1. Volume 40, Issue 1, Pages 63-74 (October 2010)
A Cytoplasmic ATM-TRAF6-cIAP1 Module Links Nuclear DNA Damage Signaling to Ubiquitin-Mediated NF-κB Activation 
Michael Hinz, Michael Stilmann, Seda Çöl Arslan, Kum Kum Khanna, Gunnar Dittmar, Claus Scheidereit 
Molecular Cell 
Volume 40, Issue 1, Pages (October 2010)
DOI: /j.molcel
Copyright © 2010 Elsevier Inc. Terms and Conditions

2. Molecular Cell 2010 40, 63-74DOI: (10.1016/j.molcel.2010.09.008)
Copyright © 2010 Elsevier Inc. Terms and Conditions

3. Figure 1
Ionizing Radiation Induces ATM Translocation to Cytosol and Plasma Membrane in a Calcium-Dependent but IKKγ-Independent Manner
(A) HepG2 cells were transfected with siRNAs against ATM or control. Cells were analyzed 2 hr after IR. Whole-cell extracts (WCE) were used to monitor NF-κB activation (EMSA) and knockdown of ATM (WB), respectively.
(B) Cells were transfected with siRNA against IKKγ or control. Cells were irradiated and crude cytoplasmic (CE) or nuclear (NE) extracts were immunoblotted with ATM, P-ATM, and IKKγ antibodies.
(C) After irradiation, cells were fractionated into membranes, cytosol, and nuclear extract (NE) and analyzed by western blotting.
(D) HeLa cells, left untreated or taken 30 min after IR, were fixed, labeled with P-ATM antibody, and counterstained with DAPI. The merge image depicts the DAPI and anti-P-ATM channels as blue and green, respectively.
(E and F) HepG2 cells were preincubated either with DMSO, ATM inhibitor, or BAPTA for 30 min and processed as in (C). Fractions were analyzed by western blotting.
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4. Figure 2
DNA Damage Triggers TRAF6/Ubc13-Mediated Generation of Polyubiquitin to Activate NF-κB
(A) HeLa cells were transfected with control siRNA or siRNAs against TRAF6 (left panel) or Ubc13 (right panel) and assayed 2 hr after IR. WCEs were analyzed for NF-κB activation (EMSA) and immunoblotted for TRAF6 and Ubc13, respectively.
(B) Wild-type (WT) or TRAF6−/− MEFs were irradiated and further incubated for 90 min. WCEs were processed as in (A).
(C) HeLa cells were transfected with siRNA against TRAF6 or control and treated with camptothecine (CPT; 10 μM) for 90 min. WCEs were processed as in (A).
(D) HepG2 cells were treated with IR or IL-1β, lysed at the times indicated (min), and immunoprecipitated with TRAF6 antibody. IP extracts were analyzed for ubiquitin (Ub) or TRAF6 by immunoblotting.
(E) HepG2 cells were transfected with siRNAs against Ubc13 or RIP-1, irradiated for 1 hr, and processed as in (D). Ubc13 and RIP-1 knockdown was confirmed by western blotting. Images separated by a black line are from the same gel.
(F) 293 cells were transfected with an NF-κB reporter plasmid and Ubc13-C87A, TRAF6Δ (aa 289–522) or MALT1-2EA (E653A/E806A), as indicated. Luciferase activity was measured 6 hr after irradiation. Error bars represent the standard error of the mean (SEM) of triplicates.
(G) 1.3E2 cells, stably transfected with WT IKKγ or mutant constructs, were irradiated. NF-κB activation was analyzed as in (A). Expression of IKKγ proteins was confirmed by western blotting.
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5. Figure 3
ATM Recruits TRAF6 to Promote TRAF6-Mediated Polyubiquitination
(A) HepG2 cells were transfected with siRNAs against PARP-1, ATM, or RIP-1. One hour after IR, cells were processed as in Figure 2D. ATM and PARP-1 knockdown was confirmed by western blotting. Images separated by a black line are from the same gel.
