2 group 20503 Kim ki yeon, 20539 Lee kun woo 20540 Lee joon hyuck, 20541 Jeong soon hyung.
E3 ligaseTRIM72 negatively regulates myogenesisby IRS-1 … · 2014-06-27 · Dr. Young-Mi Ham Miss...
Transcript of E3 ligaseTRIM72 negatively regulates myogenesisby IRS-1 … · 2014-06-27 · Dr. Young-Mi Ham Miss...
E3 ligase TRIM72 negatively regulates myogenesis by IRS-1 ubiquitination
Young-Gyu Ko
College of Life Science and BiotechnologyKorea University
MyHC immunofluorescence
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 moleculesby lipid raft proteome
지질래프트는 신호전달물질을 농축하고 있어서 새로운 신호전달물질의 동정에 매우 유리
The function of TRIM72Cell Death Differ, 2010
Lipid raft proteome papers in our lab
TRIM72, gC1qR, surface OXPHOSJBC, 2011; a paper of the weekExpert Rev. Proteomics, 2010CDD, 2010BBRC, 2010Proteomics, 2010; cover paperProteomics, 2009; issue paperProteomics, 2006; cover paper, 51 citationsDiabetologia, 2006; 82 citationsEMM, 2004; 66 citationsProteomics, 2004; 30 citationsProteomics, 2004; 108 citations
Functional proteomics of lipid rafts
TRIM72 gene analysis
Myoblasts
Myotubes
Tripartite Motif (TRIM)
E3 ligase activity
SPla and RYanodine
receptor domain
4771
Lee et al., CDD, 2010
4.4 Kb
2.4 Kb
TRIM72
Cav-3
TRIM72 is specifically expressed in skeletal and cardiac muscle
Lee et al., CDD, 2010
days after
differentiation0 1 2 3 4 5 6 7
MyoD
Myogenin
TRIM72
Cav-3
Myostatin
Coomasie
Staining
MHC
Myf5
IRS-1
0 1 2 3 4
TRIM72
Myogenin
MyoD
Cav-3
MHC
Actin
TRIM72 is highly expressed during C2C12 myogenesis
Lee et al., CDD, 2010
TRIM72 blocks Akt activation in IGF signaling.
Si-Con Si-TRIM72mins after
IGF activation
Ad-EV Ad-TRIM72
0 10
30
60120
TRIM72
p-Akt
Akt
Actin
p-MAPK
MAPK
Myoblast Myotube
0 10
30
60120
0 10
30
60
120
0 10
30
60
120
IGF
IGFR
IRS-1
PI-3-K
p-AKT
mTOR
Hyper-
trophy
Lee et al., CDD, 2010
TRIM72
mins after
IGF activation
pAkt
Akt
IP: IRS-1; IB, pTyr
IP, IGFR; IB, pTyr
IP: IRS-1; IB, PI-3K
IP: IRS-1; IB, IGFR
IP: IRS-1; IB, IRS-1
IP: IGFR; IB, IGFR
Si-Con
Si-TRIM
Si-Con
Si-TRIM
Si-Con
Si-TRIM
0 2 10
c
Myoblast
IGF
IGFR
IRS-1
PI-3-K
p-AKT
mTOR
Hyper-
trophy
TRIM72 supresses IRS-1 phosphorylation by IGF-1.
Ad-EV
Ad-TRIM
Ad-EV
Ad-TRIM
Ad-EV
Ad-TRIM
0 2 10
Myotube
Lee et al., CDD, 2010
Molecular association of TRIM72 with IRS-1
IGF
IGFR
IRS-1
PI-3-K
p-AKT
mTOR
Hyper-
trophy
TRIM72
IRS-1
TRIM72
IRS-1
TRIM72
Mock Ab
0 5 300 5 30 mins after
IGF activation
IP: anti-IRS1
IP: anti-CEMI
WCL
Lee et al., CDD, 2010
TRIM72 sequenceMSAAPGLLHQELSCPLCLQLFDAPVTAECGHSFCRACLGRVAGEPAADGTVLC
PCCQAPTRPQALSTNLQLARLVEGLAQVPQGHCEEHLDPLSIYCEQDRALVCGV
CASLGSHRGHRLLPAAEAHARLKTQLPQQKLQLQEACMRKEKSVAVLEHQLVV
EETVRQFRGAVGEQLGKMRVFLAALEGSLDCEAERVRGEAGVALRRELGSLNS
YLEQLRQMEKVLEEVADKPQTEFLMKYCLVTSRLQKILAESPPPARLDIQLPIISD
DFKFQVWRKMFRALMPALEELTFDPSSAHPSLVVSSSGRRVECSEQKAPPAGED
PRQFDKAVAVVAHQQLSEGEHYWEVDVGDKPRWALGVIAAEAPRRGRLHAVPS
QGLWLLGLREGKILEAHVEAKEPRALRSPERRPTRIGLYLSFGDGVLSFYDASD
ADALVPLFAFHERLPRPVYPFFDVCWHDKGKNAQPLLLVGPEGAEA
Ring Finger domain of TRIM72
Consensus Sequence of Ring Finger Domain, Zn2+ binding site
CX2CX(9-39)CX(1-3)HX(2-3)C/HX2CX(4-48)CX2C
C14C17C29H31C34C37C53C56
Zn2+ Zn2+
TRIM72 C14A mutant ?
