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Supplementary Figure 1
IP:GST
A
CK2ab
Rat GST-Par-4 +
GST +
35S-CK2a
35S-CK2b
43
37
B
CK2a + Rat GST-Par-4
CK2b
32P-Par-4
32P-CK2b
75
37
Autophosphorylation
Figure S1. Par-4 is a new substrate of CK2. (A) Recombinant protein GST-Par-4, CK2a and
CK2b were produced in vitro by TNT reticulocyte lysate system. After 35S labelling of CK2
subunits, a pull-down of GST-Par-4 was performed using anti-GST antibody. Shown is an
autoradiography of the immunoprecipitates run on a 10% SDS-PAGE. (B) in vitro kinase assay
using as substrate GST-Par-4 (5 mg), was performed in presence of [g-32P]ATP with 60 ng of
recombinant CK2a subunit alone or together with increasing amounts of CK2b subunit (1, 3, 7.5,
15, 30, 60 ng). Phosphorylated Par-4 and autophosphorylation of CK2b is vizualized by
autoradiography.
1
Supplementary Figure 2
A
0
CalA
30’
60’
90’
Ph-Par-4
Par-4
43
Hsp90
90
OA
CalA
B
-
20nM
0,5
1
µM
Ph-Par-4
Par-4
43
Inputs
C
WB: Par-4
43
37
WB:PP1
LC *
D
CalA
RecPP1g
Ph-Par-4
Par-4
-
+
+
+
200
+
100
µg
43
Figure S2. Potential regulation of Par-4 phosphorylation by Phosphatase PP1 (A) PC-3 cells
were treated with the phosphatase inhibitor CalA (20 nM) and, at indicated time points,
phosphorylated Par-4 (upward shift) was assessed by western blotting. (B) Cells were treated for 1h
with CalA (20 nM) or okadaic acid (OA) both at a low (0.5 µM; shown to inhibit only phosphatase
PP2) and at a high dose (1 µM; shown to inhibit both PP1 and PP2 phosphatases). Par-4 was analyzed
by western blotting. (C) To study the interaction of endogenous Par-4 with PP1 phosphatase,
immunoprecipitation of Par-4 in PC-3 cells was followed by immunodetection of PP1. IP IgG:
immunoprecipitation with a non relevant antibody (IgG mouse). Inputs: protein level in total cell
lysates. LC: light Chain (D) Cell lysates from PC-3 cells treated or not with CalA, were incubated in
presence or absence of purified phosphatase PP1 (100 or 200µg) 1,2, 45 min at 30°C. Par-4
dephosphorylation was evaluated by immunoblotting.
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Supplementary Figure 3
A
117GDEEEPDpSAPEKGR130
[MH]+ = 1595,64
117GDEEEPDpSAPEK128
[MH]+ = 1382,53
220STIpSAPEEEILNRYPR235
[MH]+ = 1954,95
P1’
P1
P2
-
B
Cycle N°
1
2
3
4
5
6
7
8
9
10
P1
CK2rec P1’
P2
Disc
Inputs
IP: GFP
C
RecCK2
+
+
+
+
Par-4 (GFP)
75
Actin
50
75
32P-Par-4
0,5
2
0,1
1,5
Figure S3. Rodent Par-4 is phosphorylated by CK2 at residues S124 and S223 (A, B) The
phosphopeptides observed in Fig.2A (phosphorylated by the recombinant CK2) were analyzed as
described in Material and Methods by MALDI-TOFF mass spectrometry (A) and Edman degradation
(B), thus identifying S124 and S223 as Par-4 phosphorylated residues. (B) Edman degradation data
showed that peptides P1/P1’ and P2 were phosphorylated at the amino-acid position number 4 and 8,
respectively. Numbers in Edman degradation data correspond to the amino-acid number in a tryptically
generated peptide starting from its N terminus. ‘Disc’ designates the amount of remaining 32P-label on
the Sequelon-AA disc after performing 10 Edman cycles. (C) After immunoprecipitation of COS cells
extracts, the wild-type or 124D or 223D mutant Par-4 (GFP-tagged) were subjected to an in vitro kinase
assay in the presence of recombinant CK2 and [g-32P]ATP. Radiolabeled phospho-Par-4 was visualized
by autoradiography. In parallel, equal expression levels of GFP-Par-4 and GFP-124D, 223D were
assessed (aliquot of the immunoprecipitates) by western blot (Inputs, right panel). Actin is the loading
3
control.
