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Supplementary Figure 1 IP: GST A CK 2 ab Rat GST-Par-4 + GST + 35 S-CK 2 a 35 S-CK 2 b 43 37 B CK 2 a + Rat GST-Par-4 CK 2 b CK 2 32 P-Par-4 32 P-CK 2 b 75 37 Autophosphorylation Figure S 1. Par-4 is a new substrate of CK 2. (A) Recombinant protein GST-Par-4, CK 2 a and CK 2 b were produced in vitro by TNT reticulocyte lysate system. After 35 S labelling of CK 2 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-32 P]ATP with 60 ng of recombinant CK 2 a subunit alone or together with increasing amounts of CK 2 b subunit (1, 3, 7. 5, 15, 30, 60 ng). Phosphorylated Par-4 and autophosphorylation of CK 2 b is vizualized by autoradiography. 1
Supplementary Figure 2 A Cal. A 0 30’ 60’ 90’ Ph-Par-4 43 Hsp 90 90 OA Cal. A B - 20 n. M 0, 5 1 µM Ph-Par-4 43 IP C G Ig -4 ar P Inputs P: I WB: Par-4 43 37 WB: PP 1 LC * D Cal. A Rec. PP 1 g Ph-Par-4 - + + + 200 + 100 µg 43 Figure S 2. Potential regulation of Par-4 phosphorylation by Phosphatase PP 1 (A) PC-3 cells were treated with the phosphatase inhibitor Cal. A (20 n. M) and, at indicated time points, phosphorylated Par -4 (upward shift) was assessed by western blotting. (B) Cells were treated for 1 h with Cal. A (20 n. M) or okadaic acid (OA) both at a low (0. 5 µM; shown to inhibit only phosphatase PP 2) and at a high dose (1 µM; shown to inhibit both PP 1 and PP 2 phosphatases). Par-4 was analyzed by western blotting. (C) To study the interaction of endogenous Par-4 with PP 1 phosphatase, immunoprecipitation of Par-4 in PC-3 cells was followed by immunodetection of PP 1. IP Ig. G: immunoprecipitation with a non relevant antibody (Ig. G mouse). Inputs: protein level in total cell lysates. LC: light Chain (D) Cell lysates from PC-3 cells treated or not with Cal. A, were incubated in presence or absence of purified phosphatase PP 1γ (100 or 200µg) 1, 2, 45 min at 30°C. Par-4 dephosphorylation was evaluated by immunoblotting. 2
Supplementary Figure 3 A 117 GDEEEPDp. SAPEKGR 130 [MH]+ = 1595, 64 117 GDEEEPDp. SAPEK 128 [MH]+ = 1382, 53 220 STIp. SAPEEEILNRYPR 235 [MH]+ = 1954, 95 P 1’ P 1 P 2 - B Cycle N° 1 2 3 4 5 6 7 8 9 10 P 1 CK 2 rec P 1’ P 2 Disc Inputs IP: GFP + 75 0, 5 2 0, 1 r-4 12 4 D 22 3 D Par-4 (GFP) + 32 P-Par-4 Pa GF P D 22 3 r-4 + GF P + Pa Rec. CK 2 12 4 D (R a t) C 75 Actin 50 1, 5 Figure S 3. Rodent Par-4 is phosphorylated by CK 2 at residues S 124 and S 223 (A, B) The phosphopeptides observed in Fig. 2 A (phosphorylated by the recombinant CK 2) were analyzed as described in Material and Methods by MALDI-TOFF mass spectrometry (A) and Edman degradation (B), thus identifying S 124 and S 223 as Par-4 phosphorylated residues. (B) Edman degradation data showed that peptides P 1/P 1’ and P 2 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 32 P-label on the Sequelon-AA disc after performing 10 Edman cycles. (C) After immunoprecipitation of COS cells extracts, the wild-type or 124 D or 223 D mutant Par-4 (GFP-tagged) were subjected to an in vitro kinase assay in the presence of recombinant CK 2 and [g-32 P]ATP. Radiolabeled phospho-Par-4 was visualized by autoradiography. In parallel, equal expression levels of GFP-Par-4 and GFP-124 D, 223 D were assessed (aliquot of the immunoprecipitates) by western blot (Inputs, right panel). Actin is the loading 3 control.
Supplementary Figure 4 CTL NF-k. B prom. Luc +TPA 120 3 D 22 4 D 12 23 A 2 4 A 12 rat) ( r-4 ck Pa Mo 80 Par-4 (GFP) 40 Hsp 90 Inputs D 23 42 A 12 23 42 12 Pa oc M r 4 0 k Luciferase intensity (a. u) * Figure S 4. Effect of Par-4 mutants on NF-k. B activity Luciferase reporter assay using an NF-k. B luciferase promoter was performed on COS cells co-transfected either with an empty vector (Mock), wild-type Par-4, 124 A 223 A or 124 D 223 D Par-4 mutants. Prior analysis, cells were treated or not by TPA (50 n. M) for 1 h to stimulate NF-k. B activity 3 . 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 4
Supplementary Figure 5 A Caspase cleavage site CK 2 site B Species P-Site -4 -3 -2 -1 0 1 2 3 4 Human G 132 E E E P D G V P E Bonobo G 132 E E E P D G L P E Macaque G 132 E E E Q D G V P E Mouse S 125 E E E P D S A R E Rat S 124 E E E P D S A P E Taurus C 216 E E E R D C A P E Zebrafish S 117 E S P T H S K Figure S 5. 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 5 caspase cleavage site and in blue, the CK 2 site conserved specifically in rodent (S 124 in rat).