(B) HepG2 cells were preincubated either with DMSO or BAPTA for 30 min and processed as in Figure 2D.
(C) Bacterially expressed GST-ATM fusion proteins were purified on glutathione agarose beads. Ten percent of the beads were analyzed by SDS-PAGE and protein staining (upper panel). Remaining beads containing GST-ATM fusion proteins (4 μg) were used to pull down 35S-labeled, in vitro-translated TRAF6. Interactions were detected by SDS-PAGE and autoradiography (lower panel).
(D) Glutathione agarose beads containing GST-ATM fusion proteins were used to pull down TRAF6 from 293 cell extracts. TRAF6 and polyubiquitin were detected by western blotting.
(E) 293 cells were transfected with FLAG-TRAF6 and GFP-ATM9 (2141–2428) or GFP-ATM9ΔT6 (2158–2428). Lysates were immunoprecipitated with control mouse IgG or anti-FLAG antibodies. Immunoblotting was performed with GFP, FLAG, and IKKα antibodies.
(F) 293 cells were transfected with HA-ubiquitin along with GFP-ATM9 or GFP-ATM9ΔT6, lysed, and immunoprecipitated with TRAF6 antibodies. Lysates and IP extracts were immunoblotted with HA, GFP, and TRAF6 antibodies.
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6. Figure 4
Requirement for ATM, TRAF6, and TAB2 in DNA Damage-Induced TAK1 Activation
(A) WT and TAK1−/− MEFs were irradiated and further incubated for 90 min. WCEs were monitored for NF-κB activation (EMSA) and immunoblotted for TAK1 and p50.
(B) HepG2 cells were treated with IR and lysed at indicated times. Lysates were immunoblotted with P-TAK1 and TAK1 antibodies.
(C and D) HepG2 cells were transfected with siRNAs against RIP-1, ATM, TRAF6, or Ubc13. Forty-five minutes after, IR cells were lysed. Immunoblotting was performed to monitor TAK1 phosphorylation and expression of the indicated proteins.
(E) Cells were transfected with siRNAs against TAB1 and TAB2 or control and treated with IR for 2 hr. WCEs were analyzed for NF-κB activation (EMSA) and protein expression.
(F) Cells were transfected with siRNAs against RIP-1 or TAB2 and processed as in (C). Control lanes (UT and siRIP1) are the same as in (C). Images separated by a black line are from the same gel.
(G) Cells were preincubated either with DMSO or BAPTA for 30 min, irradiated, and fractionated at indicated times. Membranes and cytosol were analyzed by western blotting, as indicated. Asterisks indicate Phospho-TAK1 (B, C, D, and F) or TAB2 (E and F).
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7. Figure 5
An ATM-TRAF6-cIAP1 Module Induces IKKγ Monoubiquitination in the Cytoplasm
(A) HepG2 cells were irradiated, lysed, and immunoprecipitated with IKKγ antibody. Lysates and IP extracts were immunoblotted with IKKγ or ubiquitin (Ub) antibodies.
(B) Cells were irradiated and crude cytoplasmic (CE) or nuclear (NE) extracts were processed as in (A). CE and NE were immunoblotted with PARP-1 or tubulin antibodies.
(C) Forty-five minutes after IR, HepG2 cells were fractionated and cytoplasmic extracts were applied to a Superose 6 gel filtration column. Fractions were immunoblotted for IKKα and IKKγ. Marker protein migration is indicated.
(D) 1.3E2 cells, expressing WT IKKγ or mutant constructs, were irradiated. After 45 min, cells were processed as in (A).
(E) HepG2 cells were transfected with siRNAs against ATM, TRAF6, Ubc13, or control. Forty-five minutes after IR, cells were processed as in (A). Protein knockdown was confirmed by western blotting, as indicated.
