a b c - nature research · phosphorylation sites of big3 by pka, as determined using netphos 3.1...

a b

d

c

0

20

40

60

80

100

120

-

E2

PP

1C

a a

cti

vit

y (

pm

ole

/min

)

E2 treatment time (h) 0 6 12 24

KPL-3C cells

**

**

**

E2 : 24 h

0

20

40

60

80

100

120

PP

1C

a a

cti

vit

y (

pm

ole

/min

)

E2 treatment time (h)

0 6 12 24

MCF-7 cells

***

*** ***

e

-726 -704

gttcaaGGCCAcctGGCCAacat

5’-ERE

f

Predictive PP1Ca binding region in BIG3 :

1,228 – 1,232aa (-KAVSF-)

WT

E2

FLAG-BIG3

PP1Ca

FLAG-BIG3

PP1Ca

IP : FLAG

IP : IgG

- - E2

FLAG-BIG3 DPP1Ca

HA-ERa + +

HEK293T cells

WCL FLAG-BIG3

PP1Ca

250

37

250

37

250

37

E2

-726/-704

ChIP assay

Input

IP : ERa

IP : IgG

-

Rela

tive e

xp

ressio

n

0

0.5

1

1.5

2

2.5

ERAP tamoxifen - - - +

- - + -

MCF-7 KPL-3C

E2 - + + +

- - - + - - + -

- + + + PPP1CA

5’-ERE

ER

E-l

ucif

era

se a

cti

vit

y

empty 0

0.5

1

1.5

2

2.5

3

3.5

-

E2

***

Western blotting

PP1Ca

b-actin

1.0 2.4 1.0 0.7 1.0 1.7 0.9 0.7

ERAP

tamoxifen - - - + - - + -

MCF-7 KPL-3C

E2 - + + +

- - - + - - + - - + + +

37

50

0

20

40

60

80

100

120

140

-

E2

siBIG3 siEGFP siPP1Ca

***

PP

1C

a a

cti

vit

y (

pm

ole

/min

)

KPL-3C cells

IP :

PP1Ca

BIG3

PP1Ca

*** ***

250

37

MCF-7 cells

siBIG3 siEGFP siPP1Ca

0

20

40

60

80

100

120

-

E2

**

PP

1C

a a

cti

vit

y (

pm

ole

/min

)

IP :

PP1Ca

BIG3

PP1Ca

*** **

250 37

MC

F-7

KP

L-3

C

HC

C1500

BT

-474

T47D

KP

L-1

HB

C4

ZR

-75-1

PP1Ca

b-actin

BIG3 250

37

50

FLAG (BIG3)

mock BIG3 DPP1Ca -

0.5 1.0 2.0 2.0 2.0 0

0

50

100

150

200

PP

1C

a a

cti

vit

y (

pm

ole

/min

) HEK293T cells

DNA (mg)

FLAG-

IP :

PP1Ca

WCL

PP1Ca

FLAG (BIG3)

PP1Ca

*** **

250

37

250 37

h

Position PSSM Score

318 - 338

439 - 459

496 - 516

1106 - 1126

1225 - 1245

VARTIYYIAAELVRLVGSVDS

ELSQGKGLSEGQVQLLLLRLE

SGSSAAKVVLTLSTQADRLFE

GNERSLDISISVTTDTGQTTL

VSQKAVSFIHDILTEVLTDWN

228 - 248 QLLYLECILSVLSSSSSSMHL

1449 - 1469 GLIEVWIILLEQLTAAVSNCP

24.2

74.4

42.4

26.2

20.6

43.4

83.0

Sequence

j k i

0

0.5

1

1.5

2

0

0.2

0.4

0.6

0.8

1

1.2

1.4

Bin

din

g B

IG3

Bin

din

g P

KA

cata

lyti

c NS

**

- +

PKA catalytic IPs

- - + +

H-89 E2

- +

BIG3 IPs

- - + +

1 mM H-89 10 nM E2

0

500,000

1,000,000

1,500,000

2,000,000

2,500,000

3,000,000

- +

*** ***

BIG3 IPs

PK

A a

cti

vit

y (

RL

U)

- - + +

1 mM H-89 10 nM E2

- +

PKA catalytic IPs

- - + +

*** **

m

Target site Score

T162 0.79

(Analysis by NetPhos 3.1)

S305

S689

S925

S1208

S1763

0.79

0.81

0.78

0.83

0.80

n

Score Modification

56.34 CWSLVAPH

72.15 CWSLVAPH

1C: Propionamide

3S: Phospho

-

Sequence

S1208-Peptide (RCWSLVAPH)

+ recombinant PKA for 30 min at 30˚C

Score Modification

83.14

57.63

GSGCSCTAPALSGPVAR

GSGCSCTAPALSGPVAR

Sequence

S305-Peptide (DHGRGSGCSCTAPALSGPVAR)

+ recombinant PKA for 30 min at 30˚C

2S: Phospho

4C: Carboxymethyl

6C: Propionamide

-

l

g

***

0

20

40

60

80

100

-

E2

PP

1C

a a

cti

vit

y (

pm

ole

/min

)

FLAG-BIG3-transfected HEK293T

FLAG-BIG3

* ***

* ***

NS

Supplementary Figure 1 | BIG3 regulated PP1Ca activity through its phosphorylation by PKA. (a) Expression patterns of

BIG3 and PP1Ca in breast cancer cell lines. b-actin served as a quantitative internal control. (b) Identification of the predictive

PP1Ca binding region in BIG3. The indicated FLAG-tagged BIG3 constructs (WT; full-length BIG3, DPP1Ca; BIG3 deleting

PP1Ca-binding region, 1,228-KAVSF-1,232) and HA-tagged ERa construct transfected-HEK293T cells were

immunoprecipitated using an anti-FLAG antibody. (c) The inhibitory effects of BIG3 overexpression on phosphatase activity of

PP1Ca-immunoprecipitates in FLAG-tagged BIG3 or DPP1Ca construct-transfected HEK293T cells using pNPP as a substrate.

