E3 ligase TRIM72 negatively regulates myogenesis by IRS-1 ...
E3 ligase TRIM72 negatively regulates myogenesis by IRS-1 ...
E3 ligase TRIM72 negatively regulates myogenesis by IRS-1 ...
- No tags were found...
You also want an ePaper? Increase the reach of your titles
YUMPU automatically turns print PDFs into web optimized ePapers that Google loves.
<strong>E3</strong> <strong>ligase</strong> <strong>TRIM72</strong> <strong>negatively</strong> <strong>regulates</strong><br />
<strong>myogenesis</strong> <strong>by</strong> <strong>IRS</strong>-1 ubiquitination<br />
Young-Gyu Ko<br />
College of Life Science and Biotechnology<br />
Korea University<br />
MyHC immunofluorescence
Lipid rafts are plasma membrane compartments composed of<br />
cholesterol and glycosphingolipids.
Insulin action through lipid rafts<br />
Signaling molecules found in lipid rafts; IR, <strong>IRS</strong>, Grb2,<br />
Sos, Ras, PI-3-K, TC10, Cbl, CAP, and GLUT4
Identification of novel signaling molecules<br />
<strong>by</strong> lipid raft proteome<br />
지질래프트는 신호전달물질을 농축하고 있어서 새로운 신호전달물질의 동정에 매우 유리
Functional proteomics of lipid rafts
Functional proteomics of lipid rafts<br />
The function of <strong>TRIM72</strong><br />
Cell Death Differ, 2010<br />
Lipid raft proteome papers in our lab<br />
<strong>TRIM72</strong>, gC1qR, surface OXPHOS<br />
JBC, 2011; a paper of the week<br />
Expert Rev. Proteomics, 2010<br />
CDD, 2010<br />
BBRC, 2010<br />
Proteomics, 2010; cover paper<br />
Proteomics, 2009; issue paper<br />
Proteomics, 2006; cover paper, 51 citations<br />
Diabetologia, 2006; 82 citations<br />
EMM, 2004; 66 citations<br />
Proteomics, 2004; 30 citations<br />
Proteomics, 2004; 108 citations
C2C12 Myogenesis<br />
Myoblasts<br />
Myotubes<br />
Myosin Heavy Chain
<strong>TRIM72</strong> is found in the lipid rafts of C2C12 myotubes.<br />
Lee et al., CDD, 2010
<strong>TRIM72</strong> gene analysis<br />
Myoblasts<br />
Myotubes<br />
1<br />
477<br />
Tripartite Motif (TRIM)<br />
<strong>E3</strong> <strong>ligase</strong> activity<br />
SPla and RYanodine<br />
receptor domain<br />
Lee et al., CDD, 2010
<strong>TRIM72</strong> is specifically expressed in skeletal and cardiac muscle<br />
4.4 Kb<br />
2.4 Kb<br />
<strong>TRIM72</strong><br />
Cav-3<br />
Lee et al., CDD, 2010
<strong>TRIM72</strong> is highly expressed during C2C12 <strong>myogenesis</strong><br />
0 1 2 3 4<br />
<strong>TRIM72</strong><br />
0 1 2 3 4 5 6 7<br />
days after<br />
differentiation<br />
<strong>TRIM72</strong><br />
Cav-3<br />
Cav-3<br />
Myogenin<br />
Myogenin<br />
MyoD<br />
MHC<br />
MHC<br />
Actin<br />
Myostatin<br />
MyoD<br />
Myf5<br />
<strong>IRS</strong>-1<br />
Coomasie<br />
Staining<br />
Lee et al., CDD, 2010
<strong>TRIM72</strong> is a <strong>myogenesis</strong> inhibitor<br />
Lee et al., CDD, 2010
What is a molecular target of <strong>TRIM72</strong> in IGF signaling
<strong>TRIM72</strong> blocks Akt activation in IGF signaling.<br />
Ad-EV<br />
0<br />
10<br />
30<br />
60<br />
120<br />
Ad-<strong>TRIM72</strong><br />
