2d-f), indicating that MG53-induced FAK ubiquitination is not dependent on the phosphorylation of FAK There was also an 132 observation previously that MG53-IRS-1 interaction is not altered after IGF stimulation in C2C12 myotubes (2) With all these data, I can conclude that the molecular association of MG53 to IRS-1 or FAK is independent of the phosphorylation status of substrate proteins Although MG53-dependent FAK degradation during myogenesis remains to be further studied in vivo together with downstream signalling pathways added, the documented mechanism certainly contributes explanations and clues to the involvement of FAK in important aspects of organismal disease and development 133 References Ozato, K., Shin, D M., Chang, T H and Morse, 3rd H., C TRIM family proteins and their emerging roles in innate immunity Nature Review Immunology (2008), 849-860 Lee, C S., Yi, J S., Jung, S.Y., Kim, B W., Lee, N R., Choo, H J., Jang, S Y., Han, J., Chi, S G., Park, M., Lee, J H and Ko, Y G TRIM72 negatively regulates myogenesis via targeting insulin receptor substrate-1 Cell Death and Differentiation 17 (2010), 1254-1265 Cai, C., Masumiya, H., Weisleder, N., Matsuda, N., Nishi, M., Hwang, M., Ko, J K., Lin, P., Thornton, A., Zhao, X., Pan, Z., Komazaki, S., Brotto, M., Takeshima, H., and Ma, J MG53 nucleates assembly of cell membrane repair machinery Nature Cell Biology 11 (2009), 56-64 Park, E Y., Kwon, O B., Jeong, B C., Yi, J S., Lee, C S., Ko, Y G and Song, H K Crystal structure of PRY-SPRY domain of human TRIM72 Proteins 78 (2010), 790-795 Yi, J S., Park, J S., Ham, Y M., Nguyen, N., Lee, N R., Hong, J., Kim, B W., Lee, H., Lee, C S., Jeong, B C., Song, H K., Cho, H., Kim, Y K., Lee, J S., Park, K S., Shin, H., Choi, I., Lee, S H., Park, W J., Park, S Y., Choi, C S., Lin, P., Karunasiri, M., Tan, T., Duann, P., Zhu, H., Ma, J and Ko, Y G 134 MG53-induced IRS-1 ubiquitination negatively regulates skeletal myogenesis and insulin signalling Nature Commununications (2013), 2354 Rommel, C., Clarke, B A., Zimmermann, S., Nuñez, L., Rossman, R., Reid, K., Moelling, K., Yancopoulos, G D and Glass, D J Differentiation stagespecific inhibition of the Raf-MEK-ERK pathway by Akt Science 286 (1999), 1738-1741 Murgia, M., Serrano, A L., Calabria, E., Pallafacchina, G., Lomo, T and Schiaffino, S Ras is involved in nerve-activity-dependent regulation of muscle genes Nature Cell Biology (2000), 142-147 Song, R., Peng, W., Zhang, Y., Lv, F., Wu, H K., Guo, J., Cao, Y., Pi, Y., Zhang, X., Jin, L., Zhang, M., Jiang, P., Liu, F., Meng, S., Zhang, X., Jiang, P., Cao, C M and Xiao, R P Central role of E3 ubiquitin ligase MG53 in insulin resistance and metabolic disorders Nature 494 (2013), 375-379 Ye, Y and Rape, M Building ubiquitin chains: E2 enzymes at work Nature Review Molecular Cell Biology 10 (2009), 755-764 10 Serra, C., Palacios, D., Mozzetta, C., Forcales, S V., Morantte, I., Ripani, M., Jones, D R., Du, K., Jhala, U S., Simone, C and Puri, P L Functional interdependence at the chromatin level between the MKK6/p38 and IGF1/PI3K/AKT pathways during muscle differentiation Molecular Cell 28 (2007), 200-213 135 11 Jung, S Y and Ko, Y G TRIM72, a novel negative feedback regulator of myogenesis, is transcriptionally activated by the synergism of MyoD (or myogenin) and MEF2 Biochemical