These data represent the means ± s.e.m. of three independent experiments. (d) Effects of siPP1Ca and siBIG3 on

phosphatase activity of PP1Ca immunoprecipitates using pNPP as a substrate in MCF-7 and KPL3C cells. These data

represent the means ± s.e.m. of three independent experiments. (e) Phosphatase activity of PP1Ca after E2 stimulation in

MCF-7 (left) and KPL-3C (right) cells. These data represent the means ± s.e.m. of three independent experiments. (f) Positive

0 10 nM E2 0

1.0 4.6 4.5 0.5 0.5 0.5

pS305-BIG3

pS1208-BIG3

BIG3

PKA catalytic

BIG3

PKA catalytic WCL

IP :

BIG3

10 5

MCF-7 cells

siEGFP siPKA

5 10 (min)

1.0 3.5 3.2 0.9 0.7 0.7

250

250

250

37

250

37

HC

C1500

MC

F-7

KP

L-3

C

BT

-474

T47D

KP

L-1

HB

C4

ZR

-75-1

b-actin

PKA catalytic 37

50

feedback regulation of PPP1CA transactivation. Left, Effects of tamoxifen on PPP1CA expression. For immunoblot analysis

(upper), b-actin served as a loading control. For real-time PCR analysis (lower), the data are expressed as the fold-increase

over untreated cells (set at 1.0). These data represent the mean ± s.e.m. of three independent experiments. Right, ChIP assays

of the transactivation of PPP1CA through an ERE motif in 5’ upstream (upper), and luciferase assays of the transactivation of

PPP1CA using a luciferase reporter containing an ERE motif conserved within 5’ upstream of the PPP1CA gene (lower). The

data represent the mean ± s.e.m. of three independent experiments. (g) The predicted PKA binding regions in BIG3, as

determined using Hou et al.24 and the PSIVER software. The bold letters indicate the potential PKA binding regions in BIG3. (h)

Expression patterns of PKA protein in breast cancer cell lines. (i) Statistical analysis of PKA and BIG3 binding to BIG3 (right)

and PKA (left) immunoprecipitates, respectively. These data are expressed as the fold-increase over untreated cells (set at 1.0),

and represent the mean ± s.e.m. of three independent experiments. (j) Statistical analysis of in vitro PKA activity of BIG3 and

PKA immunoprecipitates. These data represent the mean ± s.e.m. of three independent experiments. (k) The predicted

phosphorylation sites of BIG3 by PKA, as determined using NetPhos 3.1 software. (l) Phosphatase activity of PP1Ca in a

pseudo-phosphorylation mutant of BIG3 (S305E and S1208E) and alanine mutant of BIG3 (S305A and S1208A). (m) The

inhibitory effects of siPKA on BIG3 phosphorylation. Representative results are shown from one of two experiments. (n) 2DICAL

analysis of engineered peptides representing S305 and S1208 on BIG3 with recombinant PKA (recPKA). **P<0.01, ***P<0.001

(two-sided Student’s t-test)

E2 for 24 hr E2 + ERAP

pS39-PHB2 pS39-PHB2

l-Ppase + E2 + ERAP

pS39-PHB2

PHB2 PHB2 PHB2

50 mm

a

b

c

d

Supplementary Figure 2 | PHB2 is phosphorylated at S39 via PKCa. (a) Serine phosphorylation of PHB2 in ERa

immunoprecipitates of the nuclear fraction of MCF-7 cells after E2 ± ERAP treatment for 24 h. (b) Time course of serine

phosphorylation of PHB2 in MCF-7 cells after E2 ± ERAP treatment for the indicated time. (c) Representative

immunofluorescence images of PHB2 phosphorylation at S39; PHB2 (green), phosphorylated S39 PHB2 (red). (d) The

subcellular localization of PHB2 phosphorylation at S39 in the presence of E2 in the cytoplasmic (Cyto) and nuclear (Nuc)

fractions of the indicated PHB2 (WT, S39A) and ERa construct-transfected HEK293T cells. a/b-tubulin (tublin) and laminin B

(lamin) were used as loading controls for the cytoplasmic and nuclear fractions, respectively.

+ -

10 mM ERAP

pSer-PHB2

10 nM E2

Nucleus of MCF-7 cells

IP : ERa WCL

+ - + - + -

- + + -

PHB2

ERa

+

-

-

+ 37

37

75

ERAP

+ + + + + + + + + + + +

1 h 3 h 6 h 12 h 18 h 24 h

pSer-PHB2

PHB2

E2

+ + - + - - - - - + + +

MCF-7 cells

37

37

-

Cyto Cyto

- E2 -

HEK293T cells

E2

WT

Nuc

HA-PHB2 FLAG-ERa

+ +

+

Nuc

- E2 E2

S39A +

- - - + - - - + - + + -

Myc-BIG3

FLAG-ERa

HA-PHB2

FLAG-ERa

HA-PHB2

pS39-PHB2

lamin

tubulin

HA-PHB2

FLAG-ERa

WCL

IP :

FLAG

IP : IgG

10 mM ERAP 75

37

75

37

75

37

37

50 75

b

d

0

0.1

0.2

0.3

0.4***

Fre

e p

ho

sp

hate

rele

ased

fro

m p

ho

sp

ho

-PH

B2 (

A6

20)

MCF-7 cells

***

(-)

0 50 75 (mM)