0<br />
10<br />
30<br />
60<br />
120<br />
Si-Con<br />
0<br />
10<br />
30<br />
60<br />
120<br />
Si-<strong>TRIM72</strong><br />
0<br />
10<br />
30<br />
60<br />
120<br />
mins after<br />
IGF activation<br />
<strong>TRIM72</strong><br />
p-Akt<br />
IGF<br />
Akt<br />
IGFR<br />
p-MAPK<br />
<strong>IRS</strong>-1<br />
MAPK<br />
PI-3-K<br />
Myoblast<br />
Myotube<br />
Actin<br />
p-AKT<br />
mTOR<br />
Hypertrophy<br />
Lee et al., CDD, 2010
<strong>TRIM72</strong> supresses <strong>IRS</strong>-1 phosphorylation <strong>by</strong> IGF-1.<br />
0 2 10<br />
0 2 10<br />
mins after<br />
IGF activation<br />
Ad-EV<br />
Ad-TRIM<br />
Ad-EV<br />
Ad-TRIM<br />
Ad-EV<br />
Ad-TRIM<br />
Si-Con<br />
Si-TRIM<br />
Si-Con<br />
Si-TRIM<br />
Si-Con<br />
Si-TRIM<br />
c<br />
<strong>TRIM72</strong><br />
pAkt<br />
Akt<br />
IP, IGFR; IB, pTyr<br />
IP: <strong>IRS</strong>-1; IB, pTyr<br />
IGF<br />
IGFR<br />
<strong>IRS</strong>-1<br />
IP: <strong>IRS</strong>-1; IB, IGFR<br />
PI-3-K<br />
IP: <strong>IRS</strong>-1; IB, PI-3K<br />
p-AKT<br />
Myotube<br />
Myoblast<br />
IP: IGFR; IB, IGFR<br />
IP: <strong>IRS</strong>-1; IB, <strong>IRS</strong>-1<br />
mTOR<br />
Hypertrophy<br />
Lee et al., CDD, 2010
Molecular association of <strong>TRIM72</strong> with <strong>IRS</strong>-1<br />
WCL<br />
Mock<br />
Ab<br />
0 5 30 0 5 30 mins after<br />
IGF activation<br />
<strong>TRIM72</strong><br />
IP: anti-<strong>IRS</strong>1<br />
<strong>IRS</strong>-1<br />
IGF<br />
IGFR<br />
IP: anti-CEMI<br />
<strong>IRS</strong>-1<br />
<strong>TRIM72</strong><br />
<strong>IRS</strong>-1<br />
PI-3-K<br />
<strong>TRIM72</strong><br />
p-AKT<br />
mTOR<br />
Hypertrophy<br />
Lee et al., CDD, 2010
<strong>TRIM72</strong> <strong>negatively</strong> <strong>regulates</strong> <strong>myogenesis</strong> via targeting <strong>IRS</strong>-1.<br />
Lee et al., CDD, 2010
<strong>E3</strong> <strong>ligase</strong> activity <br />
Protein Ubiquitination
Ring Finger domain of <strong>TRIM72</strong><br />
Consensus Sequence of Ring Finger Domain, Zn 2+ binding site<br />
CX 2 CX (9-39) CX (1-3) HX (2-3) C/HX 2 CX (4-48) CX 2 C<br />
<strong>TRIM72</strong> sequence<br />
MSAAPGLLHQELSCPLCLQLFDAPVTAECGHSFCRACLGRVAGEPAADGTVLC<br />
PCCQAPTRPQALSTNLQLARLVEGLAQVPQGHCEEHLDPLSIYCEQDRALVCGV<br />
CASLGSHRGHRLLPAAEAHARLKTQLPQQKLQLQEACMRKEKSVAVLEHQLVV<br />
EETVRQFRGAVGEQLGKMRVFLAALEGSLDCEAERVRGEAGVALRRELGSLNS<br />
YLEQLRQMEKVLEEVADKPQTEFLMKYCLVTSRLQKILAESPPPARLDIQLPIISD<br />
DFKFQVWRKMFRALMPALEELTFDPSSAHPSLVVSSSGRRVECSEQKAPPAGED<br />
PRQFDKAVAVVAHQQLSEGEHYWEVDVGDKPRWALGVIAAEAPRRGRLHAVPS<br />
QGLWLLGLREGKILEAHVEAKEPRALRSPERRPTRIGLYLSFGDGVLSFYDASD<br />
ADALVPLFAFHERLPRPVYPFFDVCWHDKGKNAQPLLLVGPEGAEA<br />
C 14 C 17 C 29 H 31 C 34 C 37 C 53 C 56<br />
<strong>TRIM72</strong> C14A mutant <br />
<strong>TRIM72</strong> ∆RING mutant <br />
Zn 2+ Zn 2+
RING domain of <strong>TRIM72</strong> is essential for the negative regulation of <strong>myogenesis</strong>.<br />
AD-LacZ AD-TRIM AD-C14A<br />
AD-∆R<br />