and Biophysical Research Communications 396 (2010), 238-245 12 Kim, B W., Lee, C S., Yi, J S., Lee, J H., Lee, J W., Choo, H J., Jung, S Y., Kim, M S., Lee, S W., Lee, M S., Yoon, G and Ko, Y G Lipid raft proteome reveals that oxidative phosphorylation system is associated with the plasma membrane Expert Review of Proteomics (2010), 849-866 13 Pete, G., Fuller, C R., Oldham, J M., Smith, D R., D'Ercole, A J., Kahn, C R and Lund, P K Postnatal growth responses to insulin-like growth factor I in insulin receptor substrate-1-deficient mice Endocrinology 140 (1999), 5478-5487 14 Tamemoto, H., Kadowaki, T., Tobe, K., Yagi, T., Sakura, H., Hayakawa, T., Terauchi, Y., Eeki, K., Kaburagi, Y Satoh, S., Sekihara, H., Yoshioka, S., Horikoshi, H., Furuta, Y., Ikawa, Y., Kasuga, M., Yazaki, Y and Aizawa, S Insulin resistance and growth retardation in mice lacking insulin receptor substrate-1 Nature 372 (1994), 182-186 15 Araki, E., Lipes, M A., Patti, M E., Brüning, J C., Haag, B 3rd, Johnson, R S and Kahn, C R Alternative pathway of insulin signalling in mice with targeted disruption of the IRS-1 gene Nature 372 (1994), 186-190 136 16 Kawaguchi, T., Yoshida, T., Harada, M., Hisamoto, T., Nagao, Y., Ide, T., Taniguchi, E., Kumemura, H., Hanada, S., Maeyama, M., Baba, S., Koga, H., Kumashiro, R., Ueno, T., Ogata, H., Yoshimura, A and Sata, M Hepatitis C virus down-regulates insulin receptor substrates and through upregulation of suppressor of cytokine signaling American Journal of Pathology 165 (2004), 1499-1508 17 Nakao, R., Hirasaka, K., Goto, J., Ishidoh, K., Yamada, C., Ohno, A., Okumura, Y., Nonaka, I., Yasutomo, K., Baldwin, K M., Kominami, E., Higashibata, A., Nagano, K., Tanaka, K., Yasui, N., Mills, E M., Takeda, S and Nikawa, T Ubiquitin ligase Cbl-b is a negative regulator for insulin-like growth factor signaling during muscle atrophy caused by unloading Molecular and Cellular Biology 29 (2009), 4798-4811 18 Xu, X., Sarikas, A., Dias-Santagata, D C., Dolios, G., Lafontant, P J., Tsai, S C., Zhu, W., Nakajima, H., Nakajima, H O., Field, L J., Wang, R., Pan, Z Q The CUL7 E3 ubiquitin ligase targets insulin receptor substrate for ubiquitin-dependent degradation Molecular Cell 30 (2008), 403-414 19 Shi, J., Luo, L., Eash, J., Ibebunjo, C and Glass, D J The SCF-Fbxo40 complex induces IRS1 ubiquitination in skeletal muscle, limiting IGF1 signaling Developmental Cell 21 (2011), 835-847 137 20 Cai, C., Masumiya, H., Weisleder, N., Pan, Z., Nishi, M., Komazaki, S., Takeshima, H and Ma, J MG53 regulates membrane budding and exocytosis in muscle cells Journal of Biological Chemistry 284 (2009), 3314-3322 21 Cai, C., Masumiya, H., Weisleder, N., Matsuda, N., Nishi, M., Hwang, M., Ko, J K., Lin, P., Thornton, A., Zhao, X., Pan, Z., Komazaki, S., Brotto, M., Takeshima, H., and Ma, J MG53 nucleates assembly of cell membrane repair machinery Nature Cell Biology 11 (2009), 56-64 22 Weisleder, N., Takizawa, N., Lin, P., Wang, X., Cao, C., Zhang, Y., Tan, T., Ferrante, C., Zhu, H., Chen, P J., Yan, R., Sterling, M., Zhao, X., Hwang, M., Takeshima, M., Cai, C., Cheng, H., Takeshima, H., Xiao, R P and Ma, J Recombinant MG53 protein modulates therapeutic cell membrane repair in treatment of muscular dystrophy Science Translational Medicine (2012), 139ra85 23 Nguyen, N., Yi, J