10 nM E2

BIG3/PP1Ca

inhibitor

0

BIG3-immunoprecipitates

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

-

E2

MCF-7 cells

E2 : 24 h

Fre

e p

ho

sp

hate

rele

ased

fro

m p

ho

sp

ho

-PH

B2 (

A6

20)

siEGFP siPKA

*** ***

siPP1Ca

***

BIG3-immunoprecipitates

a

c

e

YGVRESVFTVE

PHB2

Phospho-PHB2

Phospho-PHB2

Phospho-PHB2 peptide + PP1Ca

Supplementary Figure 3 | Phosphatase activity of BIG3-PP1Ca regulates PHB2 phosphorylation. (a) Phosphatase

activity (left) and Western blot analyses (right) of BIG3 immunoprecipitates of PP1Ca- and PKA-depleted MCF-7 cells against

phospho-S39 PHB2 peptide. Phosphatase activity was measured using malachite green. These data represent the means ±

s.e.m. of three independent experiments. (b) The inhibitory effect of PP1Ca on PHB2 S39 phosphorylation. The indicated

PP1Ca

IP :

BIG3

WCL

BIG3

PKA catalytic

PP1Ca

pS305-BIG3

BIG3

PKA catalytic

siEGFP siPKA siPP1Ca

- E2 - E2 - E2

MCF-7 cells

pS1208-BIG3

250

250

250

37

37

250

37

37

WCL

IP :

PHB2

- E2 - E2

FLAG-BIG3 DPP1Ca WT

HA-ERa + +

FLAG-BIG3

pS39-PHB2

PHB2

HEK293T cells

1.0 1.3 1.6 2.4

PP1Ca

FLAG-BIG3

PHB2

PP1Ca

250

37

37

37

250 37

37

(-)

IP :

BIG3

WCL

pS1208-BIG3

PHB2

pS39-PHB2

BIG3

pS305-BIG3

PHB2

1.0 0.5 0.0 1.1 0.9

BIG3

IP :

PHB2

20

okadaic

10 nM E2

- 1.0

H-89

0.5 - 40

MCF-7 cells

1.0 0.5 0.0 0.8 0.6

1.0 2.5 1.7 2.9

250

250

250

37

37

250 37

(-) 10 nM E2

IP :

BIG3

WCL

pS1208-BIG3

BIG3

pS305-BIG3

PKA catalytic

1.0 1.1 0.3 0.1

PP1Ca

BIG3

PKA catalytic

PP1Ca

50 0 75 (mM) BIG3/PP1Ca

inhibitor 0

MCF-7 cells

1.0 1.0 1.0 0.7

250

250

37

37

37

37

250

250

FLAG-tagged BIG3 (WT, DPP1Ca) and HA-tagged ERa-transfected HEK293T cells, followed by immunoprecipitation with an

anti-PHB2 antibody. (c) The inhibitory effect of BIG3-PP1Ca binding inhibitor on PP1Ca phosphatase activity. MCF-7 cells

were treated with BIG3-PP1Ca binding inhibitor for 24 h in the presence of E2 and were immunoprecipitated using BIG3

antibody. (d) The effects of PKA inhibitor H-89 and PP1Ca inhibitor okadaic acid on BIG3 phosphorylation (S305 and S1208)

and PHB2 phosphorylation (S39). MCF-7 cells were treated with H-89 or okadaic acid for 24 h in the presence of E2 and were

immunoprecipitated using BIG3 or PHB2 antibody. (e) Q-TOF spectra indicating dephosphorylation of phospho-PHB2-peptide

by PP1Ca in positive ion mode. The dephosphorylation of peptide is exemplarily shown for m/z 643.3. Upper, the left spectrum

is an untreated control, and the right spectrum was acquired after dephosphorylation with PP1Ca. Lower, product ion spectrum

of the PHB2 peptide YGVRESVFTVE, with a precursor mass of m/z 643.3. *** P<0.001 (two-sided Student’s t-test).

a b

0

0.5

1

1.5

2

2.5

3

3.5

MCF-7

KPL-3C

ZR-75-1

Rela

tive e

xp

ressio

n

PKCa PKCe CAMK2

YGVREpSVFTVE

PHB2

PHB2

Phospho-PHB2

PHB2 peptide + PKCa

c

d

+ + + + + +

IB : PHB2

recombinant PP1Ca (ng) 0 6.25 12.5

pS39-PHB2 IPs

25 50 100

P

% De-phosphorylation 0 25 35 82 85 83

37 (pmole) 0 0.16 0.33 0.65 1.3 2.6

e

PKCa depeltion

PHB2

BIG3

PP1Ca E2

PKA

P

S39 P

PKCa

E2 PKA depletion

PHB2

BIG3

PP1Ca E2

PKA S39

PKCa

P

P

E2

0.01 mg PHB2

0

20

40

60

80

100

6.25 12.5 25 50 100 recombinant

PKCa (ng) + + + + +

% P

ho

sp

ho

ryla

tio

n

of

PH

B2

f

Supplementary Figure 4 | PKCa is confirmed to be responsible kinase for PHB2 S39 phosphorylation. (a) Expression

patterns of PKCa, PKCe, and CAMK2 in ERa-positive breast cancer cell lines (MCF-7, KPL-3C and ZR-75-1) using real-time

PCR. (b) The inhibitory effects of siPKA on PHB2 phosphorylation at S39 in MCF-7 cells after E2 ± ERAP treatment for 24 h.

(c) Q-TOF spectra of direct PHB2 peptide phosphorylation by PKCa in positive ion mode. The phosphorylation of peptide is

exemplarily shown for m/z 683.3. Upper, the left spectrum is an untreated control, and the right spectrum was acquired after

phosphorylation with PKCa. Lower, product ion spectrum of the phosphor-S39 PHB2 peptide YGVRE(pS)VFTVE, with a

precursor mass of m/z 683.3. (d) Statistical analysis of the ratio of phosphorylation at S39 in full-length PHB2 by PKCa. These

data are expressed as the percentage of phosphorylated band in total PHB2 band and represent the mean ± s.e.m. of four

independent experiments. (e) Dephosphorylation of PHB2 phosphorylation at S39 immunoprecipitated by phospho-specific

PHB2 (S39) antibody from MCF-7 cells treated with E2/ERAP for 24 h. (f) Schematic illustration of BIG3-PKA-PP1Ca tri-

complex under the depletion of PKA (left) and PKCa (right).

