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

Part 1: MG53-induced IRS-1 ubiquitination negatively regulates skeletal myogenesis .... RING domain of MG53 is required for the negative regulation of skeletal myogenesis .... Part 2: MG

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

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

Nguyen Thi Huynh Nga 의 理學 博士學位 論文

審査를 完了함

2014 年 8 月

Table of Contents

Table of Contents I List of Tables IV List of Figures V List of Abbreviations VIII

Abstract 1

Chapter I: General introduction & Literature review 3

1 Lipid rafts 4

2 Mitsugumin 53 10

3 Skeletal myogenesis 18

4 The ubiquitin proteasome system (UPS) 25

5 Focal adhesion kinase (FAK) 29

6 Objectives 33

References 35

Chapter II: Materials and methods 44

1 Cell culture and differentiation 45

2 Adenoviral preparation and infection 45

5 Measurement of the myogenic index 51

6 RNA interference 52

7 In vitro binding assay 52

8 Pulse-chase analysis 53

9 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

1 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

2 Part 2: MG53 ligase ubiquitinates focal adhesion kinase during skeletal myogenesis 94

2.1 FAK protein is down-regulated during skeletal myogenesis 95

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

List of Tables

Chapter I: General introduction & Literature review

Table 1 Diseases for which rafts and raft proteins are targets 6

Chapter II: Materials and methods

Table 1 List of primary antibodies for immunoblotting (IB),

immunoprecipitation (IP) and immunofluorescence (IFA) 48 Table 2 List of secondary antibodies for immunoblotting (IB) and

immunofluorescence (IFA) 51 Table 3 List of primer sequences 54

List of Figures

Chapter I: General introduction & Literature review

Figure 1 The structure of lipid rafts 4 Figure 2 Identification of TRIM72 in the lipid rafts of skeletal muscle 10 Figure 3 A schematic representation of the proposed function of MG53 in muscle membrane repair 16 Figure 4 Hierarchy of transcription factors regulating progression 19 Figure 5 Morphology of C2C12 cells in different phases 21 Figure 6 A signaling pathway mediated by IRS-1 in which skeletal muscle differentiation is inhibited by MG53 (TRIM72) 22 Figure 7 The ubiquitin system 26

Figure 8 Molecular architecture of focal contacts 30

Chapter III: Results and discussions

1 Part 1: MG53-induced IRS-1 ubiquitination negatively regulates skeletal myogenesis 60 Figure 1 RING domain-disrupting MG53 mutants 62 Figure 2 The RING domain of MG53 is required to negatively regulate

Figure 3 IRS-1 signaling and the IRS-1 expression level in overexpressed C2C12 myoblasts and MG53-knockdowned C2C12 myotubes 66 Figure 4 MG53 induces the degradation of IRS-1 but not of IRS-2 69 Figure 5 MG53 is an E3-ligase enzyme inducing IRS-1 ubiquitination

MG53- 72

Figure 6 UBE2H is an E2 enzyme for MG53 80 Figure 7 UBE2H is an E2 enzyme for MG53-mediated IRS-1

ubiquitination 83 Figure 8 MG53-mediated negative feedback regulation of skeletal myogenesis 90

2 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 1 The protein expression level of FAK decreases 101 Figure 2 FAK interacts with MG53 and UBE2H 107 Figure 3 The B-box of MG53 is a binding domain for FAK 112

Figure 4 The RING domain is required for MG53-induced FAK

degradation 115 Figure 5 FAK ubiquitination requires the RING domain of MG53 119 Figure 6 UBE2H is required for MG53-induced FAK ubiquitination.

125

List of Abbreviations

Cbl Casitas b-lineage lymphomaCav-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

HRP Horseradish Peroxidase

IGFR Insulin-like Growth Factor Recptor

IFA Immunofluorescence

IP Immunoprecipitation IRS-1 Insulin Receptor Substrate-1 MBD2 CpG-binding Domain Protein 2 MEF Myocyte enhancer factor MEFs Mouse embryonic fibroblasts MG53 Mitsugumin 53

MyHC Myosin Heavy Chain PAGE Poly-Arylamide Gel Electrophoresis

PC Phosphatidylcholine PCR 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

SOCS Suppressor of Cytokine Signaling SPRY SPla and RYanodine receptor TRIM Tripartite Motif-containing

UBE2H Ubiquitin-conjugating enzyme E2H UPS Ubiquitin Proteasome System

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 in important aspects of organismal disease and

