E3 ligase negatively regulates myogenesis by IRS-1 ubiquitination Young-Gyu Ko College of Life Science and Biotechnology Korea University MyHC immunofluorescence
Lipid rafts are plasma membrane compartments composed of cholesterol and glycosphingolipids.
Insulin action through lipid rafts Signaling molecules found in lipid rafts; IR, IRS, Grb2, Sos, Ras, PI-3-K, TC10, Cbl, CAP, and GLUT4
Identification of novel signaling molecules by lipid raft proteome 지질래프트는신호전달물질을농축하고있어서새로운신호전달물질의동정에매우유리
Functional proteomics of lipid rafts
Functional proteomics of lipid rafts The function of Cell Death Differ, 2010 Lipid raft proteome papers in our lab, gc1qr, surface OXPHOS JBC, 2011; a paper of the week Expert Rev. Proteomics, 2010 CDD, 2010 BBRC, 2010 Proteomics, 2010; cover paper Proteomics, 2009; issue paper Proteomics, 2006; cover paper, 51 citations Diabetologia, 2006; 82 citations EMM, 2004; 66 citations Proteomics, 2004; 30 citations Proteomics, 2004; 108 citations
C2C12 Myogenesis Myoblasts Myotubes Myosin Heavy Chain
is found in the lipid rafts of C2C12 myotubes. Lee et al., CDD, 2010
gene analysis Myoblasts Myotubes 1 477 Tripartite Motif (TRIM) E3 ligase activity SPla and RYanodine receptor domain Lee et al., CDD, 2010
is specifically expressed in skeletal and cardiac muscle 4.4 Kb 2.4 Kb Cav-3 Lee et al., CDD, 2010
is highly expressed during C2C12 myogenesis 0 1 2 3 4 0 1 2 3 4 5 6 7 days after differentiation Cav-3 Cav-3 Myogenin Myogenin MyoD MHC MHC Actin Myostatin MyoD Myf5 IRS-1 Coomasie Staining Lee et al., CDD, 2010
is a myogenesis inhibitor Lee et al., CDD, 2010
What is a molecular target of in IGF signaling?
blocks Akt activation in IGF signaling. Ad-EV 0 10 30 60 120 Ad- 0 10 30 60 120 Si-Con 0 10 30 60 120 Si- 0 10 30 60 120 mins after IGF activation p-akt IGF Akt IGFR p-mapk IRS-1 MAPK PI-3-K Myoblast Myotube Actin p-akt mtor Hypertrophy Lee et al., CDD, 2010
supresses IRS-1 phosphorylation by IGF-1. 0 2 10 0 2 10 mins after IGF activation Ad-EV Ad-TRIM Ad-EV Ad-TRIM Ad-EV Ad-TRIM Si-Con Si-TRIM Si-Con Si-TRIM Si-Con Si-TRIM c pakt Akt IP, IGFR; IB, ptyr IP: IRS-1; IB, ptyr IGF IGFR IRS-1 IP: IRS-1; IB, IGFR PI-3-K IP: IRS-1; IB, PI-3K p-akt Myotube Myoblast IP: IGFR; IB, IGFR IP: IRS-1; IB, IRS-1 mtor Hypertrophy Lee et al., CDD, 2010
Molecular association of with IRS-1 WCL Mock Ab 0 5 30 0 5 30 mins after IGF activation IP: anti-irs1 IRS-1 IGF IGFR IP: anti-cemi IRS-1 IRS-1 PI-3-K p-akt mtor Hypertrophy Lee et al., CDD, 2010
negatively regulates myogenesis via targeting IRS-1. Lee et al., CDD, 2010
E3 ligase activity??? Protein Ubiquitination
Ring Finger domain of Consensus Sequence of Ring Finger Domain, Zn 2+ binding site CX 2 CX (9-39) CX (1-3) HX (2-3) C/HX 2 CX (4-48) CX 2 C sequence MSAAPGLLHQELSCPLCLQLFDAPVTAECGHSFCRACLGRVAGEPAADGTVLC PCCQAPTRPQALSTNLQLARLVEGLAQVPQGHCEEHLDPLSIYCEQDRALVCGV CASLGSHRGHRLLPAAEAHARLKTQLPQQKLQLQEACMRKEKSVAVLEHQLVV EETVRQFRGAVGEQLGKMRVFLAALEGSLDCEAERVRGEAGVALRRELGSLNS YLEQLRQMEKVLEEVADKPQTEFLMKYCLVTSRLQKILAESPPPARLDIQLPIISD DFKFQVWRKMFRALMPALEELTFDPSSAHPSLVVSSSGRRVECSEQKAPPAGED PRQFDKAVAVVAHQQLSEGEHYWEVDVGDKPRWALGVIAAEAPRRGRLHAVPS QGLWLLGLREGKILEAHVEAKEPRALRSPERRPTRIGLYLSFGDGVLSFYDASD ADALVPLFAFHERLPRPVYPFFDVCWHDKGKNAQPLLLVGPEGAEA C 14 C 17 C 29 H 31 C 34 C 37 C 53 C 56 Zn 2+ Zn 2+ C14A mutant? RING mutant?
