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Figure S1 RACK1 binds to MTK1. (a) Proteins associated with MTK1. Cytoplasmic extracts were prepared from COS-7 cells transfected with a Flag-MTK1 expression vector, as well as from untransfected COS-7 cells (control). Extracts were immunoprecipitated using a Flag antibody. Polypeptides present in the precipitates were separated by 10.5% SDS-PAGE, and visualized by silver staining. The bands that are found only in the Flag-MTK1 transfected cells were excised from the gel, and identified by LC-MS-MS. In addition to RACK1 and actin, the ribosomal proteins S2, S3, and S6 were detected. Flag-MTK1 (ca 180 kDa) is above the limit of this panel. (b) The MTK1 MAPKKK, but not the TAK1 MAPKKK, binds to endogenous RACK1. Myc-tagged MTK1 (Myc-MTK1) or Myc-TAK1 was immunoprecipitated from transiently transfected COS-7 cells, and co-purified endogenous RACK1 was detected by immunoblotting (top row). (c) No MAPKK bound to endogenous RACK1. Flag-tagged MAPKKs (MEK1, MKK3/6/4/7), as well as the positive control Flag-MTK1, were individually expressed in COS-7 cells. Flag-tagged

MAPKKs, or Flag-MTK1, were immunoprecipitated from the cell extracts, and the co-precipitated endogenous RACK1 was assayed by immunoblotting (top row). (d) No MAPK was found to bind endogenous RACK1. HA-tagged MAPKs (p38α, JNK1, ERK2) were transiently transfected into COS-7 cells. HA-tagged MAPKs were immunoprecipitated from the cell extracts, and co-precipitated endogenous RACK1 was assayed by immunoblotting (top panel). (b-d) The middle and bottom rows show the levels of protein expression. (e) Schematic structure of RACK1. RACK1 consists of seven WD40 repeats. Myc-RACK1 constructs in which one or more WD40 repeats are deleted were used in (f) and (g). (f) COS-7 cells were transiently co-transfected with Flag-MTK1 and one of Myc-RACK1 deletion mutants. Immunoprecipitated Flag-MTK1 was probed for co-precipitating Myc-RACK1 (top row). (g) COS-7 cells were transfected as indicated, and immunoprecipitated Flag-MTK1-N was probed for co-precipitating proteins. For both (f) and (g), the levels of protein expression are shown in the lower rows.

a cbMyc-MTK1

Myc-TAK1

IP : MycIB : RACK1

RACK1

Myc

++-

- --

37

181

37

kDa

IP : Flag

IB : RACK1

RACK1

Flag

MTK

1

MK

K7

ME

K1

MK

K3

MK

K4

MK

K6

Flag-vector37

37

48

kDacontro

l

Flag-MTK1

actin

RACK1

kDa

48

37

25

d

48 136

180

221

269

317

93

1 2 3 4 5 6 7

WD40 repeat

RACK1

e

f g

kDa

181

IP: Flag, IB: Myc

IP: Flag, IB: Flag

Myc

Flag-MTK1

Myc-RACK1 ! 1

! 1

-3! 7

! 6

-7! 5

-7! 4

+ + + +++++-- W

T

WT

37

37

Myc-RACK1

Flag-MTK1-N

Myc-MTK1-N

IB: Flag

IB: Myc

Myc-MTK1-N

Myc-RACK1

IP: Flag

Lysate

+ ++ +++ +-

+ ++ +

WT

! 4!

1-3

! 5-

7

- -

115

115

115

37

kDa

RACK1

HA

p38

JNK

ER

K

HA-vector

IP : HA

IB : RACK1

kDa

37

37

46

Figure S1

© 2008 Macmillan Publishers Limited. All rights reserved.


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Figure S2 (a and b) RACK1 knock-down inhibits MTK1 activation. M57 cells were transfected with either a control or a RACK1 shRNAi expression vector. After 72 h incubation with puromycin, the cells were stimulated with 1 mM H2O2 for 90 min (a), or 50 ng/ml TNFα for 120 min (b). Activation-associated phosphorylation of Myc-MTK1was detected by immunoblotting using anti-P-Thr1493. The fold activation of MTK1 was determined and is shown in the bar graphs. (c) RACK1 knock-down does not affect TAK1 activation by osmotic stress. HEK293 cells were transfected with a RACK1 shRNAi expression vector. After a 36 h incubation, the cells were

super-transfected with Myc-TAK1, and incubated for an additional 36 h. The cells were stimulated with 0.5 M sorbitol, and activation-associated phosphorylation of Myc-TAK1 was detected by immunoblotting. Fold activation is shown in the bar graph below. (d and e) RACK1 expression does not affect the interaction between MTK1-N and MTK1-C fragments. COS-7 cells were transiently transfected with Myc-MTK1-N, Flag-MTK1-C, GADD45β, and HA-RACK1 (d) or shRNAi-RACK1 (e) as indicated. Flag-MTK1-N was immunoprecipitated, and co-precipitated Myc-MTK1-C was detected by immunoblotting (top rows).

a b

0

0.5

1.0

1.5

2.0

2.5

Fold

activation

RACK1shRNAi Control

- + - +Sorbitol

Myc-TAK1

P-TAK1

RACK1

actin

82

8237

37

kDa

c

ed

Myc-MTK1-C

Flag-MTK1-N

Myc-MTK1-C

HA-RACK1

GADD45!

