1. Volume 8, Issue 3, Pages 601-611 (September 2001)
Phosphorylation of Bid by Casein Kinases I and II Regulates Its Cleavage by Caspase 8 
Solange Desagher, Astrid Osen-Sand, Sylvie Montessuit, Edith Magnenat, Francis Vilbois, Alena Hochmann, Laurent Journot, Bruno Antonsson, Jean-Claude Martinou 
Molecular Cell 
Volume 8, Issue 3, Pages (September 2001)
DOI: /S (01)

2. Figure 1 Phosphorylation of Bid
(A) Western blot analysis of overexpressed Bid. HEK cells were transfected either with the empty pCI vector or pCI-Bid, and cell lysates were analyzed by Western blotting using the 224B anti-Bid antibody. For comparison, an aliquot of recombinant Bid (Bid rec.) was resolved in the same gel. Similar results were obtained with HeLa cells.
(B) In vivo phosphorylation of overexpressed Bid. HeLa cells were transfected either with the empty pCI vector or pCI-Bid, labeled with [32P]-orthophosphate, and immunoprecipitated with 224B anti-Bid antibody. Bid was visualized by both autoradiography (left) and Western blotting using the biotinylated 224B anti-Bid antibody (right). Similar results were obtained with HEK cells.
(C) In vivo phosphorylation of endogenous Bid. L929 cells were labeled with [32P]-orthophosphate. Bid was then immunoprecipitated and visualized as in B.
(D) In vitro phosphorylation of Bid. Jurkat cells were incubated with or without anti-Fas antibody (1 μg/ml) for 24 hr. Whole-cell extracts were then used to phosphorylate recombinant murine and human Bid for 5 min in the presence of 100 μM Z-VAD. The reaction mix was resolved by SDS-PAGE and analyzed by autoradiography. 32P-incorporation in the phosphorylated bands is presented below the autoradiogram. A Western blot of the extracts used for in vitro phosphorylation was done using specific antibodies directed against actin and human Bid
Molecular Cell 2001 8, DOI: ( /S (01) )

3. Figure 2 Identification of the Phosphorylation Sites of Bid
(A) Bid phosphopeptide analysis. Recombinant murine Bid was phosphorylated in vitro by cytosolic extract from Jurkat cells in the presence of [γ-33P]-ATP and subjected to trypsin digestion. The derived phosphopeptides were separated by reverse-phase HPLC, and the peaks were detected by absorption at 214 nm (left). 33P-incorporation into each peptide was counted and plotted against peptide fraction numbers (right).
(B) Amino acids sequence of murine Bid. The peptide contained in the most radioactive fraction 17 corresponded to amino acids 41–65 (solid bar). The peptide contained in the less radioactive fractions 12 and 13 corresponded to amino acids 66–88 (hatched bar).
(C) Schematic representation of mutations introduced into Bid.
(D) In vivo phosphorylation of Bid mutants. Hela D98/AH2 cells were transfected with the different forms of Bid and labeled with [32P]-orthophosphate. Bid proteins were immunoprecipitated with anti-Bid antibody (224B) and detected by autoradiography. 32P-incorporation is presented below the autoradiogram. Western blotting (not shown) of the same nitrocellulose membrane confirmed equal loading for each form of Bid. Similar results were obtained with Jurkat and HEK cells
Molecular Cell 2001 8, DOI: ( /S (01) )

4. Figure 3 Purification of CKIα as a Bid Phosphorylating Kinase
(A) The different steps of purification of CKIα. The active fractions eluted from each chromatography column are indicated with the amount of proteins recovered.
(B) SP sepharose cation exchange chromatography profile. The bound proteins were eluted with a gradient of NaCl as indicated. Fractions were assayed for phosphorylation of recombinant murine Bid. The active fraction SP#54–60 was further purified to homogeneity as shown in (A).
(C) Top: A silver-stained SDS-PAGE gel of fractions #31–42 from the final Mono S column. Bottom: The same fractions were assayed for Bid kinase activity.
(D) A Western blot showing the Jurkat cytosolic extract used for the purification (Jurkat), the most active fraction of the Mono S (MS#40), and the first two peaks of activity eluted from the SP sepharose column (SP#15–27 and SP#29–37). The antibody used is specific for CKIα
Molecular Cell 2001 8, DOI: ( /S (01) )

5. Figure 4
Identification of CKII and CKIϵ as Other Bid Phosphorylating Kinases
(A) Western blotting of Jurkat cytosolic extract (Jurkat), the most active fraction of Mono S (MS#40), the first active peaks of the SP sepharose (SP#15–27 and SP#29–37), and purified recombinant CKII using an anti-CKIIα antibody.
(B) Western blotting of the same samples with an anti-CKIϵ antibody.
(C) In vitro phosphorylation of Bid mutants by fraction SP#15–27 (2 μl) and by recombinant purified CKII (20 U; Calbiochem) for 2 min in the presence of 5 μM cold ATP
Molecular Cell 2001 8, DOI: ( /S (01) )