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 experiments FAK ubiquitination and degradation is induced by MG53 overexpression in myoblasts but abolished by MG53 or UBE2H knockdown in myotubes Furthermore, the proteasome inhibitor MG132 also blocks FAK degradation Because RING-disrupted MG53 mutants (C14A and ΔR) do not induce FAK ubiquitination and degradation, the RING domain is determined to be required for MG53-induced FAK ubiquitination Taken together, these data indicate that MG53 ubiquitinates FAK with the aid of UBE2H during skeletal myogenesis

Chapter I

General introduction & Literature review

1 Lipid rafts

Lipid rafts, introduced in the 1990s, are subdomains of the plasma

membrane that consists of a high amount of cholesterols and glycosphingolipids (1, 2) Due to the saturation of fatty-acid side chain in raft glycosphingolipids and the

stiff four-ring structure of cholesterol, the raft regions of the membrane are more

order and less fluid than non-raft regions, probably leading to phase separation (2, 3) On the other hand, the presence of cholesterol is responsible for the insolubility

of lipid rafts in nonionic detergents (3-5) Based on their distinctive biochemical

criteria, lipid rafts are isolated and brought to proteomic analyses

Figure 1 The structure of lipid rafts Adopted from (2) Lipid rafts: bringing

Lipid rafts (blue bilayer) are specialized membrane domains containing high concentrations of cholesterol, sphingomyelin and gangliosides They are also enriched in phospholipids that contain saturated fatty acyl chains (straight lines in lipid tails) This composition results in lateral phase separation and the generation

of 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 lipid rafts have the identical protein or lipid composition (Raft 1 vs Raft 2) Not shown are invaginated caveolae, a subclass of lipid rafts that contains caveolin PC, phosphatidylcholine; PE, phosphatidylethanolamine; PS, phosphatidylserine; PI, phosphatidylinositol, SPM, sphingomyelin, Chol, cholesterol; Gang, gangliosides

Adopted from (2) Lipid rafts: bringing order to chaos, Journal of Lipid Research

44 (2003), 655-667

The cell uses cholesterol as a spacer for keeping rafts together However, cholesterol is toxic and its cellular levels are kept in tight control by an intricate network of transcriptional regulation of cholesterol biosynthesis and cellular uptake

cellular efflux (6) Therefore, disturbance of these tightly regulated processes leads

to of diseases of lipid metabolism (Table 1)

Table 1 Diseases for which rafts and raft proteins are targets

Adopted from (6) Cholesterol, lipid rafts and disease, Journal of Clinical

Investigation 110 (2002), 597-603

Alzheimer disease

Parkinson disease

Muscular dystrophy

Polyneuropathies, demyelinating diseases

Autoimmune disease, chronic inflammation, vaccine response

Osteoarthritis

Gastrointestinal ulceration

Paroxysmal nocturnal hemoglobinuria

Lysosomal storage disease

Niemann-Pick disease

Tay-Sachs disease, morbus Fabry, metachromatic leukodystrophy Pilzaeus-Merzbacher disease

Postsqualene cholesterol biosynthesis disorders

Pore-forming toxins (gas gangrene)

Sepsis, septic shock

HIV-1

Measles virus

Respiratory syncytial cell virus

Filoviridae (Ebolavirus, Marburgvirus)

Papillomaviridae and polyomaviridae

detergent-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)

Figure 2 Identification of TRIM72 in the lipid rafts of skeletal muscle

Adopted from (12) TRIM72 negatively regulates myogenesis via targeting insulin

receptor substrate-1 Cell Death and Differentiation 17 (2010), 1254-1265

MG53 is highly expressed in skeletal muscle and heart

MG53 is upregulated during myogenesis

Figure 2 Identification of TRIM72 in the lipid rafts of skeletal muscle

Adopted from (12) TRIM72 negatively regulates myogenesis via targeting insulin

receptor substrate-1 Cell Death and Differentiation 17 (2010), 1254-1265

(a) C2C12 myotube-specific TRIM72 spots were identified by the

comparative 2-DE analysis of lipid raft proteins isolated from myoblasts and myotubes; C2C12 myoblasts were differentiated into myotubes for 2 days Areas

containing red circles in Supplementary Figure S1B are enlarged TRIM72 spots are indicated by dotted circles

(b) Gene analysis of TRIM72 shows a RING finger domain, a B-box, two

coiled-coil domains, and a SPRY domain

(c) Northern blotting analysis of TRIM72 in various mouse 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 of TRIM72, Cav-3, myogenin, MyHC, MyoD, and