TRIM72 ∆RING mutant ?
20 µm
AD-LacZ AD-TRIM AD-∆RAD-C14A
20
40
60
80
100
Myo
gen
icin
dex
(%
)
*
**
**
* : < 0.01** : < 0.05
MyHC
Caveolin-3
Myogenin
IRS-1
Actin
RING domain of TRIM72 is essential for the negative regulation of myogenesis.
C2C12 myotubes
TRIM72
IGF
IGFR
IRS-1
PI-3-K
p-AKT
mTOR
Hyper-
trophy
TRIM72
RING domain of TRIM72 is required for the negative regulation
of IGF signaling.
C2C12 myoblasts
TRIM72
pAkt
Akt
IP:IGFR, IB:pY
IP:IRS-1, IB:pY
IP:IRS-1, IB:PI3K
IB:IGFR, IB:IGFR
IP:IGFR, IB:IRS-1
IRS-1
Actin
0 2 5 0 2 5 0 2 5 0 2 5
IP:IRS-1, IB:IRS-1
IGF-1 (min)
pErk1/2
Erk1/2
WCL
IP: Myc, IB: Myc
IP: Flag, IB: Myc
IP: Flag, IB: Flag
IP: Myc, IB: Flag
IB: Myc (WCL)
IB: Flag (WCL)
Myc-TRIM+- - + - +Flag-TRIM- - + + - +Flag-C14A- - - - + +
IP: Myc, IB: Myc
IP: Flag, IB: Myc
IP: Flag, IB: Flag
IP: Myc, IB: Flag
IB: Myc (WCL)
IB: Flag (WCL)
Myc-TRIM+- - +Flag-∆R- - + +
RING domain-lacking TRIM72 mutants work as dominant
negative forms of endogenous TRIM72.
TRIM72 oligomerization
HEK 293 cells
RING domain does not prevent binding of TRIM72 to IRS-1
A0 1 2 3 7
IRS-1 mRNA
Actin mRNA
Differentiation days
TRIM72
Actin
Myogenin
MyHC
Cav-3
IRS-1
Flag-IRS-1++++
IP:Flag, IB:Flag
IP:Flag, IB:HA
IP:HA, IB:Flag
IP:HA, IB:HA
IB:Flag (WCL)
IB:HA (WCL)
C2C12 cells HEK 293 cells
HA-hTRIM72
Flag-hIRS-1
IB: HA
IB: Flag
- + - +
- - + +
- MG132
- + - +
- - + +
+ MG132
0
20
40
60
80
100
120
140
Flag-hIRS-1HA-hTRIM72
+ + + ++-+-
MG132- - ++
IRS-
1 Ex
pre
ssio
nLe
vel (%
)
Flag-hIRS-1HA-hTRIM72
IB : HA
IB : Flag
-+ + + +
MG132----
RING domain of TRIM72 is required for IRS-1 degradation.
HEK 293 cells
RING domain of TRIM72 is required for IRS-1 ubiquitination.
HEK 293 cells
+- + + + +- + + + + +- - - - -- - - - -
-----
++
+ HA-TRIM
IP, α-Flag; IB, α-His
IB: α-HA (WCL)
IB: α-Flag (WCL)
HA-C14AHA-∆RFlag-IRS-1His-Ub
IP, α-Flag; IB, α-Flag
In the presence of MG132
C2C12 Myotubes
- + - ++ - + -
si-TRIM72
si-Control
TRIM72
IRS-1
IP: α-IRS-1IB: α-Ub
IP: α-IRS-1IB: α-IRS-1
IP: α-IRS-1IB: α-Ub
IP: α-IRS-1IB: α-IRS-1
C2C12 Myoblasts
-MG132 +MG132
TRIM72
IRS-1
RING domain of TRIM72 is required for IRS-1 ubiquitination.