Supplementary Figure 4
*
Luciferase intensity (a.u)
CTL
NF-kB promLuc +TPA
120
80
Par-4 (GFP)
40
Hsp90
Inputs
0
Figure S4. Effect of Par-4 mutants on NF-kB activity Luciferase reporter assay using an NF-kB
luciferase promoter was performed on COS cells co-transfected either with an empty vector (Mock),
wild-type Par-4, 124A223A or 124D223D Par-4 mutants. Prior analysis, cells were treated or not by
TPA (50 nM) for 1h to stimulate NF-kB activity3 . Inputs: protein level in total cell lysates. Bars
represent the mean ±S.D. of at least six independent experiments. Mock, cell transfected with an empty
vector. * p<0,05
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Supplementary Figure 5
A
Caspase
cleavage site
CK2 site
B
Species
P-Site
-4
-3
-2
-1
0
1
2
3
4
Human
G132
E
E
E
P
D
G
V
P
E
Bonobo
G132
E
E
E
P
D
G
L
P
E
Macaque
G132
E
E
E
Q
D
G
V
P
E
Mouse
S125
E
E
E
P
D
S
A
R
E
Rat
S124
E
E
E
P
D
S
A
P
E
Taurus
C216
E
E
E
R
D
C
A
P
E
Zebrafish
S117
E
S
P
T
H
S
K
S
K
Figure S5. Par-4 Sequence conservation (A) Sequence alignment of Human Par-4 vs. Rat Par-4 (B)
Alignment between species of the sequence containing the conserved caspase targeted site. In red, the
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caspase cleavage site and in blue, the CK2 site conserved specifically in rodent (S124 in rat).
Supplementary Figure 6
A
+
TRAIL
Par-4 (GFP)
Cleaved Par-4
-
75
50
B
+
-
+
43
34
90
Hsp90
90
Hsp90
-
TRAIL
hPar-4 (Myc)
Cleaved Par-4
+
TRAIL
TRAIL
95
Cleaved PARP
28
Hsp90
90
p41/43
Cleaved Casp-8
26
p18
75
Par-4 (GFP)
40
% Apoptosis
55
Casp-8
PARP
30
GFP
26
20
10
0
*
C
Rec.Casp3
Rec.CK2
+
+
-
+
+
hPar-4 (GFP)
75
*
50
Cleaved Par-4
6
Supplementary Figure 6
Figure S6. Caspases mediated cleavage of rodent Par-4, at residue D123, is required for its full
proapoptotic activities. (A) HCT116 cells transfected either with the GFP-tagged constructs rat Par4 (wild type, D123A mutant, left panel) or with the Myc-tagged constructs human Par-4 (wild type,
D131A mutant, right panel) were treated with TRAIL (150 ng/ml, 3h) and Par-4 cleavage was
evaluated by Western-blotting using GFP or Myc antibody. (B) As already shown for the human
D131A mutant of Par-424, the caspase resistant D123A mutant of rat Par-4 impaired apoptosis
observed by PARP (left panel)/caspase-8 (right panel) cleavage (Western-blot). ) Hsp90 was used as a
loading control. (C) Caspase assay using recombinant caspase-3 (Rec.Casp3) was performed on prephosphorylated human GFP-Par-4 (in vitro kinase assay using recombinant CK2 (Rec.CK2) in the
presence of ATP as described in Fig.2) and cleaved Par-4 was detected by immunoblotting using GFP
antibody. *non-specific band
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Supplementary Figure 7
A
40
*
CTL
TRAIL
*
*
*
% Apoptosis
30
20
10
0
Mock
B
Par4
(Rat)
124A
124D
223A
223D
TRAIL
Mock Par-4 124A 124D 223A 223D
Par-4 (GFP)
Cleaved Par-4
75
50
GFP
26
PARP
Cleaved PARP
Casp8
95
28
55
Cleaved Casp8
26
Hsp90
90
Figure S7. Effect of single Par-4 mutants on pro-apoptotic activity of rodent Par-4. (A) GFP-tagged
constructs rat Par-4 (wild type, 124A/D or 223A/D mutants) were transfected in HCT116 cells and treated
with TRAIL (150ng/ml, 3h). Apoptosis were measured by Dapi staining (A) and caspase-8/PARP
cleavage (B) Hsp90 was used as a loading control. Bars represent the mean ±S.D. of at least four
independent experiments. *p<0.05.
