Volume 36, Issue 3, Pages 365-378 (November 2009) A Nuclear Poly(ADP-Ribose)-Dependent Signalosome Confers DNA Damage-Induced IκB Kinase Activation  Michael Stilmann, Michael Hinz, Seda Çöl Arslan, Anja Zimmer, Valérie Schreiber, Claus Scheidereit  Molecular Cell  Volume 36, Issue 3, Pages 365-378 (November 2009) DOI: 10.1016/j.molcel.2009.09.032 Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 1 PARP-1 Is Required for DNA Damage-Induced NF-κB Activation (A) Recombinant, C-terminally Strep-tagged IKKγ was used to pull down interaction partners from HeLa cell extracts (duplicate experiment). Interaction with PARP-1 was detected by western blotting. (B) Wild-type (WT) and Parp-1−/− MEF were irradiated and cellular fractions were prepared at indicated time points. Cytoplasmic (CE) or nuclear extracts (NE) were immunoblotted with p65, IκBα, phospho-IκBα, or PARP-1 antibodies. (C) Wild-type and Parp-1−/− MEF were irradiated (IR) and lysates analyzed with an in vitro kinase assay (KA). Precipitated IKKγ was controlled by western blotting (WB). (D) Parp-1−/− MEF were transfected with empty vector (control) or PARP-1 expression constructs. Mutant and wild-type cells were irradiated and assayed for NF-κB activation by EMSA. PARP-1 protein levels were evaluated by western blotting. (E) Macrophages (top panel) and small intestinal epithelial cells (bottom panel) from wild-type and Parp-1−/− mice were exposed to IR (10 Gy) and further incubated, as indicated. Extracts were assayed for NF-κB activity by EMSA. An EMSA with an Oct probe served as a loading control. Free probes are not shown. Molecular Cell 2009 36, 365-378DOI: (10.1016/j.molcel.2009.09.032) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 2 IKKγ Interacts with Automodified PARP-1 and PIASy (A) Wild-type MEF were γ-irradiated and protein extracts prepared at the indicated time points were subjected to IP with an IKKγ antibody. Samples were immunoblotted with antibodies against PAR, PARP-1, or IKKγ (left panel). Wild-type (untreated or preincubated with 3-AB) and Parp-1−/− MEF were analyzed as above. Specificity of the IP was confirmed with a control antibody (right panel). (Bottom panels) Protein expression in the lysates. (B) Wild-type MEF were γ-irradiated and cytoplasmic (CE) and nuclear extracts (NE) were prepared at indicated time points and analyzed by western blotting. (C) Wild-type, Parp-1−/−, and Piasy−/− MEF were assayed by EMSA for NF-κB activation following IR (2 hr) or etoposide (E; 100 μM; 2 hr) treatment. The genotype of Parp-1−/− and Piasy−/− MEF was confirmed by western blotting. An asterisk denotes the PIASy signal. (D) Ikkγ−/− MEF were transfected with empty vector or vectors encoding Flag-tagged IKKγ wild-type or deletion mutants, respectively. Extracts were prepared 10 min after IR, Flag-tagged proteins immunoprecipitated with anti-Flag antibody and samples were analyzed by western blotting with PARP-1 or Flag antibodies (top panels). Alternatively, protein extracts were subjected to PIASy IP followed by immunoblotting for Flag-tagged IKKγ constructs or PIASy, respectively (bottom panels). (Top panel) Scheme of IKKγ with kinase binding domain (KBD), leucine zipper (LZ), and zinc finger (ZF). Asterisks indicate precipitated IKKγ proteins. (E) Wild-type and Piasy−/− MEF were γ-irradiated. At indicated times, extracts were prepared and subjected to IP with an IKKγ antibody. Samples were immunoblotted with PARP-1, IKKγ, or PIASy antibodies. Bottom panels show protein expression in the lysates. Asterisks denote IKKγ and PIASy signals. Molecular Cell 2009 36, 365-378DOI: (10.1016/j.molcel.2009.09.032) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 3 Poly(ADP-ribosyl)ated PARP-1 Promotes Complex Formation of IKKγ with PIASy (A) Wild-type MEF were γ-irradiated, and extracts were prepared at indicated time points and subjected to IP with PIASy antibody. Immunoprecipitates were analyzed by western blotting with PAR, PARP-1, IKKγ, or PIASy antibodies. Bottom panels show PAR, PARP-1, and IKKγ expression in cell lysates. (B) Wild-type (untreated or preincubated with 3-AB) and Parp-1−/− MEF were processed as in (A) and analyzed with PARP-1, IKKγ, or PIASy antibodies. (C) Wild-type and Ikkγ−/− MEF were analyzed as in (A). Note that additional, weak bands close to 55 kDa in (B) and (C) are caused by the IgG heavy chain. Asterisks in (A), (B), and (C) denote the specific PIASy signal. (D) Combinations of recombinant proteins, as indicated, were incubated and subjected to IP with PIASy antibody. IP samples and input were analyzed by western blotting, as indicated. (E) Recombinant proteins were analyzed as in (D), except that IP was performed with PARP-1 antibody. Molecular Cell 2009 36, 365-378DOI: (10.1016/j.molcel.2009.09.032) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 4 PIASy Contains PAR Binding Motifs (A) PAR binding by purified recombinant IKK subunits and PIASy was determined using a slot-blot assay. Histone H2A