IP :

pS39-PHB2

MCF-7 cells

- - +

WCL

10 mM ERAP

pS39-PHB2

PKCa

pS39-PHB2

PHB2

PKCa

- + + 10 nM E2

37

75 37

37

75

siPKA

- - + - - - + + + -

+ +

siEGFP

pS39-PHB2

PHB2

PKA catalytic

1.0 0.9

10 mM ERAP 10 nM E2

37

37

37

Disease-free survival (year)

Absence (34)

Presence (48)

Log Rank = 0.009 Cu

mu

lati

ve s

urv

ival

Cytoplasmic phospho-PHB2


Low (47)

High (26) Log Rank = 0.001 C

um

ula

tive s

urv

ival

BIG3 mRNA

0

500

1000

1500

2000

2500

n = 73 P < 0.001

Normal Tumour

Inte

nsit

y

a

b


Score 0 (10)

Score 3+ (16)

Cu

mu

lati

ve s

urv

ival

BIG3 protein

Score 1+ (24)

Score 2+ (32)

Log Rank : P < 0.001

c

Supplementary Figure 5 | BIG3 overexpression was predictive of worse outcomes in ERa-positive breast cancer. (a)

mRNA expression of BIG3 in patients with ERa-positive breast cancer (left) and Kaplan-Meier curves of overall survival as a

function of BIG3 expression (right) based on the TCGA data set. (b, c) Kaplan-Meier analysis of survival associated with

BIG3 protein (b) and cytoplasmic PHB2 phosphorylation at S39 (c) in representative ERa-positive breast cancer specimens.

Uncropped images of Figure 1a Uncropped images of Figure 1b

Uncropped images of Figure 1c

Uncropped images of Figure 1d

Uncropped images of Figure 1f

Continued on the following page

250

75

50

100

37

150

250

75

50

100

37

150

PP1Ca

BIG3

IP

WCL

E2 - E2 -

MCF-7 cells

PP1Ca

BIG3

E2 -

BIG3 IgG WCL

E2 - E2 -

KPL-3C cells

E2 -

IgG BIG3

IP

250

75

50

100

37

150

250

75

50

100

37

150

PP1Ca

BIG3

siRNA BIG3

E2 - E2 -

MCF-7 cells

PP1Ca

BIG3

E2 -

Control PP1Ca

-

IP : BIG3

siRNA BIG3

E2 - E2

KPL-3C cells

E2 -

Control PP1Ca

BIG3 : IP

250

37

50

150

100

E2 - - - E2 E2 - E2

BIG3

BIG3 PKA

catalytic IgG WCL

MCF-7 cells

PKA catalytic

75

IP

250

37

50

150

100

E2 - - - E2 E2 - E2

BIG3

BIG3 PKA

catalytic IgG WCL

KPL-3C cells

PKA catalytic

75

IP

IP : BIG3

siEGFP

- E2 - E2

siPKA

pSer-BIG3

pThr-BIG3

BIG3

PKA catalytic

siEGFP

- E2 - E2

siPKA

WCL

KPL-3C cells

IP : FLAG

FLAG-BIG3 S309A T162A S1208A S925A S689A WT

- E2

150

100

50

75

250

37

- E2 - E2 - E2 - E2 - E2 - E2

S1763A

BIG3

PKA catalytic

PP1Ca

S309A T162A S1208A S925A S689A WT

- E2 - E2 - E2 - E2 - E2 - E2 - E2

S1763A

WCL

BIG3

PKA catalytic

PP1Ca

150

50

50

37

250

37

150

250

37

50

100

75

BIG3

PKA catalytic

IP : BIG3

siEGFP

- E2 - E2

siPKA

WCL

siEGFP

- E2 - E2

siPKA

MCF-7 cells

150

250

pSer

IP : BIG3

siEGFP

- E2 - E2

siPKA siEGFP

- E2 - E2

siPKA

pThr

MCF-7 cells

100

Uncropped images of Figure 2c Uncropped images of Figure 2b


Uncropped images of Figure 1g

0 12 24

10 nM E2 + - - + -

IP: BIG3

MCF-7 cells

250

150

250

150

250

150

BIG3

pS305-BIG3

WCL

pS1208-BIG3

+ - - + -

0 12 24 Time (h)

0 12 24

10 nM E2 + - - + -

IP: BIG3 IP: BIG3

+ - - + -

0 12 24 Time (h)

0 12 24

+ - - + -

IP: IgG

100

75

50

37

BIG3

250

75

50

100

37

150

PHB2

BIG3

siBIG3 siEGFP

lamin

tubulin

E2 - - - E2 E2 - E2

Cyto Nunc Cyto Nunc

MCF-7 cells

50

37

25

pS39-PHB2

IP : HA-Ab

75

50

37

75

50

37

S39A -

- E2 - E2

WT S39A WT - HA-PHB2

- E2 - E2 - E2 - E2

HA-Ab Phospho-PHB2 (Ser39)

HA-PHB2 WT S39A

10 mM ERAP - - - + - +

IP: IGF-1Rb

HEK293T cells

250

150

100

75

50

37

25

HA-PHB2

S39A

- - 150

75

+ - - +

10 nM E2 + - + + - + - + + - + +

Myc-ERa + + + + FLAG-BIG3 + + + +

WT

100

WT S39A

- - - + - +

WCL

250

150

100

75

50

37

25

150

75

+ - + + - +

+ + + +

100 IGF-1Rb

FLAG-BIG3

Myc-ERa

HA-PHB2

IGF-1Rb

FLAG-BIG3

Myc-ERa

pS39-PHB2

P-IGF-1Rb

(Y1135/Y1136)