S., Park, H., Lee, J S and Ko, Y G Mitsugumin 53 (MG53) ligase ubiquitinates focal adhesion kinase during skeletal myogenesis Journal of Biological Chemistry 289 (2014), 3209-3216 24 Quach, N L., Biressi, S., Reichardt, L F., Keller, C and Rando, T A Focal Adhesion Kinase Signaling Regulates the Expression of Caveolin and beta Integrin, Genes Essential for Normal Myoblast Fusion Molecular Biology of the Cell 20 (2009), 3422-3435 138 25 Clemente, C F., Corat, M A., Saad, S T and Franchini, K G Differentiation of C2C12 myoblasts is critically regulated by FAK signaling American Journal of Physiology – Regulatory, Integrative and Comparative Physiology 289 (2005), R862-870 26 Burattini, S., Ferri, P., Battistelli, M., Curci, R., Luchetti, E and Falcieri, E C2C12 murine myoblasts as a model of skeletal muscle development: morpho-functional characterization European Journal of Histochemistry 48 (2004), 223-233 27 Yaffe, D and Saxel, O Serial passaging and differentiation of myogenic cells isolated from dystrophic mouse muscle Nature 270 (1997), 725-727 28 Schiaffino, S., Gorza, L., Sartore, S., Saggin, L and Carli, M Embryonic myosin heavy-chain as a differentiation marker of developing human skeletalmuscle and Rhabdomyosarcoma - a monoclonal-antibody study Experimental Cell Research 163 (1986), 211-220 29 Wright, W E., Sassoon, D A and Lin, V K Myogenin, a factor regulating myogenesis, has a domain homologous to Myod Cell 56 (1989), 607-617 30 Edmondson, D G and Olson, E N A gene with homology to the Myc similarity region of Myod1 is expressed during myogenesis and is sufficient to activate the muscle differentiation program Genes and Development (1989), 628-640 139 31 Song, K S., Scherer, P E., Tang, Z L., Okamoto, T., Li, S W., Chafel, M., Chu, C., Kohtz, D S and Lisanti, M P Expression of caveolin-3 in skeletal, cardiac and smooth muscle cells - Caveolin-3 is a component of the sarcolemma and co-fractionates with dystrophin and dystrophin-associated glycoproteins Journal of Biological Chemistry 271 (1996), 15160-15165 32 Koishi, K., Zhang, M., McLennan, I S and Harris, A J MyoD protein accumulates in satellite cells and is neurally regulated in regenerating myotubes and skeletal muscle fibers Developmental Dynamics 202 (1995), 244-254 33 Ciechanover, A., Orian, A and Schwartz, A L The ubiquitin-mediated proteolytic pathway: Mode of action and clinical implications Journal of Cellular Biochemistry 34 (2000), 40-51 34 Hershko, A., Ciechanover, A and Varshavsky, A The ubiquitin system Nature Medicine (2000), 1073-1081 35 Rafiq, K., Guo, J F., Vlasenko, L., Guo, X J., Kolpakov, M A., Sanjay, A., Houser, S R and Sabri, A c-Cbl ubiquitin ligase regulates focal adhesion protein turnover and myofibril degeneration induced by neutrophil protease cathepsin G Journal of Biological Chemistry 287 (2012), 5327-5339 140 36 Matsuda, T., Ikeda, O and Sekine, Y STAP-2 regulates integrin-mediated Tcell adhesion through protein degradation of FAK Faseb Journal 22 (2008), 1071.10 37 Martinez-Noel, G., Muller, U and Harbers, K Identification of molecular determinants required for interaction of ubiquitin-conjugating enzymes and RING finger proteins European Journal of Biochemistry 268 (2001) 59125919 38 Eleniste, P P and Bruzzaniti, A Focal adhesion kinases in adhesion structures and disease