Phosphorylation by Protein Kinase CK2 Pei-Feng Li, Jincheng Li, Eva-Christina Müller, Albrecht Otto, Rainer Dietz, Rüdiger von Harsdorf  Molecular Cell  Volume 10, Issue 2, Pages 247-258 (August 2002) DOI: 10.1016/S1097-2765(02)00600-7

Figure 1 CK2 Phosphorylates ARC In Vitro and In Vivo (A) In vitro phosphorylation of rARC by CK2. Purified rARC was at 0.5 μg. Heparin was at 10 μg/ml. (B) ARC was translated using the reticulocytes lysate system. PCR3.1 is the control empty plasmid. Heparin was at 10 μg/ml. (C) CK2 coimmunoprecipitation with ARC. HEK293 cells were transfected with ARC plasmids and harvested 48 hr after transfection. Cell lysates were immunoprecipitated with the anti-CK2-α subunit antibody, and the immunoprecipitates were analyzed by immunoblotting using anti-ARC antibody. (D) ARC is in vivo labeled by [32P]orthophosphate. HEK293 cells were transfected with ARC plasmids. Twenty-four hours after transfection cells were incubated with 1 mCi/ml [32P]orthophosphate for 6 hr. ARC was immunoprecipitated with the anti-ARC antibody. The precipitated ARC was separated by SDS-PAGE, transferred to a nitrocellulose membrane, and subjected to autoradiography. Loaded ARC is shown by immunoblotting of the same membrane using anti-ARC antibody. Molecular Cell 2002 10, 247-258DOI: (10.1016/S1097-2765(02)00600-7)

Figure 2 ARC Is Phosphorylated at T149 by CK2 (A) ARC phosphorylation detected by mass spectrometry. Upper panel: Nanoelectrospray spectrum of ARC with the doubly charged nonphosphorylated (m/z = 722.36) and phosphorylated (m/z = 762.32) peptides. Lower panel: MS/MS spectrum of the peptide 149pTPEEPELEAEATK (pT-phosphothreonine residue) with m/z = 762.32 Da. The base peak with m/z =713.37 Da is the result of the loss of the neutral H3PO4 (98 Da). Both the N-terminal b-ions and the C-terminal y-ions in the spectrum containing the phosphothreonine are produced by consecutive fragmentation reactions breaking the amino bond and losing the H3PO4, or vice versa. The mass of the y-ions corresponds to the nonphosphorylated C-terminal part, whereas the b-ions confirm the phosphorylation of T149. (B) WtARC and its mutants were translated using the reticulocytes lysate system. The products were analyzed by immunoblotting using the antibody against phospho-T149. Heparin was at 10 μg/ml. (C) Constructs of wtARC and its mutants were transfected into HEK293 cells. Samples were analyzed by immunoblotting using the antibody against phospho-T149. DRB was at 75 μM. Molecular Cell 2002 10, 247-258DOI: (10.1016/S1097-2765(02)00600-7)

Figure 3 Endogenous ARC Is Phosphorylated by CK2 (A) Detection of the phosphorylation status of endogenous ARC. Rat hearts and skeletal muscle were from Wistar rats. Neonatal rat cardiomyocyte cultures were prepared from 1-day-old Wistar rats. (B) CK2 coimmunoprecipitation with endogenous ARC. The lysates of neonatal rat cardiomyocytes or HEK293 cells were immunoprecipitated with the anti-CK2-α subunit antibody, and the immunoprecipitates were analyzed by immunoblotting using anti-ARC antibody. HEK293 cells, in which endogenous ARC expression is undetectable, served as a negative control. (C) DRB is able to inhibit endogenous ARC phosphorylation. Cardiomyocytes were treated with DRB and harvested for immunoblotting 48 hr after treatment. (D) Effect of CK2 antisense oligonucleotides on endogenous ARC phosphorylation. The mixtures of CK2-α or CK2-β oligonucleotides with lipofectin were separately prepared according to the kit's instructions, but they were simultaneously added to cardiomyocytes as described in the Experimental Procedures. Cells were harvested for immunoblotting 48 hr after transfection. Immunoblotting using anti-actin antibody confirmed equivalent amounts of protein loading. (E) Cardiomyocyte cytosolic extracts could phosphorylate rARC. The endogenous ARC in the cytosolic extracts was immunodepleted by anti-ARC antibody from cardiomyocyte cytosolic extracts. The extracts were immunodepleted with anti-CK2-α antibody to yield cell extracts without CK2. The cell extracts (10 μg) or the immunoprecipitates were incubated with 0.5 μg rARC in kinase buffer for 30 min at 30°C. Heparin was at 10 μg/ml. The immunoprecipitates performed with anti-actin antibody were used as a control. Samples were analyzed by immunoblotting using the antibody against phospho-T149 or ARC. Molecular Cell 2002 10, 247-258DOI: (10.1016/S1097-2765(02)00600-7)