150

100

75

37

25


Uncropped images of Figure 2f

Uncropped images of Figure 2e

150

100

75

250

150

100

75

250

pS305-BIG3

pS1208-BIG3

Mock WT S305A S1208A

FLAG-BIG3

75

250

37

50

- E2 - E2 E2 -

HEK293T cells

PKA catalytic

- E2

FLAG-BIG3

IP : FLAG

150

100

75

37

50

PP1Ca

E2 - E2 E2 - - E2 -

Mock S305A WT S1208A

HEK293T cells

IP : FLAG

FLAG-BIG3

50

150

100

75

25

37

250

50

37

FLAG-BIG3

PKA catalytic

PP1Ca

pS1208-BIG3

pS39-PHB2

pS305-BIG3

E2

50

150

100

75

37

- -

siEGFP

E2

siEGFP

E2 E2 - - E2 E2 - -

siPP1Ca siPKA siPP1Ca siPKA

IP : PHB2

250

MCF-7 cells

PP1Ca PKA catalytic

E2

25

37

- - siEGFP

E2

siEGFP

E2 E2 - - E2 E2 - - siPP1Ca siPKA siPP1Ca siPKA

WCL

50

250

E2

BIG3

50

150

100

75

25

37

PHB2

MCF-7 cells

- -

siEGFP

E2

siEGFP

E2 E2 - - E2 E2 - -

siPP1Ca siPKA siPP1Ca siPKA

IP : PHB2 WCL


Uncropped images of Figure 3a

100

75

50

37

siPKCa

- - + - -

- + + + -

+

+

siEGFP

10 mM ERAP

10 nM E2

siPKCa

- - + - -

- + + + -

+

+

siEGFP

IP : PHB2 WCL

MCF-7 cells

PHB2

PKCa

100

75

50

37 PHB2

PKCa

Cytoplasm

Nucleus

75

50

37

siPKCa

- - + - -

- + + + -

+

+

siEGFP

ERAP

E2

siPKCa

- - + - -

- + + + -

+

+

siEGFP

Cytoplasm Nucleus

IP : PHB2

tubulin

lamin

pS39-PHB2

WCL


+ + + + + +

IB : PHB2

recombinant PKCa (ng) 0 6.25 12.5 37

25

PHB2

0.01 mg PHB2

P

25 50 100

+ + + + + +

0 6.25 12.5 25 50 100

PHB2 : PKCa

IB : pS39-PHB2

Uncropped images of Figure 3c


+ + + + + +

IB : PHB2

recombinant PP1Ca (ng) 0 6.25 12.5 37

PHB2

Phospho-PHB2

P

25 50 100

+ + + + + +

0 6.25 12.5 25 50 100

IB : pS39-PHB2

25


siEGFP siPKA siPKCa

10 nM E2 - - + - + +

IP: BIG3

MCF-7 cells

250

150

100

75

50

37

25

BIG3

siEGFP

PHB2

- +

WCL

siPKA siPKCa - + - +

6 h

ERa

100

75

50

37 PKA catalytic

37

25

PKCa

pS39-PHB2

75

50

50

37

siEGFP siPKA siPKCa

10 nM E2 - - + - + +

IP: BIG3

MCF-7 cells

250

150

100

75

50

37

25

BIG3

siEGFP

PHB2

- +

WCL

siPKA siPKCa - + - +

12 h

100

75

PKA catalytic

37

25

pS39-PHB2

PKCa

75

50

ERa


siEGFP siPKA siPKCa

10 nM E2 - - + - + +

IP: BIG3

MCF-7 cells

250

150

100

75

50

37

25

BIG3

PHB2

siEGFP

- +

WCL

siPKA siPKCa

- + - +

24 h

75

50

ERa

siEGFP siPKA siPKCa

E2 - - + - + +

IP: BIG3

MCF-7 cells

100

75

50

37

siEGFP

- +

WCL

siPKA

- + +

24 h

37

25

PKA catalytic

pS39-PHB2

PKCa

-

siPKCa

Uncropped images of Supplementary Figure 1a Uncropped images of Supplementary Figure 1b

FLAG-BIG3

100

75

50

37

250

150

- E2 - E2

WT DPP1Ca

IP : FLAG

+ + HA-ERa

PP1Ca

FLAG-BIG3

E2 - E2

WT DPP1Ca

IP : IgG

+ +

- E2 - E2

WT DPP1Ca

WCL

+ +

- 250

37

50

150

100

BIG3

b-actin

75

PP1Ca

MC

F-7

KP

L-3

C

ZR

-75-1

T47D

BT

-474

HC

C1500

KP

L-1

HB

C4

Uncropped images of Supplementary Figure 1c

siPP1Ca siBIG3 siPP1Ca siEGFP

75

50

250

150

siBIG3

- E2 - E2

siEGFP

37

- E2

IP : PHB2

100

- E2 - E2 - E2

WCL

KPL-3C cells

BIG3

PP1Ca

Uncropped images of Supplementary Figure 1d

HEK293T cells

mock BIG3 DPP1Ca

DNA (mg)

FLAG- -

0.5 1.0 2.0 2.0 2.0 0 0.5 1.0 2.0 2.0 2.0 0

mock BIG3 DPP1Ca -

IP : PP1Ca WCL

75

50

250

150

37

100

PP1Ca

FLAG-BIG3

75

50

PP1Ca

250

150

BIG3

siBIG3

- E2 - E2

-

37

-

- E2 - E2

siPP1Ca siPP1Ca

- E2 - E2

siBIG3

IP : PP1Ca WCL

100

MCF-7 cells


50

37

37 PKA catalytic

b-actin

50

PP1Ca

MC

F-7

KP

L-3

C

ZR

-75-1

T47D

BT

-474

HC

C1500

KP

L-1

HB

C4

Uncropped images of Supplementary Figure 1f

ERAP

tamoxifen - - - + - - + -

MCF-7 KPL-3C

E2 - + + +

- - - + - - + - - + + +

- - - + - - + -

MCF-7 KPL-3C

- + + +

- - - + - - + - - + + +

75

50

37

75

50

37

Long exposure Short exposure

b-actin

PP1Ca

Uncropped images of Supplementary Figure 1h


Uncropped images of Supplementary Figure 1m

10 nM E2 0 10 5

MCF-7 cells

siEGFP siPKA

0 5 10

BIG3

PKA catalytic

PHB2

75

150

100

250

50

37

0 10 5

siEGFP siPKA

0 5 10 (min)