0 1 2 3 4
Differentiation days
TRIM72
MyoD
Myogenin
Caveolin-3
MyHC
IRS-1
Actin
WT KO
TRIM72 disruption enhances MyoD-driven myogenesis in MEFs.
MyoD-driven myotubes from TRIM72+/+ and -/- MEFs
0
1
2
3
4
5
Nucl
ei N
o.
WT KO
TRIM72
MyoD
Caveolin-3
Myogenin
MyHC
IRS-1
Actin
WT
KO
TRIM72 disruption abolishes IRS-1 ubiquitination
in MyoD-driven myotubes of MEFs.
MyoD-driven myotubes from TRIM72+/+ and -/- MEFs
TRIM72
IRS-1
IP: α-IRS-1IB: α-Ub
IP: α-IRS-1IB: α-IRS-1
WT
KO
WT
KO
MG132- - + +
UbcH2
UbcH3
UbcH8
UbcH10
Cav-3
GAPDH
MyHC
MyoD
UbcH7
UbcH5b
0 1 2 3 4 5
Differentiation days
UbcH6
Ubc12
Ubc13
1.0
2.0
3.0
4.0
0 1 2 3 4 5
**
*
UbcH2/GAPDH
Differentiation days
UbcH2 mRNA and protein are increased during C2C12 myogenesis
Real-time PCR
Actin
Cav-3
IRS1
Mgn
TRIM72
UBCH2
0 1 2 3 4 5
Differentiation days
IP:MBPIB:His
IB:MBP
IB:His
Molecular association of TRIM72 with UbcH2
Purified TRIM72 and E2 enzymes In vitro binding assay
TRIM72
UbcH2
UbcH2
TRIM72
IP:TRIM72
IP:UbcH2
Flag
Myc
Myc
Flag
Myc
Flag
Flag-UbcH2+-+-
Myc-WT- - + +
IP:IF
lag
IP:IM
ycW
CL
Molecular association of TRIM72 with UbcH2
Exogenous IPin 293 cells
Endogenous IPin C2C12 myotubes
Si-control Si-UbcH2
10
20
30
40
50
60
Myo
gen
ic index
(%
)
*
Mgn
Actin
UbcH2
Cav-3
IRS-1
TRIM72
MyHC
- + - + Si-UbcH2+ - + - Si-control
UbcH2
IRS-1
Ubiquitin
IRS-1 IP:IR
S-1
WCL
UbcH2 knockdown enhances myogenesis
by inhibiting IRS-1 ubiquitination.
C2C12 myotubes
Si-controlHA-TRIM72
Si-UbcH2HA-TRIM72
MyHCHADAPI
123456789
si-c
ontr
ol
HA-T
RIM
72
si-U
bch
2H
A-T
RIM
72
The
num
ber
of
nucl
eus
In H
A-p
osi
tive
cel
ls
TRIM72 inhibition of myogenesis is released by UbcH2 knockdown
C2C12 myotubes
TRIM72 is highly expressed in soleus muscle.
Jung and Ko, BBRC, 2010
soleus gastrocnemius
Red muscle White muscle
Smaller Larger
Resistant Sensitive
Color
Size
Fatigue
TRIM72 High Low
WT KO
WT KO0.0
5.0
10.0
15.0
Wei
ght
(mg)
*<0.05
2.0
4.0
6.0
8.0
10.0
12.0
14.0
16.0
18.0WT
KO
Perc
enta
ge
(%)
Cross Sectioned Area (mm2)
Skeletal muscle size is increased in TRIM72-disrupted mice.
Mouse soleus muscle
Actin
Akt
Erk
pAkt
pERK
TRIM72
0 5 10 0 5 10 Insulin (min)
C2C12 Myoblasts
Mouse soleus muscle
IRS-1 expression level is elevated in TRIM-disrupted skeletal muscle
+- - + Insulin
IRS-1
pAkt
Akt
pERK
TRIM72
ERK
Actin
pY
IRS-1 IP:IR
S-1
WT KO
0
100
200
300
400
500
0 15 30 45 60
WT
KO
0
20
40
60
80
100
120
0 15 30 45 60
WT
KO
Plasma glucose (mg/dl)
Time (min) Time (min)
Plasma glucose (% reduction)
Glucose tolerance test Insulin tolerance test
TRIM72 is a therapeutic target for type 2 diabetes
High fat diet-fed mice
*p<0.05 **
*
*
*
*p<0.05