20 µm<br />
Myogenic index (%)<br />
100<br />
80<br />
60<br />
40<br />
20<br />
**<br />
* : < 0.01<br />
** : < 0.05<br />
*<br />
**<br />
<strong>TRIM72</strong><br />
MyHC<br />
Caveolin-3<br />
Myogenin<br />
<strong>IRS</strong>-1<br />
Actin<br />
C2C12 myotubes
RING domain of <strong>TRIM72</strong> is required for the negative regulation<br />
of IGF signaling.<br />
0 2 5 0 2 5 0 2 5 0 2 5<br />
IGF-1 (min)<br />
<strong>TRIM72</strong><br />
pAkt<br />
Akt<br />
pErk1/2<br />
WCL<br />
Erk1/2<br />
<strong>IRS</strong>-1<br />
Actin<br />
IP:<strong>IRS</strong>-1, IB:pY<br />
IGF<br />
IGFR<br />
<strong>IRS</strong>-1<br />
PI-3-K<br />
<strong>TRIM72</strong><br />
IP:<strong>IRS</strong>-1, IB:<strong>IRS</strong>-1<br />
IP:<strong>IRS</strong>-1, IB:PI3K<br />
IP:IGFR, IB:<strong>IRS</strong>-1<br />
IP:IGFR, IB:pY<br />
IB:IGFR, IB:IGFR<br />
p-AKT<br />
mTOR<br />
Hypertrophy<br />
C2C12 myoblasts
RING domain-lacking <strong>TRIM72</strong> mutants work as dominant<br />
negative forms of endogenous <strong>TRIM72</strong>.<br />
- + - + - +<br />
- - + + - +<br />
- - - - + +<br />
Myc-TRIM<br />
Flag-TRIM<br />
Flag-C14A<br />
- + - +<br />
- - + +<br />
Myc-TRIM<br />
Flag-∆R<br />
IP: Myc, IB: Myc<br />
IP: Myc, IB: Myc<br />
IP: Myc, IB: Flag<br />
IP: Myc, IB: Flag<br />
IP: Flag, IB: Flag<br />
IP: Flag, IB: Flag<br />
IP: Flag, IB: Myc<br />
IP: Flag, IB: Myc<br />
IB: Myc (WCL)<br />
IB: Myc (WCL)<br />
IB: Flag (WCL)<br />
IB: Flag (WCL)<br />
<strong>TRIM72</strong> oligomerization<br />
HEK 293 cells
RING domain does not prevent binding of <strong>TRIM72</strong> to <strong>IRS</strong>-1<br />
Differentiation days<br />
0 1 2 3 7<br />
A<br />
<strong>IRS</strong>-1 mRNA<br />
Actin mRNA<br />
+<br />
+<br />
+<br />
+<br />
Flag-<strong>IRS</strong>-1<br />
<strong>TRIM72</strong><br />
Myogenin<br />
MyHC<br />
Cav-3<br />
IP:Flag, IB:Flag<br />
IP:Flag, IB:HA<br />
IP:HA, IB:Flag<br />
IP:HA, IB:HA<br />
<strong>IRS</strong>-1<br />
IB:Flag (WCL)<br />
Actin<br />
IB:HA (WCL)<br />
C2C12 cells<br />
HEK 293 cells
RING domain of <strong>TRIM72</strong> is required for <strong>IRS</strong>-1 degradation.<br />
- - - -<br />
+ + + +<br />
-<br />
MG132<br />
Flag-h<strong>IRS</strong>-1<br />
HA-h<strong>TRIM72</strong><br />
IB : Flag<br />
IB : HA<br />
140<br />
- MG132<br />
- - + +<br />
- + - +<br />
+ MG132<br />
- - + +<br />
- + - +<br />
Flag-h<strong>IRS</strong>-1<br />
HA-h<strong>TRIM72</strong><br />
IB: Flag<br />
IB: HA<br />
<strong>IRS</strong>-1 Expression<br />
Level (%)<br />
120<br />
100<br />
80<br />
60<br />
40<br />
20<br />
0<br />
+ + + +<br />
-<br />
+<br />
-<br />
+<br />
- - + +<br />
Flag-h<strong>IRS</strong>-1<br />
HA-h<strong>TRIM72</strong><br />
MG132<br />
HEK 293 cells
RING domain of <strong>TRIM72</strong> is required for <strong>IRS</strong>-1 ubiquitination.<br />
-<br />
-<br />
-<br />
+<br />
-<br />
- - - - + -<br />
- - - - -<br />
-<br />
+<br />
- + + + + +<br />
- + + + + +<br />
HA-TRIM<br />
HA-C14A<br />
HA-∆R<br />
