stat3 undergoes acetylation dependent mitochondrial ... · translocation to regulate pyruvate...
TRANSCRIPT
STAT3 Undergoes Acetylation‐dependent Mitochondrial Translocation to Regulate Pyruvate Metabolism
Yan S. Xu1, Jinyuan J. Liang1, Yumei Wang2, Xiang‐zhong J. Zhao1,2, Li
a b
, y g , g , g g , ,Xu3, Ye‐yang Xu1, Quanli C. Zou2, Junxun M. Zhang2, Cheng‐e Tu2, Yan‐geCui2, Wei‐hong Sun2, Chao Huang1,4, Jing‐hua Yang1,*, Y. Eugene Chin2,*
Seq # b y +1--- -- ------ ------ --I 1 114.1 - 8 K 2 242.2 906.5 7 Q 3 370.2 778.4 6 F 4 517.3 650.3 5 L 5 630.4 503.3 4 Q 6 758.4 390.2 3 S 7 845.5 262.1 2 R 8 - 175.1 1
peptide=IKQFLQSR, (510.3 d)
Seq # b y +1--- -- ------ ------ --I 1 114.1 - 8 *K 2 284.2 948.5 7 Q 3 412.2 778.4 6 F 4 559.3 650.3 5 L 5 672.4 503.3 4 Q 6 800.5 390.2 3 S 7 887.5 262.1 2 R 8 - 175.1 1
peptide=IKQFLQSR, (531.3 d)
Anti‐aK87 AB
Anti‐aK707 AB
Anti‐aK709 AB
K87
K707
K709
c
IP:
293Ts
‐Flag‐STAT3IB: Flag
‐Flag‐SIRT5IB: Flag
IP:
‐Myc‐STAT3IB: Myc
‐Flag‐SIRT5IB: Flag
293Ts
IP:‐INSRIB: INSR
‐CBPIB: CBP
MEFs
IP:‐STAT3 IB:STAT3
‐aK87 IB:aK87
SS/R
‐aK‐STAT3 IB: aK (pan)
IP:‐Myc‐STAT3
IB: Myc‐Myc‐PDC E1
IB: Myc293Ts
IP:
‐Flag‐STAT3IB: Flag
‐Myc‐PDC E1IB: Myc293Ts
MEFs
IP:
MEFs
‐STAT3IB: STAT3‐PDC E1IB: PDC E1
‐aK685 IB: aK685
‐aK707 IB: aK707
‐aK709 IB: aK709
‐pY705 IB: pY705
‐pS727 IB: pS727
MEFs
Figure S1. (a) Peptide slot blot analysis of the antibodies prepared for STAT3 acetylation on K87, K707 and K709. (b) y and b numbers of the mass spectrum in Fig. 2b. (c) IgG controls were detedted for Immunoprecipitationand co‐immunoprecipitation experiments involved in this text.
a b c CBP‐HA: + +
INSR: + +INS: ‐ +
‐INSR STAT3
STAT3:‐STAT3
STAT3: ‐ + +CBP: ‐ ‐ +
1 6 1 23 4
d e
‐CBP‐HA
INSR
‐CBP‐HA
‐INSR Input
IP:CBP 1 1.7STAT3
‐Porin
Mit
1 49.4 44.3 36 31
‐STAT3
‐Porin
‐aK87
‐Tubulin
Mit
Cyt
1 6.1 23.4
1 29.0 54.9
d e
‐STAT3
STAT3:
Cyt Mit
‐Porin
‐Tubulin
1 1.3 0.3 0.6‐aK‐STAT3
‐STAT3
‐Porin
(pan)1 1.2 2.4
1 12.2 27.7
Figure S2. (a) INSR and CBP were overexpressed in 293T cells. HA‐CBP was immunoprecipitated followed by Western blotting analysis with anti‐INSR and anti‐HA. The bottom panel gave the input of INSR and HA‐CBP. (b) 293T ll t f t d ith EV STAT3 STAT3 d CBP STAT3
Porin
Mit
293T cells were transfected with EV, STAT3, or STAT3 and CBP. STAT3 acetylation on lysine 87 was detected with a specific antibody via Western blotting. (c) 293T cells were transfected with EV, wild‐type STAT3 and STAT3 with the N‐terminus, C‐terminus, or both N‐ and C‐termini deleted (i.e., 1‐738, 28‐770, and 28‐738). Mitochondria were isolated from the 293T transfectants and STAT3 and Porin levels were shown by Western293T transfectants and STAT3 and Porin levels were shown by Western blotting. (d) STAT3‐K87R mutant or STAT3‐WT was transiently transfectedinto 293T cells. Cytoplasmic and mitochondrial fractions were prepared for STAT3 localization analysis via Western blotting with anti‐Myc. (e)Mitochondrial lysates were prepared for STAT3 acetylation analysis from 293T cells transfected with EV, STAT3 or Cox4‐STAT3.
Fig. 1a
170170130
Figure S3. Full scans of western blotting data in all figures.
