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

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 moleculesby lipid raft proteome

지질래프트는 신호전달물질을 농축하고 있어서 새로운 신호전달물질의 동정에 매우 유리

Functional proteomics of lipid rafts

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

C2C12 Myogenesis

Myoblasts Myotubes

Myosin Heavy Chain

TRIM72 is found in the lipid rafts of C2C12 myotubes.

Lee et al., CDD, 2010

TRIM72 gene analysis

Myoblasts

Myotubes

Tripartite Motif (TRIM)

E3 ligase activity

SPla and RYanodine

receptor domain

4771


4.4 Kb

2.4 Kb

TRIM72

Cav-3

TRIM72 is specifically expressed in skeletal and cardiac muscle


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


TRIM72 is a myogenesis inhibitor


What is a molecular target of TRIM72 in IGF signaling?

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


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


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


TRIM72 negatively regulates myogenesis via targeting IRS-1.


E3 ligase activity ???

Protein Ubiquitination

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



C2C12 Myoblasts

－MG132 ＋MG132

TRIM72

IRS-1

RING domain of TRIM72 is required for IRS-1 ubiquitination.

0 1 2 3 4


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



WT

KO

WT

KO

MG132- - + +

E3 ligase activity ???

Protein Ubiquitination

UbcH2

UbcH3

UbcH8

UbcH10

Cav-3

GAPDH

MyHC

MyoD

UbcH7

UbcH5b

0 1 2 3 4 5


UbcH6

Ubc12

Ubc13

1.0

2.0

3.0

4.0

0 1 2 3 4 5

**

*

UbcH2/GAPDH


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


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

E3 ligase TRIM72 negatively regulates myogenesisby IRS-1 ubiquitination.

by Ub

Suggestion; TRIM72 is a negative muscle size regulator.

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

Korea University

e3 ligasetrim72 negatively regulates myogenesisby irs-1 … · 2014-06-27 · dr. young-mi ham miss...

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