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Supplementary Figure 8
A
Par-4 (GFP)
Par-4(124-332)
75
GFP
26
Hsp110
100
Conditioned
Media
Cell
lysate
24h
B
Mock
= 18% apoptosis
= 40% apoptosis
Par-4 (124-332)
Par-4 (Rat)
= 22% apoptosis
Figure S8. The rat Par-4 cleaved 124-332 amino acid fragment is secreted and induces apoptosis
in the surrounding cells. (A) COS cells were transfected with GFP-tagged rat Par-4 wild type or Par4(124-332). After 16 hours, cells were washed and fresh medium was added for 3h. Conditioned media
were concentrated and the presence of Par-4 was determined by immunoblotting using GFP antibody.
Non secreted Hsp110 was used as a control. (B) Concentrated conditioned media from COS cells
transfected with GFP-tagged Par-4 and Par-4(124-332) were added to PC-3 cells for 24h. As a control,
we used conditioned medium from cells transfected with a GFP construct. Apoptosis was visualized by
microscopy and percentages of cell death evaluated by FACS analysis.
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Supplementary Figure 9
A
CalA
-
+
+
+
TBB
Ph-Par-4
hPar-4
-
+
-
+
<
43
90
Hsp90
B
CalA
-
+
+
TBB
-
-
+
Ph231-hPar-4
43
Ph-Par-4
hPar-4
43
Hsp90
90
Figure S9. CK2 inhibition impairs human Par-4 phosphorylation induced by phosphatase
inhibitor CalA (A,B) PC-3 cells were treated or not with the phosphatase inhibitor CalA (20 nM, 1h)
and pre-treated or not by CK2 inhibitor TBB (40 mM, 30 min). (A) inhibition of the endogenous Par-4
phosphorylation (upward shift) was assessed by western blotting. (B) inhibition of the human Par-4
phosphorylation on serine 231 was determined by western blotting using the anti-phosphoserine231
antibody. Hsp90 served a as loading control.
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Casp8 cleavage
PARP cleavage
120
TRAIL
100
80
TRAIL
Normalized ratio
Cleaved C8/C8(a.u)
A
Normalized ratio
Cleaved PARP/ PARP (a.u)
Supplementary Figure 10
60
80
60
40
40
20
20
0
0
TRAIL
Normalized ratio
Cleaved PARP/ PARP (a.u)
PARP cleavage
80
B
60
90
Cleaved PARP
28
Casp8
55
Cleaved
Casp8
26
CK2a
43
Par-4
43
Hsp90
90
TRAIL
40
20
Normalized ratio
Cleaved C8/C8(a.u)
PARP
0
Casp8 cleavage
30
TRAIL
20
10
0
Figure S10. Anti-apoptotic role of CK2 is dependent on Par-4 in the human resistant
prostate cancer cells. PC-3 cells were transfected with different CK2 siRNA (siCK2T from
ThermoFischer or siCK2AB from Ambion) together with scrambled siRNA fluorescently labeled
with FAM (Scr siRNA) or Par-4 siRNA (siPar-4sc from Santa-cruz) for 48h hours. Then, cells
were treated or not with TRAIL (3h, 500ng/ml). Apoptosis were measured by PARP/caspase-8
cleavage. The downregulation of Par-4 and CK2α proteins was confirmed by immunoblot using
the corresponding antibodies. Hsp90 was used as a loading control. Bar graphs show semiquantified densitometry from Caspase-8 and PARP Western blot analysis of the Figure 7B (A) and
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panel (B).
Supplementary Figure 11
Figure S11. Hypothetic model of negative regulation of Par-4 by CK2. In the resistant prostate cancer
cells, which are characterized by a high CK2 activity, apoptotic stimuli favours CK2 induced Par-4
phosphorylation both in human and in rodent (S231 in human and S124/S223 in rat Par-4), thereby
dramatically reducing the apoptotic functions of the full length and the cleaved form of Par-4.
Interestingly, the inhibitory effect of CK2 slightly differs among species. While rat Par-4 is mainly
phosphorylated by CK2 at S124 thereby inhibiting caspase-mediated Par-4 cleavage (D123) and apoptosis
(right panel), the caspase-mediated cleavage of human Par-4 (D131) is not affected by CK2
phosphorylation (left panel). Conversely, and in contrast to the rat ortholog residue S223, the
phosphorylation at residue S231 of human Par-4 is critical to inhibit the pro-apoptotic functions of Par-4
(both full length and cleaved form).