and BSA were used as positive and negative controls, respectively. Protein loading was determined by Ponceau S staining. (B) Schematic presentation of PIASy. SAP (SAF-A/B, Acinus, and PIAS) motif, RING-like domain (RLD), and PAR binding sites are indicated. (C) Alignment of PAR binding sequences. The sequences of other PAR binding motifs were taken from Pleschke et al. (2000) and Haince et al. (2007). Amino acids mutated to Ala in ATM-1, PIASy-1, and PIASy-2 are indicated by circles. (D) PAR binding of ATM and PIASy peptides (wild-type or mutants, as indicated in C) determined by slot-blot assay. Peptide loading was controlled by Sypro Ruby staining. (E) Piasy−/− MEF were transfected with empty vector or vectors encoding murine HA-tagged PIASy wild-type or point mutant M2 (as indicated in C), respectively. Extracts were prepared from untreated cells or 10 min after IR, as indicated. IP was carried out with anti-HA affinity matrix. Precipitates were analyzed by western blotting with PARP-1, IKKγ, or HA antibodies. The unspecific signal in the IKKγ western blot is caused by IgG. Bottom panels show protein expression in cell lysates. Molecular Cell 2009 36, 365-378DOI: (10.1016/j.molcel.2009.09.032) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 5 PARP-1 Signalosome Requirement for IKKγ SUMOylation and IKK Activation (A) Lysates prepared from γ-irradiated wild-type, Parp-1−/−, and Piasy−/− MEF at the time points indicated were subjected to IP with an IKKγ antibody. Samples were analyzed by western blotting with SUMO-1 or IKKγ (sc-8330) antibodies. (B) Wild-type MEF were pretreated with DMSO or EB47 for 90 min and processed as in (A). (C) Piasy−/− MEF were transfected with empty vector or vectors encoding HA-tagged PIASy-wild-type or PIASy-M2. Extracts were prepared 60 min after IR and processed as in (A). In (B) and (C), lysates were prepared with modified RIPA buffer. SUMOylated species are indicated by an asterisk. (D) Piasy−/− MEF were transfected as in (C). Whole cell extracts were prepared 90 min after IR and assayed for NF-κB activation by EMSA. PIASy expression and IκBα phosphorylation were detected by western blotting. Molecular Cell 2009 36, 365-378DOI: (10.1016/j.molcel.2009.09.032) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 6 ATM Is Part of the PARP-1 Signalosome (A) Wild-type MEF were exposed to IR. Lysates were prepared at indicated time points and used for IP with PIASy or IKKγ antibodies or as input control. Precipitates were analyzed for ATM, RIP1, PARP-1, IKKγ, and PIASy by western blotting. (Asterisk) Note that PIASy is poorly recovered in IKKγ IP, perhaps due to epitope shielding. (B) Wild-type and Parp-1−/− MEF were irradiated. Lysates were prepared and used for IP with IKKγ antibody. IKKγ, coprecipitated ATM, and phospho-ATM (pS1981) were analyzed by western blotting of IP samples and input lysates. (C) Piasy−/− MEF were transfected with empty vector or vectors encoding HA-tagged PIASy-wild-type or PIASy-M2. Extracts were prepared from untreated or irradiated cells, as indicated. IP was carried out with anti-HA affinity matrix. Precipitates were analyzed by western blotting with antibodies against ATM, phospho-ATM (pS1981), or HA. Bottom panels show protein expression in input lysates. Molecular Cell 2009 36, 365-378DOI: (10.1016/j.molcel.2009.09.032) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 7 Equivalent Loss of Survival Signaling by Impairment of Single PARP-1 Signalosome Components (A) Wild-type, Parp-1−/−, Piasy−/−, and Ikkγ−/− MEF were γ-irradiated. Twenty-four hours later, induction of apoptosis was examined with annexin-V staining and flow cytometry. Data are presented as mean ± SEM of triplicates. Statistical significance was determined with the Student's t test. (B) Wild-type and Parp-1−/− MEF were γ-irradiated and total RNA was extracted at indicated time points. mRNA profiles of bcl-XL and β-actin were analyzed by semiquantitative RT-PCR (top panel). Signal intensities were quantified (bottom panel). (C) Wild-type and Parp-1−/− MEF were treated with etoposide (100 μM). Total RNA was extracted at indicated time points and analyzed as in (B). (D) Wild-type, Piasy−/−, and Ikkγ−/− MEF were preincubated with 3-AB (10 mM) for 90 min and irradiated, as indicated, and analyzed as in (A). (E) Schematic summary. DNA damage-induced IKK and NF-κB activation is triggered by PARP-1-mediated PAR formation. PARP-1 is recruited to DNA strand breaks and undergoes PAR automodification. Upon dissociation into the nucleoplasm, PARP-1 rapidly recruits PIASy, IKKγ, and ATM. The signalosome is stabilized by a network of direct protein-protein interactions as well as by PAR binding of PIASy and ATM through PAR binding motifs. PAR degradation by PARG causes subsequent destabilization of the signalosome, resulting in IKKγ SUMOylation, and NF-κB activation. Molecular Cell 2009 36, 365-378DOI: (10.1016/j.molcel.2009.09.032) Copyright © 2009 Elsevier Inc. Terms and Conditions