Supplementary Figure 6 t) Ra A ( 3 r-4 12 a D A P + TRAIL Par-4 (GFP) Cleaved Par-4 - -4 TRAIL h. Par-4 (Myc) Cleaved Par-4 + 75 50 B 1 A 13 D - + 43 34 90 Hsp 90 ar h. P + TRAIL M oc k Pa r-4 D (ra 12 t) M 3 A oc k Pa r-4 (r D 12 at) 3 A D 1 2 Pa 3 A r G -4 ( FP R at ) D 1 23 Pa A r G -4 ( FP R at ) TRAIL PARP Cleaved PARP 95 28 Hsp 90 90 % Apoptosis 40 55 Casp-8 p 41/43 Cleaved Casp-8 26 p 18 75 Par-4 (GFP) 30 GFP 26 20 10 12 D 3 A ar -4 * G FP -P oc M -10 k 0 C Rec. Casp 3 Rec. CK 2 + + - + + h. Par-4 (GFP) 75 * 50 Cleaved Par-4 6
Supplementary Figure 6 Figure S 6. Caspases mediated cleavage of rodent Par-4, at residue D 123, is required for its full proapoptotic activities. (A) HCT 116 cells transfected either with the GFP-tagged constructs rat Par 4 (wild type, D 123 A mutant, left panel) or with the Myc-tagged constructs human Par-4 (wild type, D 131 A mutant, right panel) were treated with TRAIL (150 ng/ml, 3 h) and Par-4 cleavage was evaluated by Western-blotting using GFP or Myc antibody. (B) As already shown for the human D 131 A mutant of Par-424, the caspase resistant D 123 A mutant of rat Par-4 impaired apoptosis observed by PARP (left panel)/caspase-8 (right panel) cleavage (Western-blot). ) Hsp 90 was used as a loading control. (C) Caspase assay using recombinant caspase-3 (Rec. Casp 3) was performed on prephosphorylated human GFP-Par-4 (in vitro kinase assay using recombinant CK 2 (Rec. CK 2) in the presence of ATP as described in Fig. 2) and cleaved Par-4 was detected by immunoblotting using GFP antibody. *non-specific band 7
Supplementary Figure 7 A 40 * CTL TRAIL * * * % Apoptosis 30 20 10 0 Mock B Par 4 (Rat) 124 A 124 D 223 A 223 D TRAIL Mock Par-4 124 A 124 D 223 A 223 D Par-4 (GFP) Cleaved Par-4 75 50 GFP 26 PARP 95 Cleaved PARP 28 Casp 8 55 Cleaved Casp 8 26 Hsp 90 90 Figure S 7. Effect of single Par-4 mutants on pro-apoptotic activity of rodent Par-4. (A) GFP-tagged constructs rat Par-4 (wild type, 124 A/D or 223 A/D mutants) were transfected in HCT 116 cells and treated with TRAIL (150 ng/ml, 3 h). Apoptosis were measured by Dapi staining (A) and caspase-8/PARP cleavage (B) Hsp 90 was used as a loading control. Bars represent the mean ±S. D. of at least four independent experiments. *p<0. 05. 8
Par-4 (GFP) Par-4(124 -332) Pa A r. M 4(1 oc 24 k -3 32 Pa ) r 4 (R M oc at) k Pa r 4 Pa (12 r- 4 -3 4 (R 32 at ) ) Supplementary Figure 8 75 GFP 26 Hsp 110 100 Conditioned Media Cell lysate 24 h B Mock = 18% apoptosis = 40% apoptosis Par-4 (124 -332) Par-4 (Rat) = 22% apoptosis Figure S 8. 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 Par 4(124 -332). After 16 hours, cells were washed and fresh medium was added for 3 h. Conditioned media were concentrated and the presence of Par-4 was determined by immunoblotting using GFP antibody. Non secreted Hsp 110 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 24 h. 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. 9
Supplementary Figure 9 A Cal. A - + + + TBB Ph-Par-4 h. Par-4 - + < 43 90 Hsp 90 Cal. A B - + + TBB - - + Ph 231 -h. Par-4 43 Ph-Par-4 h. Par-4 43 Hsp 90 90 Figure S 9. CK 2 inhibition impairs human Par-4 phosphorylation induced by phosphatase inhibitor Cal. A (A, B) PC-3 cells were treated or not with the phosphatase inhibitor Cal. A (20 n. M, 1 h) and pre-treated or not by CK 2 inhibitor TBB (40 m. M, 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-phoserine 231 antibody. Hsp 90 served a as loading control. 10