Flag-<strong>IRS</strong>-1<br />
His-Ub<br />
IP, α-Flag; IB, α-His<br />
IP, α-Flag; IB, α-Flag<br />
IB: α-HA (WCL)<br />
IB: α-Flag (WCL)<br />
In the presence of MG132<br />
HEK 293 cells
RING domain of <strong>TRIM72</strong> is required for <strong>IRS</strong>-1 ubiquitination.<br />
-MG132<br />
+MG132<br />
+ - + -<br />
- + - +<br />
si-Control<br />
si-<strong>TRIM72</strong><br />
<strong>TRIM72</strong><br />
<strong>IRS</strong>-1<br />
<strong>TRIM72</strong><br />
<strong>IRS</strong>-1<br />
IP: α-<strong>IRS</strong>-1<br />
IB: α-Ub<br />
IP: α-<strong>IRS</strong>-1<br />
IB: α-Ub<br />
C2C12 Myoblasts<br />
IP: α-<strong>IRS</strong>-1<br />
IB: α-<strong>IRS</strong>-1<br />
IP: α-<strong>IRS</strong>-1<br />
IB: α-<strong>IRS</strong>-1<br />
C2C12 Myotubes
<strong>TRIM72</strong> disruption enhances MyoD-driven <strong>myogenesis</strong> in MEFs.<br />
WT<br />
KO<br />
Differentiation days<br />
0 1 2 3 4<br />
<strong>TRIM72</strong><br />
MyoD<br />
Myogenin<br />
Caveolin-3<br />
MyHC<br />
<strong>IRS</strong>-1<br />
Actin<br />
Nuclei No.<br />
5<br />
4<br />
3<br />
2<br />
1<br />
WT<br />
KO<br />
<strong>TRIM72</strong><br />
MyoD<br />
Caveolin-3<br />
Myogenin<br />
MyHC<br />
0<br />
WT<br />
KO<br />
<strong>IRS</strong>-1<br />
Actin<br />
MyoD-driven myotubes from <strong>TRIM72</strong> +/+ and -/- MEFs
<strong>TRIM72</strong> disruption abolishes <strong>IRS</strong>-1 ubiquitination<br />
in MyoD-driven myotubes of MEFs.<br />
- - + +<br />
MG132<br />
WT<br />
KO<br />
WT<br />
KO<br />
<strong>IRS</strong>-1<br />
<strong>TRIM72</strong><br />
IP: α-<strong>IRS</strong>-1<br />
IB: α-Ub<br />
IP: α-<strong>IRS</strong>-1<br />
IB: α-<strong>IRS</strong>-1<br />
MyoD-driven myotubes from <strong>TRIM72</strong> +/+ and -/- MEFs
<strong>E3</strong> <strong>ligase</strong> activity <br />
Protein Ubiquitination
UbcH2 mRNA and protein are increased during C2C12 <strong>myogenesis</strong><br />
Differentiation days<br />
0 1 2 3 4 5<br />
UbcH2<br />
UbcH3<br />
UbcH5b<br />
UbcH6<br />
UbcH2/GAPDH<br />
4.0<br />
3.0<br />
2.0<br />
1.0<br />
**<br />
*<br />
Real-time PCR<br />
UbcH7<br />
UbcH8<br />
UbcH10<br />
Ubc12<br />
0 1 2 3 4 5<br />
Differentiation days<br />
Differentiation days<br />
0 1 2 3 4 5<br />
UBCH2<br />
Ubc13<br />
<strong>TRIM72</strong><br />
Cav-3<br />
Mgn<br />
MyHC<br />
<strong>IRS</strong>1<br />
MyoD<br />
Cav-3<br />
GAPDH<br />
Actin
Molecular association of <strong>TRIM72</strong> with UbcH2<br />
IP:MBP<br />
IB:His<br />
IB:MBP<br />
IB:His<br />
Purified <strong>TRIM72</strong> and E2 enzymes<br />
In vitro binding assay
Molecular association of <strong>TRIM72</strong> with UbcH2<br />
-<br />
+<br />
-<br />
+<br />
Flag-UbcH2<br />
- - + +<br />
Myc-WT<br />
Flag<br />
Myc<br />
Myc<br />
Flag<br />
Flag<br />
Myc<br />
WCL IP:IMyc IP:IFlag<br />
UbcH2<br />
<strong>TRIM72</strong><br />
<strong>TRIM72</strong><br />
UbcH2<br />
IP:<br />
<strong>TRIM72</strong><br />
IP:<br />
UbcH2<br />
Exogenous IP<br />
in 293 cells<br />
Endogenous IP<br />