Myf5 during C2C12 myogenesis Ponceau S staining was used for a loading control

(g) Immunofluorescence analysis of TRIM72 and Cav-3 in mouse

gastrocnemius skeletal muscle obtained from 12-week-old male mice

(h) Enrichment of TRIM72 and Cav-3 in detergent-resistant lipid rafts

Detergent-resistant lipid rafts were isolated from 4-days-differentiated C2C12 myotubes and mouse gastrocnemius muscle on the basis of detergent insolubility

and low density, fractionated after a discontinuous sucrose gradient, and analyzed

by western blotting of TRIM72 and Cav-3

(i) TRIM72 was postnatally expressed in mouse skeletal muscle and heart

Mouse skeletal muscle and heart were obtained at embryonic (E) and postnatal (P) stages and analyzed by western blotting

Adopted from (12) TRIM72 negatively regulates myogenesis via targeting

insulin receptor substrate-1 Cell Death and Differentiation 17 (2010), 1254-1265

MG53 consists of a really interesting new gene (RING)-finger domain, a box, two coiled-coil domains and a SPRY (SPla and RYanodine receptor) domain

B-(Figure 2b) Its expression is highly up-regulated during the myogenesis of C2C12

and satellite cells, because its promoter contains two E-boxes and myocyte enhancer factor-binding sites for the myogenic transcription factors, MyoD and

MEF, respectively (12, 13)

MG53 is most widely known for its role as an important component of the membrane repair pathway in muscle Recent studies have indicated that MG53 is a promising therapeutic agent for diseases arising from compromised sarcolemmal

membrane integrity, especially for muscular dystrophy (14, 15)

MG53 is recruited to lipid rafts, where it associates with and inactivates IRS-1, leading to the negative feedback regulation of skeletal myogenesis In

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 repair defects (15)

Figure 3 A schematic representation of the proposed function of MG53

in muscle membrane repair Adopted by (15) MG53 nucleates assembly of cell

membrane repair machinery Nature Cell Biology 11 (2009), 56-64

Through interaction with phosphatidylserine, MG53 is tethered to plasma membrane and intracellular vesicles in cells with intact plasma membrane Upon membrane damage, entry of the oxidized milieu of the extracellular space into the reduced environment within the cell results in oligomeriztion of MG53 at the injury site This oligomerization acts as a nucleation site for recruitment of MG53-tethered intracellular vesicles toward the injury site Local elevation of intracellular

Ca2+ at the injury site facilitates fusion of intracellular vesicles with the plasma

membrane to reseal the damaged membrane Adopted from (15) MG53 nucleates

assembly of cell membrane repair machinery Nature Cell Biology 11 (2009),

56-64

3 Skeletal myogenesis

Among the four main tissues of the body - epithelium, muscle, connective and nervous tissues - muscular tissue amounts the most, including involuntary ones: smooth and cardiac muscles and voluntary muscle or skeletal muscle Skeletal muscle comprises up to 40% of the human body mass and is a major organ that is necessary for locomotion and glucose homeostasis Adult skeletal muscle mass is plastically regulated by recruiting satellite cells to preexisting muscle fibres

under hypertrophic conditions such as resistance and endurance exercise (19, 20)

Skeletal myogenesis (Figures 4 and 5), controlled by extrinsic and intrinsic

regulatory mechanisms, is a complex cascade of progressions turning the multipotent mesodermal precursors to mononucleated myoblasts, then to

multinucleated myotubes and myofibers through differentiation and fusion (21, 22)

Figure 4 Hierarchy of transcription factors regulating progression

through the myogenic lineage Adopted from (21) Building Muscle: Molecular

Regulation of Myogenesis Cold Spring Harbor Perspectives

in Biology (2012), DOI 10.1101/cshperspect.a008342

Muscle progenitors that are involved in embryonic muscle differentiation skip the quiescent satellite cell stage and directly become myoblasts Some progenitors remain as satellite cells in postnatal muscle and form a heterogeneous population of stem and committed cells Activated committed satellite cells

master regulators of early lineage specification, whereas Myf5 and MyoD commit cells to the myogenic program Expression of the terminal differentiation genes, required for the fusion of myocytes and the formation of myotubes, are performed

by both myogenin (MyoG) and MRF4 Adopted from (21) Building Muscle:

Molecular Regulation of Myogenesis Cold Spring Harbor Perspectives in Biology

Figure 5 Morphology of C2C12 cells in different phases Adopted from (23)

C2C12 murine myoblasts as a model of skeletal muscle development:

morpho-functional characterization

There have been a lot of studies in which skeletal muscle differentiation is tightly regulated by a variety of hormones, growth factors and cytokines In particular, insulin-like growth factor-1 (IGF-1) has a key role in the regulation of skeletal muscle size Indeed, IGF-1 knockout mice exhibit muscle hypoplasia and

die shortly after birth due to impaired respiration (25-27)