WCL IP : BIG3

pS305-BIG3 pS1208-BIG3

10 nM E2 0 10 5

MCF-7 cells

siEGFP siPKA

0 5 10 0 10 5

siEGFP siPKA

0 5 10 (min)

IP : BIG3 IP : BIG3

150

250

Uncropped images of Supplementary Figure 2a Uncropped images of Supplementary Figure 2b

37

50

25

37

75 - + + -

Nucleus of MCF-7 cells

IP : ERa

10 mM ERAP 10 nM E2

+ +

WCL

PHB2

pSer-PHB2

- - + - - +

ERa

+ -

+ -

50

pSer-PHB2

PHB2

50

75

37

50

37

75

b-actin

- + + - + + + +

10 mM ERAP

10 nM E2 - + - + + + + +

1 h

MCF-7 cells

- + + - + + + +

3 h 6 h 12 h 18 h 24 h

b-actin


Cyto Nuc

10 mM ERAP - - - + - +

IP: FLAG

HEK293T cells

75

50

37

37

25

FLAG-ERa

HA-PHB2

-

IP: IgG

+ - + - -

Cyto Nuc

10 nM E2 + - + + - + - + + + - +

FLAG-ERa Myc-BIG3

+ + + +

HA-PHB2 WT S39A

Cyto Nuc

ERAP - - - + - +

WCL

-

75

50

37

25

FLAG-ERa

HA-PHB2

+ - + - -

Cyto Nuc

E2 + - + + - + - + + + - +

FLAG-ERa Myc-BIG3

+ + + +

HA-PHB2 WT S39A

75

pS39-PHB2

50

37

37

25

FLAG-ERa

HA-PHB2

lamin

50

100

37

75

50 tubulin

Uncropped images of Supplementary Figure 2c

Uncropped images of Supplementary Figure 3a

E2 - - E2 E2 E2 - - E2 E2 - - siEGFP siEGFP siPP1Ca siPKA siPP1Ca siPKA

BIG3

50

150

100

75

25

37 PKA catalytic

MCF-7 cells

IP : BIG3 WCL

250

pS1208-BIG3 pS305-BIG3

E2

150

- - siEGFP

E2

siEGFP

E2 E2 - - E2 E2 - - siPP1Ca siPKA siPP1Ca siPKA

IP : BIG3

250

100

- siEGFP siPP1Ca siPKA siEGFP siPKA siPP1Ca

37

50

PP1Ca

E2 - - E2 E2 E2 - E2 E2 - -

IP : BIG3 WCL

Uncropped images of Supplementary Figure 3c Uncropped images of Supplementary Figure 3b

- E2 - E2

WT DPP1Ca

IP : PHB2

- E2 - E2

wt DPP1Ca

WCL

HEK293T cells

250

100

75

37

150

50

- E2 - E2

WT

IP : PHB2

DPP1Ca FLAG-BIG3

PP1Ca 37

50

PHB2

FLAG-BIG3

pS39-PHB2

MCF-7 cells

0 50

(-) 10nM E2

75

250

50

0 75

BIG3/PP1Ca

-Peptide (mM) 75 0 0 50 50 75 0

IP : BIG3

150

100

BIG3

WCL

37

PKA catalytic

250

150

50

pS1208-BIG3

PP1Ca

pS305-BIG3

37

25

0

(-) 10nM E2 (-) 10nM E2

0

10nM E2

50 75 0

WCL

0

10nM E2

50 75 0

IP : BIG3 (-)

(-)

PKA catalytic


Uncropped images of Supplementary Figure 4b

20 20

H-89 okadaic okadaic

pS305-BIG3

75

250

10 nM E2

- 1

H-89

- 0.5 - 40 -

MCF-7 cells

1 0.5 40

IP : BIG3

150

100

pS1208-BIG3

- 10 nM E2 -

250

150

BIG3

IP : BIG3 WCL

PHB2

37

25

37

pS39-PHB2

20 20

H-89 okadaic okadaic

10 nM E2

- 1

H-89

- 0.5 - 40 - 1 0.5 40

IP : PHB2

- 10 nM E2 -

PHB2

WCL

pS39-PHB2

PHB2

37

50

50

37

- - - +

- + - + 10 mM ERAP

10 nM E2 - + + +

siEGFP

MCF-7 cells

siPKA

PKA catalytic

+ + + + + +

IB : PHB2

recombinant PP1Ca (ng)

0 6.25 12.5

37

PHB2

pS39-PHB2 IPs

P

25 50 100

25


IP :

pS39-PHB2 WCL

BIG3

PKCa

WCL

pS39-PHB2 PHB2

- - + - - + - + - 10 mM ERAP 10nM E2

50

150

100

75

25

37

MCF-7 cells

250

- - + + - + + + +

Supplementary Figure 6 | Full-length images of immunoblots. Uncropped images of scanned immunoblots in Figures and

Supplementary figures with size marker indications (kDa)