in C2C12 myotubes
UbcH2 knockdown enhances <strong>myogenesis</strong><br />
<strong>by</strong> inhibiting <strong>IRS</strong>-1 ubiquitination.<br />
Si-control<br />
Si-UbcH2<br />
+ - + -<br />
- + - +<br />
Si-control<br />
Si-UbcH2<br />
Myogenic index (%)<br />
60<br />
50<br />
40<br />
30<br />
20<br />
10<br />
*<br />
UbcH2<br />
MyHC<br />
Cav-3<br />
Mgn<br />
<strong>IRS</strong>-1<br />
UbcH2<br />
<strong>IRS</strong>-1<br />
WCL<br />
Ubiquitin<br />
<strong>IRS</strong>-1<br />
IP:<strong>IRS</strong>-1<br />
<strong>TRIM72</strong><br />
Actin<br />
C2C12 myotubes
<strong>TRIM72</strong> inhibition of <strong>myogenesis</strong> is released <strong>by</strong> UbcH2 knockdown<br />
Si-control<br />
HA-<strong>TRIM72</strong><br />
Si-UbcH2<br />
HA-<strong>TRIM72</strong><br />
9<br />
8<br />
7<br />
6<br />
5<br />
4<br />
3<br />
2<br />
1<br />
si-control<br />
HA-<strong>TRIM72</strong><br />
si-Ubch2<br />
HA-<strong>TRIM72</strong><br />
The number of nucleus<br />
In HA-positive cells<br />
MyHC<br />
HA<br />
DAPI<br />
C2C12 myotubes
<strong>TRIM72</strong> is highly expressed in soleus muscle.<br />
soleus<br />
gastrocnemius<br />
Color Red muscle White muscle<br />
Size Smaller Larger<br />
Fatigue<br />
<strong>TRIM72</strong><br />
Resistant<br />
High<br />
Sensitive<br />
Low<br />
Jung and Ko, BBRC, 2010
Skeletal muscle size is increased in <strong>TRIM72</strong>-disrupted mice.<br />
15.0<br />
*<br />
<strong>IRS</strong>-1 expression level is elevated in TRIM-disrupted skeletal muscle<br />
WT<br />
- +<br />
KO<br />
- + Insulin<br />
0 5 10 0 5 10 Insulin (min)<br />
<strong>TRIM72</strong><br />
pAkt<br />
Akt<br />
pERK<br />
Erk<br />
Actin<br />
<strong>TRIM72</strong><br />
<strong>IRS</strong>-1<br />
pAkt<br />
Akt<br />
pERK<br />
ERK<br />
Actin<br />
C2C12 Myoblasts<br />
pY<br />
<strong>IRS</strong>-1<br />
IP:<strong>IRS</strong>-1<br />
Mouse soleus muscle
<strong>TRIM72</strong> is a therapeutic target for type 2 diabetes<br />
Plasma glucose (mg/dl)<br />
500<br />
400<br />
Glucose tolerance test<br />
WT<br />
KO<br />
*p
<strong>E3</strong> <strong>ligase</strong> <strong>TRIM72</strong> <strong>negatively</strong> <strong>regulates</strong> <strong>myogenesis</strong><br />
<strong>by</strong> <strong>IRS</strong>-1 ubiquitination.<br />
<strong>by</strong> Ub
Suggestion; <strong>TRIM72</strong> is a negative muscle size regulator.
Korea University<br />
Lab of Cell Signal Transduction<br />
Dr. Jae-Sung Yi<br />
Dr. Chang-Seok Lee<br />
Dr. Young-Mi Ham<br />
Miss Na-Rae Lee<br />
Ms. Nga Nguyen<br />
Mr. Jing Hong<br />
Mr. June-Seop Lee<br />
Mr. Jeong-Woo Lee<br />
Mr. Dong-Min Yoo
![Microsoft PowerPoint - src.ppt [\310\243\310\257 \270\360\265\345]](https://img.yumpu.com/47977670/1/190x134/microsoft-powerpoint-srcppt-310243310257-270360265345.jpg?quality=85)
![Microsoft PowerPoint - src.ppt [\310\243\310\257 \270\360\265\345]](https://img.yumpu.com/44373305/1/190x134/microsoft-powerpoint-srcppt-310243310257-270360265345.jpg?quality=85)
Change language