RING domain of is essential for the negative regulation of myogenesis. AD-LacZ AD-TRIM AD-C14A AD- R 20 µm Myogenic index (%) 100 80 60 40 20 ** * : < 0.01 ** : < 0.05 * ** MyHC Caveolin-3 Myogenin IRS-1 Actin C2C12 myotubes
RING domain of is required for the negative regulation of IGF signaling. 0 2 5 0 2 5 0 2 5 0 2 5 IGF-1 (min) pakt Akt perk1/2 WCL Erk1/2 IRS-1 Actin IP:IRS-1, IB:pY IGF IGFR IRS-1 PI-3-K IP:IRS-1, IB:IRS-1 IP:IRS-1, IB:PI3K IP:IGFR, IB:IRS-1 IP:IGFR, IB:pY IB:IGFR, IB:IGFR p-akt mtor Hypertrophy C2C12 myoblasts
RING domain-lacking mutants work as dominant negative forms of endogenous. - + - + - + - - + + - + - - - - + + Myc-TRIM Flag-TRIM Flag-C14A - + - + - - + + Myc-TRIM Flag- R IP: Myc, IB: Myc IP: Myc, IB: Myc IP: Myc, IB: Flag IP: Myc, IB: Flag IP: Flag, IB: Flag IP: Flag, IB: Flag IP: Flag, IB: Myc IP: Flag, IB: Myc IB: Myc (WCL) IB: Myc (WCL) IB: Flag (WCL) IB: Flag (WCL) oligomerization HEK 293 cells
RING domain does not prevent binding of to IRS-1 Differentiation days 0 1 2 3 7 A IRS-1 mrna Actin mrna + + + + Flag-IRS-1 Myogenin MyHC Cav-3 IP:Flag, IB:Flag IP:Flag, IB:HA IP:HA, IB:Flag IP:HA, IB:HA IRS-1 IB:Flag (WCL) Actin IB:HA (WCL) C2C12 cells HEK 293 cells
RING domain of is required for IRS-1 degradation. - - - - + + + + - MG132 Flag-hIRS-1 HA-h IB : Flag IB : HA 140 - MG132 - - + + - + - + + MG132 - - + + - + - + Flag-hIRS-1 HA-h IB: Flag IB: HA IRS-1 Expression Level (%) 120 100 80 60 40 20 0 + + + + - + - + - - + + Flag-hIRS-1 HA-h MG132 HEK 293 cells
RING domain of is required for IRS-1 ubiquitination. - - - + - - - - - + - - - - - - - + - + + + + + - + + + + + HA-TRIM HA-C14A HA- R Flag-IRS-1 His-Ub IP, α-flag; IB, α-his IP, α-flag; IB, α-flag IB: α-ha (WCL) IB: α-flag (WCL) In the presence of MG132 HEK 293 cells
RING domain of is required for IRS-1 ubiquitination. -MG132 +MG132 + - + - - + - + si-control si- IRS-1 IRS-1 IP: α-irs-1 IB: α-ub IP: α-irs-1 IB: α-ub C2C12 Myoblasts IP: α-irs-1 IB: α-irs-1 IP: α-irs-1 IB: α-irs-1 C2C12 Myotubes
disruption enhances MyoD-driven myogenesis in MEFs. WT KO Differentiation days 0 1 2 3 4 MyoD Myogenin Caveolin-3 MyHC IRS-1 Actin Nuclei No. 5 4 3 2 1 WT KO MyoD Caveolin-3 Myogenin MyHC 0 WT KO IRS-1 Actin MyoD-driven myotubes from +/+ and -/- MEFs
disruption abolishes IRS-1 ubiquitination in MyoD-driven myotubes of MEFs. - - + + MG132 WT KO WT KO IRS-1 IP: α-irs-1 IB: α-ub IP: α-irs-1 IB: α-irs-1 MyoD-driven myotubes from +/+ and -/- MEFs