Lysate

IP: Flag

Myc-MTK1-C

Flag-MTK1-N

HA-RACK1

GADD45!

+ ++ +++ +-

++

- - -- - -

kDa48

115

48

37

18 RACK1

GADD45!

Myc-MTK1-C

Flag-MTK1-N

Myc-MTK1-CIP: Flag

Lysate

shRNAi-RACK1

GADD45!

Flag-MTK1-N

Myc-MTK1-C +

- - +- - +

+++

+

++

+

+

+

kDa

48

115

48

37

18

- + - +

Control RACK1shRNAi

kDa

181

18137

37

TNF"

P-MTK1

Myc-MTK1

RACK1

actin

0

2

4

6

8

Fold

activation

- + - +

Control RACK1shRNAi

P-MTK1

Myc-MTK1

RACK1

actin

H2O2 kDa

181

18137

37

0

0.5

1.0

1.5

2.0

Fold

activation

Figure S2




Figure S3 (a) Subcellular localization of endogenous RACK1 (red) and endogenous MTK1 (green) before (-) and after (AsO2

-) arsenite treatment. HeLa cells were treated with 0.5 mM arsenite for 50 min and endogenous RACK1 and MTK1 were detected by immunostaining. (b and c) Recruitment of RACK1 into cytoplasmic stress granules (SGs) by arsenite. (b) HeLa cells

were transfected with DsRed-RACK1, and, after 36 h, treated with 0.5 mM arsenite. RACK1 localization was visualized by the DsRed fluorescence. (c) The intensity of the DsRed fluorescence at four SG-like spots and at four cytoplasmic areas were measured. The ratio of the intensity at SGs and in the cytoplasm was calculated. The scale bars represent 10 µm.

AsO2-

-

MTK1RACK1 Mergea

b c

SG

s /

Cyto

pla

sm

0

1

3

2

4

0 10 20 30 40 50

Time (min)

Arsenite: 0.5 mM

0 min

50 min

10 min

20 min

15 min

25 min 30 min

35 min 40 min

Figure S3




Figure S4 Recruitment of RACK1 into stress granules (SGs). (a and b) RACK1 is recruited into SGs induced by the ER-stress inducer thapsigargin or by the defective initiation factor mutant eIF2α(S51D). HeLa cells were treated with 10 µM thapsigargin for 50 min (a), or transfected with eIF2α(S51D) (b). Endogenous RACK1 and the typical SG component TIA1 were detected by immunofluorescence. (c) Ribosome-nonbinding RACK1(DE) mutant is not recruited into SGs. HeLa cells were transfected with Myc-RACK1(DE), and 36 h after transfection, Myc-RACK1 or

Myc-RACK1(DE) was detected by immunostaining. DE = R38D K40E. (d) MMS treatment does not induce a formation of SGs. HeLa cells were transfected with DsRed-RACK1 and 36 h after transfection, cells were treated with 100 µg/ml MMS for 180 min. Neither formation of SGs nor re-localization of DsRed-RACK1 into SG spots was observed. (e) MTK1 is not recruited into SGs. HeLa cells were co-transfected with Myc-MTK1 and HA-G3BP. Myc-MTK1 (green) and HA-G3BP (red) were detected by immunofluorescent staining. The scale bars represent 10 µm.

b

c

e

da

Thapsig

arg

in

RACK1 TIA1 Merge

-

+

eIF2!

(WT)

RACK1 TIA1 Merge

eIF2!

(S51D)

Myc-RACK1 (WT) Myc-RACK1 (DE)

DsRed-RACK1

MMS (180min)

Myc-MTK1

HA-G3BP

Merge

Figure S4




Figure S5 (a and b) G3BP-mediated SG formation suppresses MTK1 activation. (a) HeLa cells were transfected as indicated. After a 36 h incubation, immunoprecipitated Myc-MTK1 was probed for activation-associated phosphorylation (top row). (b) COS-7 cells were transfected as indicated. After a 36 h incubation, Myc-MTK1 was immunoprecipitated and probed for co-precipitating GADD45β (top row). (c-e) Arsenite pretreatment suppresses MTK1 activation by MMS. (c) M57 cells were treated with 0.5 mM arsenite. Immunoprecipitated Myc-MTK1 was probed

for activation-associated phosphorylation of MTK1. (d) M57 cells were pretreated with arsenite for 30 min followed by a 3 h treatment with MMS as indicated. Immunoprecipitated Myc-MTK1 was probed for activation-associated phosphorylation of MTK1 at T1493 (top row). (e) HEK293 cells were pretreated with arsenite for 30 min followed by a 5 h treatment with MMS as indicated. Immunoprecipitated endogenous MTK1 was probed for activation-associated phosphorylation at T1493 (top row). For (a-e), the levels of protein expression are shown in the lower rows.

ba

P-MTK1

Myc-MTK1

Myc-GADD45!