Supplementary Table 1. Mascot Search Results MS/MS Fragmentation of GSGCSCTAPALSGPVAR (upper)

and CWSLVAPH (lower) found in BIG3_HUMAN in Sprot

Nucleus Cytoplasm

1 2 2 1 5.00 + 44 Pre < 2 cm n0 I

2 0 2 1 8.09 48 Post 2.1 - 5 cm n0 I

7 1 2 2 6.48 44 Pre < 2 cm n0 IIA

9 2 3 1 10.00 69 Post 2.1 - 5 cm n1 IIB

10 2 2 1 6.92 + 58 Post 2.1 - 5 cm n1 II

12 2 0 1 7.17 63 Post 2.1 - 5 cm n0 IIA

20 1 3 2 10.00 48 Pre 2.1 - 5 cm n0 I

22 1 0 1 8.81 71 Post < 2 cm n0 IIA

29 0 0 0 7.02 82 Post < 2 cm n0 IIA

30 3 3 1 5.83 + 47 Pre 2.1 - 5 cm n0 IIA

32 1 0 0 6.84 + 70 Post < 2 cm n1 IIB

35 1 2 1 4.51 + 55 Post < 2 cm n0 II

36 1 3 1 7.22 69 Post 2.1 - 5 cm n0 IIA

37 1 3 1 8.05 58 Post < 2 cm n0 I

39 3 0 0 6.73 42 Pre 2.1 - 5 cm n0 I

41 1 3 1 9.48 45 Pre 2.1 - 5 cm n0 I

43 3 0 1 8.24 51 Post < 2 cm n0 I

44 3 1 2 5.75 58 Post 2.1 - 5 cm n0 I

45 2 3 2 8.01 48 Pre 2.1 - 5 cm n1 I

46 2 0 0 5.62 46 Pre 2.1 - 5 cm n0 I

47 2 0 0 5.54 40 Pre 2.1 - 5 cm n0 I

48 3 1 0 1.17 + 51 Post 2.1 - 5 cm n0 IIA

49 1 3 1 10.00 49 Pre < 2 cm n0 I

50 2 2 1 7.05 55 Post < 2 cm n0 I

51 2 0 1 6.38 44 Pre < 2 cm n0 I

55 2 0 1 6.13 55 Post 2.1 - 5 cm n0 I

57 0 1 1 7.06 71 Post 2.1 - 5 cm n1 IIA

58 2 0 0 5.83 46 Pre < 2 cm n0 I

61 2 3 1 9.06 67 Post < 2 cm n0 IIA

62 2 3 2 9.98 48 Pre < 2 cm n1 I

70 2 3 1 10.00 51 Post 2.1 - 5 cm n0 I

73 2 3 2 10.00 59 Post < 2 cm n0 I

74 3 0 0 7.11 + 55 Post < 2 cm n1 I

76 2 0 1 5.62 55 Post < 2 cm n0 I

77 3 0 1 6.21 + 63 Post < 2 cm n0 I

78 1 0 0 6.08 72 Post < 2 cm n0 I

79 0 3 2 5.57 54 Post 2.1 - 5 cm n0 II

82 3 0 0 9.10 + 45 Pre 2.1 - 5 cm n0 II

85 1 3 1 7.17 50 Pre 2.1 - 5 cm n1 II

86 2 3 0 6.99 50 Pre < 2 cm n0 I

88 1 3 2 9.81 46 Pre < 2 cm n0 I

89 0 2 1 3.00 52 Pre 2.1 - 5 cm n0 II

92 3 0 0 10.00 + 40 Pre 2.1 - 5 cm n0 II

93 3 0 1 5.18 + 49 Pre < 2 cm n0 I

95 2 0 0 4.99 58 Post < 2 cm n0 I

96 2 0 0 4.00 78 Post 2.1 - 5 cm n0 II

100 3 0 0 5.14 + 46 Pre 2.1 - 5 cm n0 II

101 2 0 0 3.08 52 Pre < 2 cm n0 I

102 3 0 0 5.66 44 Pre < 2 cm n0 I

103 2 0 1 4.98 58 Post < 2 cm n0 I

117 1 0 0 4.49 48 Pre < 2 cm n0 I

118 1 1 0 4.68 64 Post < 2 cm n1 II

120 2 0 0 6.63 + 57 Post < 2 cm n0 II

122 2 1 1 3.12 53 Post 2.1 - 5 cm n0 I

124 0 3 0 10.00 39 Pre 2.1 - 5 cm n0 I

125 2 0 0 5.20 70 Post < 2 cm n0 II

129 2 0 0 2.42 + 68 Post 2.1 - 5 cm n0 II

135 2 0 1 4.67 70 Post < 2 cm n0 I

136 1 0 0 5.38 47 Post < 2 cm n0 I

138 1 0 0 6.75 51 Pre < 2 cm n0 IIA

145 2 0 0 4.80 56 Post < 2 cm n0 I

146 1 3 2 9.99 58 Post < 2 cm n0 I

155 2 0 0 4.66 68 Post 2.1 - 5 cm n0 II

156 0 2 1 9.05 46 Pre < 2 cm n0 I

159 3 0 0 2.25 + 40 Pre 2.1 - 5 cm n0 II

161 1 1 1 5.43 48 Pre 2.1 - 5 cm n0 I

163 1 3 1 9.34 49 Pre < 2 cm n0 I

168 1 3 1 7.81 51 Post 2.1 - 5 cm n0 II

170 0 2 2 8.08 63 Post 2.1 - 5 cm n1 II

174 1 0 1 5.72 43 Pre 2.1 - 5 cm n0 II

176 3 3 2 5.51 80 Post 2.1 - 5 cm n0 II

180 3 0 0 5.00 78 Post 2.1 - 5 cm n0 II

181 2 0 1 5.66 70 Post < 2 cm n0 I

182 3 0 0 5.60 68 Post < 2 cm n0 I

187 1 3 2 7.73 58 Post < 2 cm n0 I

192 2 3 1 8.04 71 Post 2.1 - 5 cm n0 II

193 3 0 0 5.37 46 Pre 2.1 - 5 cm n0 II

195 0 3 0 7.96 49 Post < 2 cm n0 I

198 2 0 0 4.93 54 Post < 2 cm n0 I

203 1 0 1 6.68 50 Pre < 2 cm n0 I

205 1 1 1 5.26 44 Pre 2.1 - 5 cm n0 II

208 1 3 2 10.00 47 Pre < 2 cm n1 I

Tumour

size

Lymph node

metastasisStage

PHB2 intensity (0-3)Case

BIG3 intensity

(0-3)DFI (year) Recurrence Age Menopause


Supplementary Table 2. Clinical characteristic and results of immunohistochemistry of ERa-positive breast cancer

specimens.