Figure 4 ARC Phosphorylation at T149 Is Required for Its Antiapoptotic Effect (A) ARC is unable to prevent caspase-8-induced apoptosis in the presence of DRB. HEK293 cells were cotransfected with the constructs of caspase-8 and ARC. Cell death was assessed by trypan blue exclusion 48 hr after transfection. (B) CK2 activity was significantly decreased upon DRB treatment. CK2 activity was detected 48 hr after transfection. (C–E) ARC149A loses the ability to block caspase-8-, Fas/CD95-, or TNFR1-induced apoptosis. HEK293 cells were cotransfected with the plasmid constructs of caspase-8 (C), Fas/CD95 (D) or TNFR1 (E), and wtARC or its mutants. Cell death was assessed by trypan blue exclusion. (F and G) Inhibition of endogenous ARC phosphorylation by DRB or by CK2 antisense oligonucleotides sensitizes cardiomyocytes to undergoing apoptosis. Cardiomyocytes were pretreated with DRB (F) or CK2-α and CK2-β oligonucleotides (G) for 48 hr. To inhibit both CK2-α and CK2-β, the corresponding CK2-α and CK2-β oligonucleotides were simultaneously added to cardiomyocytes, but their mixtures with lipofectin were separately prepared as described in the Experimental Procedures. Cells were then treated with FasL (100 ng/ml) or TNF-α (15 ng/ml). Cell death was assessed by trypan blue exclusion 24 hr after treatment. Molecular Cell 2002 10, 247-258DOI: (10.1016/S1097-2765(02)00600-7)

Figure 5 T149 Phosphorylation Controls Binding of ARC to Caspase-8 In Vivo but Not In Vitro (A) Detection of in vitro association between caspase-8 and wtARC or its mutants. Caspase-8 and wtARC or its mutants were cotranslated using the reticulocytes lysate system. Immunoprecipitation using anti-caspase-8 antibody was followed by immunoblotting using anti-ARC antibody. (B) Detection of in vivo association between caspase-8 and wtARC or its mutants. HEK293 cells were cotransfected with constructs of caspase-8 and wtARC or its mutants, and harvested 48 hr after transfection. Immunoprecipitation using anti-caspase-8 antibody was followed by immunoblotting using anti-ARC antibody. DRB was at 75 μM. Molecular Cell 2002 10, 247-258DOI: (10.1016/S1097-2765(02)00600-7)

Figure 6 T149 Phosphorylation Targets ARC to Mitochondria (A) Subcellular localization of wtARC and its mutants detected by immunoblotting. HEK293 cells were transfected with constructs of wtARC or its mutants and harvested 48 hr after transfection. Cytochrome oxidase subunit V (COX) served as a mitochondrial marker. (B) Distributions of wtARC and ARC149A detected by immunofluorescence. HEK293 cells were transfected with constructs of ARC or its mutants, labeled with MitoTracker (red), stained with anti-ARC antibody, and monitored by FITC-labeled secondary antibody (green). The overlay of red and green yields orange and/or yellow. Original magnification, 600×. Molecular Cell 2002 10, 247-258DOI: (10.1016/S1097-2765(02)00600-7)

Figure 7 Analysis of ARC and Caspase-8 Binding in HM and in Cytosol (A) Detection of caspase-8 and its cleaved product. HEK293 cells were cotransfected with constructs of caspase-8 and wtARC or its mutants. Subcellular fractions were prepared 48 hr after transfection. The antibody against the catalytic domain of caspase-8 was used for immunoblotting. (B and C) Analysis of caspase-8 activity (B) and cytochrome c release (C). HEK293 cells were cotransfected with constructs of caspase-8 and wtARC or its mutant and harvested 48 hr after transfection. (D) Analysis of the association between endogenous ARC and caspase-8. Cardiomyocytes were pretreated with DRB (75 μM) or CK2-α and CK2-β antisense oligonucleotides for 48 hr and then harvested for preparation of subcellular fractions. Immunoprecipitation using anti-caspase-8 antibody was followed by immunoblotting using anti-ARC antibody. IgG light chain (IgG L) was used to illustrate protein loading. Molecular Cell 2002 10, 247-258DOI: (10.1016/S1097-2765(02)00600-7)