GFP-G3BP

Myc-MTK1

Myc-GADD45!

GFP-G3BP

+

++

--+

-+

+ -

++

kDa

181

181

25

82Lysate

IP: MycIB: GADD45!

GADD45!

GFP-G3BP

Myc-MTK1

Myc-MTK1

GFP-G3BP

GADD45!

+

++

--+

-+

+ -

++

kDa

18

181

18

82

c

d e

P-MTK1

Myc-MTK1

MMS

Arsenite --

--+

+ ++

kDa

181

181

P-MTK1

MTK1

MMS

Arsenite --

--+

+ ++

kDa181

181

Phospho-MTK1

Myc-MTK1

Arsenite 0 15 30 45 60 905 (min)

181

181

kDa

Figure S5




Figure S6 RACK1 is recruited into SGs induced by hypoxia. (a) HeLa, HEK293, and COS-7 cells transfected with DsRed-RACK1 were incubated under hypoxic conditions (0.5% oxygen) for 4 h. DsRed-RACK1 was detected

by fluorescence. (b) HeLa cells were incubated under hypoxic conditions for 24 h. Endogenous RACK1 and the typical SG component eIF4E were detected by immunofluorescent staining. The scale bars represent 10 µm.

a

b

HEK293 COS7

Hypoxia(4 h)

HeLa

MergeeIF4ERACK1

-

Hypoxia

Figure S6




Figure S7 (a-d) SG formation by hypoxia inhibits etoposide-induced apoptosis. HEK293 cells (a and b), or HEK293 cells stably expressing RACK1(DE) (c and d), were treated with 50 µM etoposide and incubated either under normoxia (-) or hypoxia (+). After a 24 h incubation, cells were stained with Annexin V-Cy3, and visualized by fluorescence microscopy. About 500 cells were scored to calculate the percentage of apoptotic cells. (e and f) RACK1 knock-down suppresses etoposide-induced apoptosis in HEK293 cells. (e) HEK293 cells were transfected with a pSIH-H1-copGFP expression vector for RACK1 shRNAi (or a control

vector for Luciferase RNAi). After a 24 h incubation, the cells were super-transfected with the same vector and incubated for an additional 24 h. Cells were then treated with 50 µM etoposide for 24 h. Cells that were transfected by the shRNAi vectors were visualized by GFP expression, whereas apoptotic cells were visualized by Annexin V-Cy3 staining. (f) Percentage of apoptotic cells among GFP-positive cells were calculated. Error bars indicate s.e.m. of three independent experiments. RACK1 knock-down by the shRNAi vector was confirmed by western blotting (data not shown). The scale bars represent 50 µm.

c d

a b

Ap

op

totic c

ells

(%

)

0

10

20

30

+ +- --

Etoposide

Hypoxia + +-- -- -+

+ ++

Etoposide

Hypoxia

Phasecontrast

Annexin V

Ap

op

totic c

ells

(%

)

0

10

20

30+ RACK1

(DE)

+ +- --

Etoposide

Hypoxia + +-- -- -+

+ ++

Phasecontrast

Annexin V

Etoposide

Hypoxia

0

10

20

30

Apopto

tic c

ells

(%

)shRNAi-Control ++ - -shRNAi-RACK1 - + +-

Etoposide + +- -e f

Etoposide

shRNAi-Control

shRNAi-RACK1

GFP

Annexin V

- - + +- -+ +

- -+ +

Figure S7




Figure S8 Full scan images of key gel/western data.

Myc-RACK1

Flag-MTK1

Myc-RACK1

Flag-MTK1

IP : FlagIB : Myc

-++

+ +-

82

kDa

63

48

36

25

82

63

48

36

25

181

115

82

63

Fig 1b

kDa

Myc-MTK1

GADD45!

GADD45!

P-MTK1

RACK1

actin

shRNAi Control

- + - +RACK1

181

181

19

14

37

48

37

48

Fig 2a

Flag-MTK1-N

GADD45!

Samples

RACK1

63

48

37

25

19

14

115

181

82

M InputkDa

Fig 2h

Fig 2d

Phospho-MTK1

Myc-MTK1

pHA-RACK1 (µg)

HA-RACK1

Myc-MTK1- 0.1 0.25 0.5 1.0

+ + ++ + kDa

181

48

181

37

Fig 4a

P-MTK1

Myc-MTK1

SampleskDa

181

181

GFP-G3BP

Myc- GADD45!

InputkDa

Myc-MTK1

82

63

37

25

181115

48

82

63

37

25

181115

48

Fig 4a

Fig 4d

P-p38

Etoposide

Hypoxia + +- -+- +- kDa

37

25

p38 37

25

Fig 4d

JNK1

P-JNK

+ +- -+- +-

63

48

37

kDa

48

37

Etoposide

Hypoxia

Fig 4d

Long exposure

37

kDa

Pro-caspase3

+ +- -+- +-

25

19

37

Short exposure

kDa

Cleaved

25

19

Etoposide

Hypoxia

Figure S8


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