Journal of Signal Transduction (2012), doi:10.1155/2012/296450 39 Infusino, G A., Jacobson, J R 2012 Endothelial FAK as a therapeutic target in disease Microvascular Research 83 (2012), 89-96 40 Kim, B and Feldman, E L Insulin-like growth factor I prevents mannitolinduced degradation of focal adhesion kinase and Akt Journal of Biological Chemistry 277 (2002), 27393-27400 41 Kim, B., van Golen, C M and Feldman, E L Degradation and dephosphorylation of focal adhesion kinase during okadaic acid-induced apoptosis in human neuroblastoma cells Neoplasia (2003), 405-416 42 Mao, H., Li, F., Ruchalski, K., Mosser, D D., Schwartz, J H., Wang, Y and Borkan, S C Hsp72 inhibits focal adhesion kinase degradation in ATP- 141 depleted renal epithelial cells Journal of Biological Chemistry 278 (2003), 18214-18220 43 Chan, K T., Bennin, D A and Huttenlocher, A Regulation of Adhesion Dynamics by Calpain-mediated Proteolysis of Focal Adhesion Kinase (FAK) Journal of Biological Chemistry 285 (2010), 11418-11426 44 Mueller, C., Emmrich, J., Jaster, R., Braun, D., Liebe, S and Sparmann, G Cis-hydroxyproline-induced inhibition of pancreatic cancer cell growth is mediated by endoplasmic reticulum stress World Journal of Gastroenterology 12 (2006), 1569-1576 45 Leskinen, M J., Lindstedt, K A., Wang, Y F and Kovanen, P T Mast cell chymase induces smooth muscle cell apoptosis by a mechanism involving fibronectin degradation and disruption of focal adhesions Arteriosclerosis Thrombosis and Vascular Biology 23 (2003), 238-243 46 Furuta, Y., Ilic, D., Kanazawa, S., Takeda, N., Yamamoto, T and Aizawa, S Mesodermal defect in late-phase of gastrulation by a targeted mutation of focal adhesion kinase, Fak Oncogene 11 (1995), 1989-1995 47 Luo, S W., Zhang, C., Zhang, B., Kim, C H, Qiu, Y Z., Du, Q S., Mei, L and Xiong, W C Regulation of heterochromatin remodelling and myogenin expression during muscle differentiation by FAK interaction with MBD2 Embo Journal 28 (2009), 2568-2582 142 48 Puri, P L and Sartorelli, V Regulation of muscle regulatory factors by DNAbinding, interacting proteins, and post-transcriptional modifications Journal of Cellular Physiology 185 (2000), 155-173 49 Kim, S S., Rhee, S., Lee, K H., Kim, J H., Kim, H S., Kang, M S and Chung, C H Inhibitors of the proteasome block the myogenic differentiation of rat L6 myoblasts Febs Letter 433 (1998), 47-50 50 Lecker, S H., Solomon, V., Mitch, W E and Goldberg, A L Muscle protein breakdown and the critical role of the ubiquitin-proteasome pathway in normal and disease states Journal of Nutrition 129 (1999), 227s-237s 51 DeConto, F., Missorini, S., Arcangeletti, C., Pinardi, F., Montarras, D., Pinset, C., Vassy, J., Geraud, G., Chezzi, C and Scherrer, K Prosome cytodistribution relative to desmin and actin filaments in dividing C2.7 myoblasts and during myotube formation in vitro Experimental Cell Research 233 (1997), 99-117 52 Gardrat, F., Montel, V., Raymond, J and Azanza, J L Proteasome and myogenesis Molecular Biology Reports 24 (1997), 77-81 53 Li, Y P., Lecker, S H., Chen, Y., Waddell, I D., Goldberg, A L and Reid, M B TNF-alpha increases ubiquitin-conjugating activity in skeletal muscle by up-regulating UbcH2/E220k Faseb Journal 17 (2003), 1048-1057 143 54 Fang, S and Weissman, A M A field guide to ubiquitylation Cellular and