SUPPLEMENTAL DATA
Par-4 phosphorylation is regulated by phosphatase PP1
Par-4 is a highly phosphorylated protein and the addition of a broad inhibitor of phosphatases
like Calyculin A induced an upward shift which increases in a time dependent manner
(Fig.Suppl. Fig.2A). Knowing that several substrates of CK2 are dephosphorylated by
PP1(ref.46), we compared the efficacy of CalA with that of Okadaic Acid (OA). The latter
inhibitor has the ability to inhibit PP2A at low doses (<0.5µM) whereas it blocks both PP1 and
PP2A at high doses (>1µM). We observed that OA had no effect on human endogenous Par-4
phosphorylation at low doses, thus suggesting the involvement of PP1 (Fig.Suppl. Fig.2B). In
that respect, we observed that both endogenous human Par-4 and PP1 co-immunoprecipitate
(Fig.Suppl. Fig.2C). To further confirm that dephosphorylation of Par-4 is PP1-dependent, we
added to the lysate of cells treated with CalA, recombinant PP1 enzyme and evaluated Par-4
phosphorylation status. Indeed, the addition of recombinant PP1 that overcome the CalA
inhibitory effect leads to an efficient dephosphorylation of Par-4 (Fig.Suppl. Fig. 2D).
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SUPPLEMENTAL EXPERIMENTAL PROCEDURES
35S
metabollic labeling
In vitro translation of the subunit GST-CK2 a or b and GST-Par-4 were performed using the
TNT assay (Promega, Charbonnières-les-Bains, France) following the manufacturers
instruction: 0,5 µg of each gene was transcribed by phage T7 polymerase and
35S-labeled
methionine (10 µCi) was incorporated into the translated proteins. To test the yield, each
reaction was submitted to autoradiography. For immunoprecipitation, the CK2 subunits were
incubated with GST-Par-4 or GST alone pre-associated with glutathion beads in PBS, 0,3%
triton, BSA 5 mg/ml, 0,01% tween, protease inhibitors cocktail (Roche diagnostic) for 90 min.
After washes, immunocomplexes were denaturated in Laemmli buffer and resolved in SDSPAGE 12%. The gels were stained in Coomassie blue and dried before exposure to
autoradiography.
Protein Phosphatase Assay
5x105 cells were lysed in 50 mM Tris-HCl (pH 7.5), 1% NP40, 1mM EDTA, 100 mM NaCl.
80µg of protein were added to a final volume of 100 µl in 20 mM Hepes (pH 7.4), 2 mM MnCl
2,5mM DTT with 100 or 200µg of recombinant phosphatase PP1g
(ref.2)
and incubated 45 min
30°C.
Secretion analysis
The medium of transfected Cos cells was replaced by fresh free medium and incubated 3 hours.
Conditioned media were harvested and concentrated using microcon 10 or Amicon ultracel-10k
following the manufacturer’s instruction (Millipore). For western-blot, Laemmli Buffer was
added and samples were run on 10% SDS-PAGE. For induction of apoptosis, conditioned media
was added on PC-3 cells for 48h and apoptosis was assessed by FACS.
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SUPPLEMENTAL REFERENCES
1. Eriksson, J. E., Toivola, D. M., Sahlgren, C., Mikhailov, A. and Harmala-Brasken, A. S.
Strategies to assess phosphoprotein phosphatase and protein kinase-mediated -mediated
regulation of the cytoskeleton. Methods Enzymol., 1998; 298, 542-69.
2. Renglin, A., Harmala-Brasken, A. S., Eriksson, J. E. and Onfelt, A. Mitotic aberrations induced
by carbaryl reflect tyrosine kinase inhibition with coincident up-regulation of serine/threonine
protein phosphatase activity: implications for coordination of karyokinesis and cytokinesis.
Mutagenesis, 1999; 14, 327-33.
3. Spencer, W., Kwon, H., Crepieux, P., Leclerc, N., Lin, R. and Hiscott, J. Taxol selectively
blocks microtubule dependent NF-kappaB activation by phorbol ester via inhibition of
IkappaBalpha phosphorylation and degradation. Oncogene,1999; 18, 495-505.
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