130
95
72
55
aK‐STAT3
(Pan)ST
170130
95
72
55
43
aK‐STAT3
(Pan)STA
130
170
95
130
TAT395
72
55
AT3
Fig. 1c
aK‐S
aK6
aK7
aK7
pY7
pS7
STA
72
55
43
aK‐STAT
(PanSTA
T3(Pan)
aK68685
aK70 7707
aK70 9709
pY705705
pS72 7727
STAT 3
AT3
T3n) 5 7 9 5 7 3
Fig. 1d
170130
95
72
170130
95
170130aK‐S
STAT
CBP
72
5572
55
95
72
STAT3
(Pan)
T3
Fig. 1e
170130
170130
95
72
55
130
95
72
55
170
130
95
aK‐STAT3
(Pan)
STAT3
CBP
Fig. 1f
17013095
HA
725543
34
Po
95
7255
43
A‐CBP
34 orin
Fig. 1g
170130
95
72
INSR IP: CBP
170130
95
72
CBP IP: CBP170130
95
72
INSR
Fig. 1h
1701309572
55
STAT3
957255433426
17
Tubulin/Porin/H3
Fig. 2a
17
170130957255
S a pS
Tubulin
55
43
34
TAT3
K685
S727
n/Porin/H3
17013095
Tubu95725543
34
26
ulin/Porin/H3
STAT3
aK685
pS727
Fig. 2b
170130957255
43
170130957255
43
STAT3
β‐actin
34
2634
26
Fig. 2d
170130 Sa p p P
Fig. 2e
1701309572 a S H
D
95
72
55
43
STAT3
K685
aK87
pY705
pS727
Porin 55
43
aK87
STAT3
DAC6
Fig. 2f
170130
95
72
55
43
STAT3
aK87
Tubulin/Porin
Fig. 2g
17095725543
34
26
170
130
95
STAT3
Porin
Fig. 2h
1701309572
170130957255
My
2672
55
43
34
5543
34
26
yc‐STAT3
Porin
Fig. 2i
17013095725543
17013095725543
Mit: STA
T
Cyt: STAT
Fig. 2j
1701309572554334
170957255433426
34 34
T3 T3
Fig. 3a
170 P34 26
17
STAT395
72
130 PDC E1
PDC E2
PDC E3
STAT3
Fig. 3b
72 72
95
17013095725543
34
PDC E 1
17013095725543
34
Myc‐STA
T3
17013095725543
34
PDC E 1 Input
*
Fig. 3d
13095725543
34
13072554334
Cox4‐STAT3
Tubulin/Porin
26
17
Fig. 3g
1309572Flag‐STA
T3
Myc‐PD
C E1
13095
721309572
55
55
Flag‐STAT3
Myc‐PD
C E1
Fig. 4a
aKSTA
T3 (pan
STAT3
1309572
55
34
130957255
130957255
3n)
Fig. 4b
170130
95
72
55
aK‐STAT3 (pan)
aK685
STAT3
43
34 SIRT5
170957255433426
17
Tubulin
Porin
H3
17
Fig. 4c
STAT3S
STAT
Fig. 4d
170
3 Input
SIRT5
T3
170
130
95
aK‐STAT3 (pan)
170
95
72
55
130
Flag‐STAT3
43
34
CBP‐HA
SIRT3/SIRT5
95
72
55
130
Fig. 4e
170
9572
55
130
aK‐STAT3 (pan)
aK685 170
957255
130
STAT
Flag‐SI
170
957255
170
9572
55
130 aK707
aK709
T3 RT543
34
Fig. 4g
95
7255
aK685
aK707
aK709
STAT3
SIRT3/SIRT
170
95130
T5
Fig. 7b
aK‐ST ST
Po
Fig. 7f
170
9572
130
Fla P
72
55
43
TAT3 (pan)
TAT3
orin 55
43
34
26
ag‐SIRT5
Porin
Fig. 7e 170
957255
130
43
aK‐STAT3 (pan)
STAT3
Tubulin/Porin
STAT3
Fig 7jFig. 7j
170
9572
55
130
43
34
aK685
STAT3
SIRT5
Porin
26
170
9572
130
aK68
STAT
Tubu
55
43
3426
85 T3
ulin
Supplementary Fig. 1c
170
95130
a aK aK aK p p
72
55
43
34
26
STAT3
K87
K685
K707
K709
pY705
pS727
170
Ac‐STA
T3
170
9572
55
130
43
34
26
Flag‐STAT3
Flag‐SIRT5
Myc‐STA
T3
Myc‐PD
C E1
Myc‐STA
T3
Flag‐SIRT5
Flag‐STAT3
Myc‐PD
C E1
170
9572
55
43
34
CBP INSR
170
130
95
72
95
55
43
STAT3 PD
C E1
130
72
34
2672
Supplementary Fig. 2a
170170
95
72
130 INSR
CBP‐HA
INSR Input
CBP‐HA Input
Supplementary Fig. 2b
170
957255
43
34
170
95
72
55
43
34
STAT3
aK87
170
9572
55
43
34
STAT3
pp y g
17095725543
3426
Porin
26 26 26
17095725543
34
26
Tubulin
170130957255
43
17095
55433426
STAT3
Tubulin/P o
Supplementary Fig. 2d
17
17
Supplementary Fig. 2c
55
1309572
7255
M
43
34
26
26 orin
17
7255
43
34
26
55
43
34
26
17
Myc‐STA
T3
Porin
Supplementary Fig. 2e
17095
170
95
72
55
43
Ac‐STA
T3
STAT3
Porin
170
95
72
55
43
95725543
34
26
17