Molecular Life Sciences 61 (2004), 1546-1561 55 Liu, E., Cote, J F and Vuori, K Negative regulation of FAK signaling by SOCS proteins Embo Journal 22 (2003), 5036-5046 56 Sekine, Y., Tsuji, S., Ikeda, O., Sugiyma, K., Oritani, K., Shimoda, K., Muromoto, R., Ohbayashi, N., Yoshimura, A., and Matsuda, T Signaltransducing adaptor protein-2 regulates integrin-mediated T cell adhesion through protein degradation of focal adhesion kinase Journal of Immunology 179 (2007), 2397-2407 57 Kawaguchi, T., Yoshida, T., Harada, M., Hisamoto, T., Nagao, Y., Ide, T., Taniguchi, E., Kumemura, H., Hanada, S., Maeyama, M., Baba, S., Koga, H., Kumashiro, R., Ueno, T., Ogata, H., Yoshimura, A and Sata M Hepatitis C virus down-regulates insulin receptor substrates and through upregulation of suppressor of cytokine signaling American Journal of Pathology 165 (2004), 1499-1508 58 Nakao, R., Hirasaka, K., Goto, J., Ishidoh, K., Yamada, C., Ohno, A., Okumura, Y., Nonaka, I., Yasutomo, K., Baldwin, K M., Kominami, E., Higashibata, A., Nagano, K., Tanaka, K., Yasui, N., Mills, E M., Takeda, S and Nikawa, T Ubiquitin ligase Cbl-b is a negative regulator for insulin-like 144 growth factor signaling during muscle atrophy caused by unloading Molecular and Cellular Biology 29 (2009), 4798-4811 59 Jehn, B M., Dittert, I., Beyer, S., von der Mark, K and Bielke, W c-Cbl binding and ubiquitin-dependent lysosomal degradation of membraneassociated Notch1 Journal of Biological Chemistry 277 (2002), 8033-8040 60 Nastasi, T., Bongiovanni, A., Campos, Y., Mann, L., Toy, J N., Bostrom, J., Rottier, R., Hahn, C., Conaway, J W., Harris, A J and d'Azzo, A Ozz-E3, a muscle-specific ubiquitin ligase, regulates beta-catenin degradation during myogenesis Developmental Cell (2004), 269-282 61 Alloush, J and Weisleder, N TRIM Proteins in Therapeutic Membrane Repair of Muscular Dystrophy Jama Neurology 70 (2013), 928-931 62 Cai, C., Masumiya, H., Weisleder, N., Matsuda, N., Nishi, M., Hwang, M., Ko, J K., Lin, P., Thornton, A., Zhao, X., Pan, Z., Komazaki, S., Brotto, M., Takeshima, H., and Ma, J MG53 nucleates assembly of cell membrane repair machinery Nature Cell Biology 11 (2009), 56-64 63 Narayanan, T and Subramaniam, S Community structure analysis of gene interaction networks in duchenne muscular dystrophy Plos One (2013) e67237 145 64 Swaney, D L., Beltrao, P., Starita, L., Guo, A L., Rush, J., Fields, S., Krogan, N J and Villen, J Global analysis of phosphorylation and ubiquitylation cross-talk in protein degradation Nature Methods 10 (2013), 676-682 146 [...]... UPS Ubiquitin Proteasome System X Abstract The striated muscle- specific mitsugumin 53 (MG53) also known as tripartite motif-containing 72, acts in the negative regulation of skeletal myogenesis by targeting insulin receptor substrate 1 (IRS-1) Indeed, possessing the catalytic RING-finger domain in its N-terminus, MG53 is postulated to be a novel ubiquitin E3 ligase that induces IRS-1 ubiquitination with... Polymerase Chain Reaction PE Phosphatidylethanolamine PI Phosphatidylinositol PI(3)K Phosphoinositide 3-kinase PI(3)P Phosphatidylinositol 3-phosphate PS phosphatidylserine PTK Protein Tyrosine Kinase RING Really Interesting New Gene si-RNA small interfering RNA IX SOCS Suppressor of Cytokine Signaling SPRY SPla and RYanodine receptor TRIM Tripartite