129 2 0 0 2.42 + 68 Post 2.1 - 5 cm n0 II

135 2 0 1 4.67 70 Post < 2 cm n0 I

136 1 0 0 5.38 47 Post < 2 cm n0 I

138 1 0 0 6.75 51 Pre < 2 cm n0 IIA

145 2 0 0 4.80 56 Post < 2 cm n0 I

146 1 3 2 9.99 58 Post < 2 cm n0 I

155 2 0 0 4.66 68 Post 2.1 - 5 cm n0 II

156 0 2 1 9.05 46 Pre < 2 cm n0 I

159 3 0 0 2.25 + 40 Pre 2.1 - 5 cm n0 II

161 1 1 1 5.43 48 Pre 2.1 - 5 cm n0 I

163 1 3 1 9.34 49 Pre < 2 cm n0 I

168 1 3 1 7.81 51 Post 2.1 - 5 cm n0 II

170 0 2 2 8.08 63 Post 2.1 - 5 cm n1 II

174 1 0 1 5.72 43 Pre 2.1 - 5 cm n0 II

176 3 3 2 5.51 80 Post 2.1 - 5 cm n0 II

180 3 0 0 5.00 78 Post 2.1 - 5 cm n0 II

181 2 0 1 5.66 70 Post < 2 cm n0 I

182 3 0 0 5.60 68 Post < 2 cm n0 I

187 1 3 2 7.73 58 Post < 2 cm n0 I

192 2 3 1 8.04 71 Post 2.1 - 5 cm n0 II

193 3 0 0 5.37 46 Pre 2.1 - 5 cm n0 II

195 0 3 0 7.96 49 Post < 2 cm n0 I

198 2 0 0 4.93 54 Post < 2 cm n0 I

203 1 0 1 6.68 50 Pre < 2 cm n0 I

205 1 1 1 5.26 44 Pre 2.1 - 5 cm n0 II

208 1 3 2 10.00 47 Pre < 2 cm n1 I

Supplementary Table 3. The sequences of each primer set.

Genes

5’-CGGAATTCATGGAAGAAATCCTGAGGA AGC-3′ (forward)

5′-ATAGTTTAGCGGCCGCACAATGATGTCATAGACACGG-3′ (reverse)

BIG3

5’-GTGCGGGCAGCCCTCAGTCAA-3′ (forward)

5′-TTGACTGAGGGCTGCCCGCAC-3′ (reverse)

BIG3 mutant

(T162A)

5’-TCAGGCTGCGCCTGCACTGCG-3′ (forward)

5′-CGCAGTGCAGGCGCAGCCTGA-3′ (reverse)

BIG3 mutant

(S305A)

5’-GGCCGAGGAGAAGGCTGCTCC-3′ (forward)

5’-GGAGCAGCCTTCTCCTCGGCC-3′ (reverse)

BIG3 mutant

(S305E)

5’-CGGCTCCTGGCCCTCTCCAAT-3′ (forward)

5′-ATTGGAGAGGGCCAGGAGCCG-3′ (reverse)

BIG3 mutant

(S689A)

5’-GCACGGCTGGCCTGCGCTCTA-3′ (forward)

5′-TAGAGCGCAGGCCAGCCGTGC-3′ (reverse)

BIG3 mutant

(S925A)

5’-CGCTGCTGGGCCCTTGTGGCC-3′ (forward)

5′-GGCCACAAGGGCCCAGCAGCG-3′ (reverse)

BIG3 mutant

(S1208A)

5’-CGCTGCTGGGAACTTGTGGCC-3′ (forward)

5’-GGCCACAAGTTCCCAGCAGCG-3′ (reverse)

BIG3 mutant

(S1208E)

5’-AGATACATCGCCATGCAGAAC-3′ (forward)

5′-GTTCTGCATGGCGATGTATCT-3′ (reverse)

BIG3 mutant

(S1763A)

The underlines indicate the recognition sites of restriction enzymes.

Double-underlines indicate the mutation sites.

Cloning

Sequence Purpose

5′-CGGAATTCCAGACCGTGCATCATGGCCCAGAACTTGAAGGA-3′ (forward)

5′-CCGCTCGAGTTTCTTACCCTTGATGAGGCTGT-3′ (reverse)

PHB2

5′-GTGCGCGAAGCCGTGTTCACC-3′ (forward)

5′-GGTGAACACGGCTTCGCGCAC-3′ (reverse)

PHB2

(S39A)

5’-CCCAAGAATGAAAGCAAGCA-3’ (forward)

5’-CCGAAACTCCAAAGGAAAGG-3’ (reverse)

Real-time PCR

Real-time PCR

PKCa

5’-TCAAGCAGCACCCATTCTTC-3’ (forward)

5’-TCAGGGCATCAGGTCTTCAC-3’ (reverse)

PKCe

5’-TCAGGGCATCAGGTCTTCAC-3’ (forward)

5’-GGGGAGAGAGGCAATGAAGA-3’ (reverse)

CAMK2

5’-AACTTAGAGGTGGGGAGCAG-3’ (forward)

5’-CACAACCATGCCTTACTTTATC-3’ (reverse)

b2-microglobulin

Real-time PCR

Real-time PCR

Cloning

Cloning

Cloning

Cloning

Cloning

Cloning

Cloning

Cloning

Cloning

Cloning

a b c - nature research · phosphorylation sites of big3 by pka, as determined using netphos 3.1...

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