Figure 6 A signaling pathway mediated by IRS-1 in which skeletal muscle

differentiation is inhibited by MG53 (TRIM72) Adopted from (12) TRIM72

negatively regulates myogenesis via targeting insulin receptor substrate-1

The above model shows that MG53 (TRIM72) expression is mediated by IGF/IGFR/IRS-1/PI(3)K/Akt/MyoD signaling pathway After its expression, MG53

interacts with and negatively regulates IRS-1 Adopted from (12) TRIM72

negatively regulates myogenesis via targeting insulin receptor substrate-1 Cell

Death and Differentiation 17 (2010), 1254-1265

Indeed, IGF-1 leads to the consecutive activation of IGF receptor (IGFR), insulin receptor substrate (IRS), phosphatidylinositol 3-kinase (PI3K), Akt, mammalian target of rapamycin (mTOR) and S6 kinase (S6K), which together

orchestrate skeletal myogenesis and hypertrophy (28-31)

Studies with skeletal muscle-specific knockout and transgenic mice for Akt, mTOR or S6K show that the Akt-mTOR-S6K signalling axis is essential for

skeletal muscle hypertrophy and regeneration (32, 33) IGF-1-mediated Akt

activation blocks the transcription factor forkhead box family proteins (FOXO1 and

FOXO3A) by phosphorylating and sequestering them in the cytoplasm (34, 35)

It is also well known that an important cellular event in myogenesis is the adhesion of cells to the extracellular matrix (ECM) proteins Focal adhesion kinase (FAK), a major cytoplasmic tyrosine kinase activated on cell-ECM adhesion, plays

a pivotal role in regulating myogenesis FAK alters heterochromatin reorganization

C2C12 cells (36) In mouse primary myoblast, FAK has an essential role in

morphological muscle differentiation by regulation the expression of profusion

genes including caveolin 3 and the 1D integrin subunit (37)

4 The ubiquitin proteasome system (UPS)

Precise temporal and spatial control of protein synthesis, processing and degradation plays a fundamental role in regulating skeletal muscle structure and

function (38, 39) In fact, the turnover rates and steady-state concentrations of all cellular proteins are controlled by protein degradation (39) The most widely

known degradation process is proteolysis via the ubiquitin proteasome system (UPS) Initially, free ubiquitin (Ub) is activated by the formation of a thiol-ester linkage between the E1 ubiquitin activating enzyme and the carboxyl terminus of ubiquitin The activated ubiquitin is then transferred to one of several different E2 ubiquitin-conjugating enzymes A specific E3 ubiquitin protein ligase interacts with the E2 to transfer the ubiquitin to its specific substrate The polyubiquitinated

substrate protein is then susceptible to degradation by the proteasome complex (40, 41)

Figure 7 The ubiquitin system Adopted from (42) RING domain E3 ubiquitin

ligases Annual Review of Biochemistry 78 (2009), 399-434

(a) Ubiquitin (Ub) and ubiquitin-like proteins are activated for transfer by

E1 (ubiquitin-activating enzyme)

(b) Activated ubiquitin is transferred in thioester linkage from the

active-site cysteine of E1 to the active-active-site cysteine of an E2 ubiquitin-conjugating enzyme

(c) The E2Ub thioester next interacts with an E3 ubiquitin ligase, which

effects transfer of Ub from E2~Ub to a lysine residue of a substrate Monoubiquitinated substrate can either dissociate from E3

(d) or can acquire additional Ub modifications in the form of multiple

single attachments (not shown) or a ubiquitin chain

(e) The chain can be knit together via different lysine residues of ubiquitin

Whereas monoubiquitin and some types of chains (e.g., those assembled via Lys63

of ubiquitin) serve mainly to alter the function of the modified protein

(f) (by changing its structure, binding partners, cellular localization, ),

polyubiquitin chains assembled via the Lys48 residue of ubiquitin typically direct the appended substrate to the proteasome for degradation

(g) The biological outcome of ubiquitination—be it degradation or

signaling—is normally dictated by ubiquitin receptors (UbR) that bind and interpret

the ubiquitin signal

Adopted from (42) RING domain E3 ubiquitin ligases Annual Review of

Biochemistry 78 (2009), 399-434

So far, more than 500 E3 ubiquitin ligases have been identified in humans

and substrate specificity is largely determined by them (40, 41) The majority of

them can be divided into two categories based on specific structural motifs: those possessing the HECT (homologous to the E6-AP carboxyl terminus) domain and

those containing the RING (really interesting new gene)-finger domain (41, 43)