Motif-containing Ub Ubiquitin UBE2H Ubiquitin-conjugating... of an E2-conjugating enzyme, UBE2H Molecular manipulations that disrupt the E3- ligase function of MG53 by overexpressing of RING domain-disrupting MG53 mutants (MG53 C14A and R) abolished IRS-1 ubiquitination and enhance skeletal myogenesis Recent studies reveal that skeletal muscles derived from the MG53 knockout mice show an elevated IRS-1 level and fortified insulin signaling Therefore, targeting... it associates with and inactivates IRS-1, leading to the negative feedback regulation of skeletal myogenesis In 14 addition, MG53 acts as a major regulator for membrane repair by interacting with dysferlin-1, caveolin-3 and cavin-1, forming membrane repair machinery after acute membrane damage in skeletal and cardiac muscles (Figure 3) (14-18) Indeed, the muscle fibres of MG53−/− mice show membrane... protein involved in important aspects of organismal disease and development I found that the FAK protein level gradually decreases while its mRNA level is constant during myogenesis in C2C12 cells and MyoD1 overexpressing mouse embryonic fibroblasts (MEFs) In addition, the FAK protein is in complex with its ubiquitin-conjugating E2 enzyme UBE2H and the E3 enzyme MG53 in endogenous and exogenous immunoprecipitation... and phagosomal proteins are surprisingly found in lipid rafts and their novel functions have been investigated, therefore (6, 7) The detergent- 8 resistant lipid rafts accumulate many different receptors and their downstream signalling molecules, making the lipid rafts as a center for signal transduction activity (8-11) 9 2 Mitsugumin 53 Mitsugumin 53 (MG53), also known as tripartite motif-containing... the interaction between MG53 and IRS-1 can provide novel insights into treatment of metabolic diseases that are associated with insulin resistance Besides inducing the ubiquitination of IRS-1 during skeletal myogenesis, MG53 is also showed to have a second target, the focal adhesion kinase (FAK) which is a crucial enzyme functioning at the crossroads of signal transduction and a scaffold protein involved... a liquid-ordered domain Bulk plasma membrane (gray) contains less cholesterol, sphingomyelin, and gangliosides, and more phospholipids with unsaturated acyl chains As a result, it is more fluid than lipid rafts A variety of proteins partition into lipid rafts: glycosylphosphatidylinositol-anchored proteins; transmembrane proteins (TM); dually acylated proteins (Acyl) As shown in the diagram, not all...  Cbl Casitas b-lineage lymphoma Cav-3 Caveolin-3 CC Coiled-coil domain ECM Extracellular Matrix ERK Extracellular Signal-regulated Kinase FAK Focal Adhesion Kinase FAT Focal Adhesion Targeting FERM 4.1 Protein/Ezrin/Radixin/Moesin FOXO Forkhead O Box FRNK FAK-related non-kinase domain GFP Green Fluorescent Protein GPI Glycosylphosphatidylinositol GSK3 Glycogen Synthase Kinase 3 HEK Human Embryonic... organs (d) Western blotting of TRIM72 and caveolin-3 (Cav-3) in various organs obtained from 12-week-old male mice (e) Reverse transcription-polymerase chain reaction (RT-PCR) analysis of TRIM72, myogenin, MyoD, Cav-3, and myosin heavy chain (MyHC) during C2C12 myogenesis by using actin as a loading control C2C12 myoblasts were differentiated to myotubes for the indicated times (f) Western blotting