E3 ligase activity??? Protein Ubiquitination
UbcH2 mrna and protein are increased during C2C12 myogenesis Differentiation days 0 1 2 3 4 5 UbcH2 UbcH3 UbcH5b UbcH6 UbcH2/GAPDH 4.0 3.0 2.0 1.0 ** * Real-time PCR UbcH7 UbcH8 UbcH10 Ubc12 0 1 2 3 4 5 Differentiation days Differentiation days 0 1 2 3 4 5 UBCH2 Ubc13 Cav-3 Mgn MyHC IRS1 MyoD Cav-3 GAPDH Actin
Molecular association of with UbcH2 IP:MBP IB:His IB:MBP IB:His Purified and E2 enzymes In vitro binding assay
Molecular association of with UbcH2 - + - + Flag-UbcH2 - - + + Myc-WT Flag Myc Myc Flag Flag Myc WCL IP:IMyc IP:IFlag UbcH2 UbcH2 IP: IP: UbcH2 Exogenous IP in 293 cells Endogenous IP in C2C12 myotubes
UbcH2 knockdown enhances myogenesis by inhibiting IRS-1 ubiquitination. Si-control Si-UbcH2 + - + - - + - + Si-control Si-UbcH2 Myogenic index (%) 60 50 40 30 20 10 * UbcH2 MyHC Cav-3 Mgn IRS-1 UbcH2 IRS-1 WCL Ubiquitin IRS-1 IP:IRS-1 Actin C2C12 myotubes
inhibition of myogenesis is released by UbcH2 knockdown Si-control HA- Si-UbcH2 HA- 9 8 7 6 5 4 3 2 1 si-control HA- si-ubch2 HA- The number of nucleus In HA-positive cells MyHC HA DAPI C2C12 myotubes
is highly expressed in soleus muscle. soleus gastrocnemius Color Red muscle White muscle Size Smaller Larger Fatigue Resistant High Sensitive Low Jung and Ko, BBRC, 2010
Skeletal muscle size is increased in -disrupted mice. 15.0 * <0.05 Weight (mg) 10.0 5.0 0.0 WT WT KO KO Percentage (%) 18.0 16.0 14.0 12.0 10.0 8.0 6.0 4.0 WT KO 2.0 Cross Sectioned Area (mm 2 ) Mouse soleus muscle
IRS-1 expression level is elevated in TRIM-disrupted skeletal muscle WT - + KO - + Insulin 0 5 10 0 5 10 Insulin (min) pakt Akt perk Erk Actin IRS-1 pakt Akt perk ERK Actin C2C12 Myoblasts py IRS-1 IP:IRS-1 Mouse soleus muscle
is a therapeutic target for type 2 diabetes Plasma glucose (mg/dl) 500 400 Glucose tolerance test WT KO *p<0.05 * * 300 200 100 0 0 15 30 45 60 Time (min) Plasma glucose (% reduction) 120 * 100 80 60 Insulin tolerance test *p<0.05 * * WT KO 40 20 0 0 15 30 45 60 Time (min) High fat diet-fed mice
E3 ligase negatively regulates myogenesis by IRS-1 ubiquitination. by Ub
Suggestion; is a negative muscle size regulator.
Korea University Lab of Cell Signal Transduction Dr. Jae-Sung Yi Dr. Chang-Seok Lee Dr. Young-Mi Ham Miss Na-Rae Lee Ms. Nga Nguyen Mr. Jing Hong Mr. June-Seop Lee Mr. Jeong-Woo Lee Mr. Dong-Min Yoo