6. Figure 5
CKI and CKII Are the Main Bid Phosphorylating Kinases in Cells
(A) Site specificity of CKI and CKII. Bid mutants were phosphorylated in vitro using 16 ng recombinant CKI, 10 U recombinant CKII, or 2 μg whole-cell extract from Jurkat cells. The relative phosphorylation levels of Bid are presented below the autoradiogram.
(B) Inhibitors of CKs inhibit phosphorylation of Bid. Whole Jurkat cell extract was used for in vitro phosphorylation of recombinant murine and human Bid in the absence or presence of different kinase inhibitors: 50 μM CKI-7, 7 μg/ml heparin, 100 μM GTP, 50 μM DRB, 100 μM U0126, 1 μM wortmannin, 1 μM bisindolylmaleimide (Bis I), or 1 μM staurosporine (stauro). The inhibitors were preincubated with the extract for 20 min and were present during the phosphorylation reaction.
(C) Immunodepletion of CKIα reduces in vitro phosphorylation of Bid. Whole Jurkat cell extract was immunodepleted with the anti-CKIα antibody or an unrelated antibody (Ctrl) and was used to phosphorylate recombinant murine and human Bid in vitro (IVP, left). A Western blot of the extracts used for phosphorylation was done using specific antibodies directed against actin and CKIα (WB, right).
(D) In vivo phosphorylation of Bid by CKIα and CKIIα. Murine Bid was expressed in MCF-Fas cells together with CKs. Phosphorylation was estimated by Western blotting with the phospho-specific anti-Bid antibody. The same blot was stripped and developed with 224A anti-Bid antibody
Molecular Cell 2001 8, DOI: ( /S (01) )

7. Figure 6 CKI and CKII Protect Cells from Fas-Induced Apoptosis
(A) Overexpression of CKs reduces Fas-induced apoptosis. HeLa D98/AH2 cells were transfected with Bcl-2 or CKs together with GFP for 48 hr. Cells were then treated with 0.5 μg/ml anti-Fas antibody for 3 hr, fixed, and analyzed by fluorescence microscopy. The percentage of cell death was scored by morphology of green cells. Data are the mean ± SEM of three independent experiments. *p < 0.01; significantly different from the control (ANOVA followed by Student-Newman-Keuls' test).
(B) Inhibitors of CKs accelerate Fas-induced apoptosis. HeLa D98/AH2 cells were treated with anti-Fas antibody (0.5 μg/ml) in the absence or presence of 250 μM CKI-7 or 50 μM DRB. DNA-fragmentation was assessed using the Cell Death Detection ELISA kit (Boehringer Mannhein). The increase in DNA fragmentation was calculated by comparing the absorbance of treated cells to the absorbance of control cells. Data are means ± SD of triplicate determinations and are representative of three independent experiments.
(C) Acceleration of apoptosis by CK inhibitors is specific to Fas-mediated cell death. Cells were induced to undergo apoptosis in the absence or presence of 250 μM CKI-7 or 50 μM DRB: HeLa cells were incubated with 100 μM cisplatin for 13 hr or exposed to 100 J/m2 UVC light and further cultured for 22 hr; PC12 cells were deprived of serum for 15 hr; HeLa D98/AH2 cells were treated with 100 ng/ml soluble Fas ligand for 4 hr. Apoptosis was assessed by DNA-fragmentation ELISA.
(D) Phosphorylation of Bid prevents spontaneous apoptosis. HeLa cells were transfected for 48 hr with GFP together with either wild-type Bid or Bid S61A-S64A. The cells were fixed and analyzed by fluorescence microscopy. Data are the means ± SEM of three independent experiments. *p < 0.01 significantly different from the control (ANOVA followed by Bonferroni's test)
Molecular Cell 2001 8, DOI: ( /S (01) )

8. Figure 7
Phosphorylation of Bid Decreases Its Susceptibility to Caspase 8 Cleavage
(A) Phosphorylation of Bid inhibits its cleavage by caspase 8 in vitro. Recombinant murine Bid was incubated with a mix of the fractions SP#15–27 and MS#40 containing CKI and CKII in the absence or presence of ATP. Then recombinant active caspase 8 was added. Bid cleavage was analyzed by Western blotting using the 224B anti-Bid antibody. Data shown are representative of four independent experiments.
(B) Phosphorylation of Bid has no effect on its cleavage by granzyme B. Recombinant Bid was phosphorylated by recombinant CKI or CKII, then cleaved by caspase 8 or granzyme B, and analyzed as in (A).
(C) A nonphosphorylateable mutant of Bid is more sensitive to Fas-triggered cleavage than wild-type Bid. HeLa D98/AH2 cells were transiently transfected with either S61A-S64A Bid or wild-type Bid for 48 hr. Cells were then treated with 20 ng/ml anti-Fas antibody for indicated times, collected, and analyzed by Western blotting using 224A anti-Bid antibody. Data shown are representative of three independent experiments.
(D) Inhibitors of CKs accelerate Fas-inducedBid cleavage. HeLa D98/AH2 cells were treated with 0.5 μg/ml anti-Fas antibody for 5 hr in the absence or presence of 250 μM CKI-7, 50 μM DRB, 20 μM U0126, or 10 μM Bis I. Cleavage of endogenous Bid was assessed by Western blotting using the anti-human Bid antibody.
(E) Inhibitors of PKC and CKs abolish protection of Bid cleavage by PMA. Jurkat cells were incubated for 8 hr with or without 1 μg/ml anti-Fas antibody in the presence or absence of 30 nM PMA. Cells treated with both PMA and anti-Fas were incubated with or without 250 μM CKI-7, 50 μM DRB, or 10 μM Bis I and analyzed as described in (D)
Molecular Cell 2001 8, DOI: ( /S (01) )