Casp 8 cleavage PARP cleavage TRAIL 100 TRA. . . 80 Normalized ratio Cleaved C 8/C 8(a. u) 120 60 80 60 40 40 20 20 28 Casp 8 55 Cleaved Casp 8 26 K 2 A B cr C 4 s c/ si Pa r- -4 T/ si C K 2 A B Cleaved PARP Pa r si C K 90 Normalized ratio Cleaved PARP/ PARP (a. u) Sc sc r /S cr C K 2 A Pa B/ r- Sc 4 s r c/ C K 2 A 4 r. Pa PARP cleavage B TRAIL B /S Sc r 2 A B /s i. C K 2 A 4 s c Pa r- Pa r -4 T/ si C K 2 A B /S Sc r si C K B 0 cr 0 80 60 TRAIL 40 20 0 Casp 8 cleavage Normalized ratio Cleaved C 8/C 8(a. u) A Normalized ratio Cleaved PARP/ PARP (a. u) Supplementary Figure 10 30 TRAIL 20 90 B 2 A cr /S c/ s i. C K B 2 A Pa r- 4 s K r. Pa Sc r Hsp 90 0 si C 43 Sc r Par-4 10 sc / 43 4 CK 2 a Figure S 10. Anti-apoptotic role of CK 2 is dependent on Par-4 in the human resistant prostate cancer cells. PC-3 cells were transfected with different CK 2 si. RNA (si. CK 2 T from Thermo. Fischer or si. CK 2 AB from Ambion) together with scrambled si. RNA fluorescently labeled with FAM (Scr si. RNA) or Par-4 si. RNA (si. Par-4 sc from Santa-cruz) for 48 h hours. Then, cells were treated or not with TRAIL (3 h, 500 ng/ml). Apoptosis were measured by PARP/caspase-8 cleavage. The downregulation of Par-4 and CK 2α proteins was confirmed by immunoblot using the corresponding antibodies. Hsp 90 was used as a loading control. Bar graphs show semiquantified densitometry from Caspase-8 and PARP Western blot analysis of the Figure 7 B (A) 11 and panel (B).
Supplementary Figure 11 Figure S 11. Hypothetic model of negative regulation of Par-4 by CK 2. In the resistant prostate cancer cells, which are characterized by a high CK 2 activity, apoptotic stimuli favours CK 2 induced Par-4 phosphorylation both in human and in rodent (S 231 in human and S 124/S 223 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 CK 2 slightly differs among species. While rat Par-4 is mainly phosphorylated by CK 2 at S 124 thereby inhibiting caspase-mediated Par-4 cleavage (D 123) and apoptosis (right panel), the caspase-mediated cleavage of human Par-4 (D 131) is not affected by CK 2 phosphorylation (left panel). Conversely, and in contrast to the rat ortholog residue S 223, the phosphorylation at residue S 231 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 PP 1 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. 2 A). Knowing that several substrates of CK 2 are dephosphorylated by PP 1(ref. 46), we compared the efficacy of Cal. A with that of Okadaic Acid (OA). The latter inhibitor has the ability to inhibit PP 2 A at low doses (<0. 5µM) whereas it blocks both PP 1 and PP 2 A 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 PP 1 (Fig. Suppl. Fig. 2 B). In that respect, we observed that both endogenous human Par-4 and PP 1 co-immunoprecipitate (Fig. Suppl. Fig. 2 C). To further confirm that dephosphorylation of Par-4 is PP 1 -dependent, we added to the lysate of cells treated with Cal. A, recombinant PP 1 enzyme and evaluated Par-4 phosphorylation status. Indeed, the addition of recombinant PP 1 that overcome the Cal. A inhibitory effect leads to an efficient dephosphorylation of Par-4 (Fig. Suppl. Fig. 2 D). 13
SUPPLEMENTAL EXPERIMENTAL PROCEDURES 35 S metabollic labeling In vitro translation of the subunit GST-CK 2 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 T 7 polymerase and 35 S-labeled methionine (10 µCi) was incorporated into the translated proteins. To test the yield, each reaction was submitted to autoradiography. For immunoprecipitation, the CK 2 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 SDS-PAGE 12%. The gels were stained in Coomassie blue and dried before exposure to autoradiography. Protein Phosphatase Assay 5 x 105 cells were lysed in 50 m. M Tris-HCl (p. H 7. 5), 1% NP 40, 1 m. M EDTA, 100 m. M Na. Cl. 80µg of protein were added to a final volume of 100 µl in 20 m. M Hepes (p. H 7. 4), 2 m. M Mn. Cl 2, 5 m. M DTT with 100 or 200µg of recombinant phosphatase PP 1 g (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-10 k 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 48 h and apoptosis was assessed by FACS. 14
SUPPLEMENTAL REFERENCES 1. Eriksson, J. E. , Toivola, D. M. , Sahlgren, C. , Mikhailov, A. and Harmala-Brasken, A. Strategies to assess phosphoprotein phosphatase and protein kinase-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-kappa. B activation by phorbol ester via inhibition of Ikappa. Balpha phosphorylation and degradation. Oncogene, 1999; 18, 495 -505. 15