(F) HepG2 cells were transfected with siRNA against cIAP1 or control and monitored for monoubiquitinated IKKγ, IKKβ, or TAK1 phosphorylation and IKKβ, TAK1, or cIAP1 expression by western blotting.
(G) 293 cells, transfected with FLAG-TRAF6-WT or FLAG-TRAF6-C70A, were left untreated or irradiated. Cytoplasmic extracts were immunoprecipitated with anti-FLAG. Lysate and IP extracts were immunoblotted with cIAP1 and TRAF6 antibodies.
(H) 293 cells were transfected with HA-cIAP1 and treated as in (G). Cytoplasmic extracts were immunoprecipitated with HA antibody. Lysate and IP extracts were immunoblotted with TRAF6, IKKγ, and HA antibodies. Asterisks indicate monoubiquitinated IKKγ (A–F).
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8. Figure 6
DNA Damage-Induced IKK Activation Requires IKKγ Ubiquitination at K285
(A) 293 cells were transfected with control plasmid or FLAG-TRAF6. Lysates were immunoprecipitated with anti-IKKγ and immunoblotted with IKKγ, ubiquitin, and FLAG antibodies. Asterisks indicate monoubiqutinated IKKγ.
(B) 293 cells were transfected with FLAG-IKKγ and MYC-TRAF6 and lysed with a chloroacetamide containing buffer. Lysates were immunoprecipitated with anti-FLAG M2 Affinity Gel (Sigma). FLAG-IKKγ was eluted with 0.1 M glycine HCl (pH 3.5) according to the manufacturer's protocol. Ten percent of IP extracts was used for anti-IKKγ immunoblotting to confirm IKKγ ubiquitination (not shown), while 90% was used for mass spectrometry. The MS/MS spectrum of an ubiquitinated IKKγ peptide is shown. Fragment ions are indicated.
(C) HepG2 cells were left untreated (UT) or irradiated for 45 min. Cytoplasmic extracts were analyzed by mass spectrometry using multiple reaction monitoring. A series of MRM transitions were followed for the eluting ubiquitinated IKKγ peptide. Intensities were quantified by summing up the intensities of three transitions and averaged over two independent measurements. SEM is indicated by error bars.
(D) 293 cells were transfected with FLAG-IKKγ-WT or K285R mutant and FLAG-TRAF6 or HA-cIAP1 constructs, as indicated. Lysed cells were subjected to immunoblotting with anti-FLAG or anti-HA antibodies.
(E) IKKγ−/− cells, transfected with control plasmid, FLAG- IKKγ-WT, or K285R mutant, were assayed 2 hr after IR, as indicated. WCEs were analyzed for NF-κB activation (EMSA) and immunoblotted for P-IKK, IKKβ, IKKγ, and P-IκBα, respectively; asterisk indicates specific NF-κB DNA binding.
(F) IKKγ−/− MEFs, stably transfected with murine IKKγ-WT or K278R mutant constructs, were treated with TNFα or IL-1β for 15 min. NF-κB activation was analyzed by EMSA (upper panel). For detection of IKKγ monoubiquitination, lysates were processed as in Figure 5A.
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9. Figure 7
PARP-1/PIASy-Mediated SUMOylation Primes IKKγ for Subsequent Cytoplasmic Monoubiquitination
(A) HepG2 cells were transfected with siRNAs against PARP-1, PIASy, or control. Forty-five minutes after IR, cells were lysed and IKKγ was immunoprecipitated. IP extracts were immunoblotted for IKKγ. Protein depletion was confirmed by western blotting, as indicated.
(B) WT, PARP-1−/−, and PIASy−/− MEFs were irradiated. Lysates were immunoblotted for IKKγ, PARP-1, and PIASy.
(C) Cells were irradiated and crude cytoplasmic (CE) or nuclear (NE) extracts immunoprecipitated with anti-IKKγ. IP extracts were analyzed with SUMO1 or IKKγ antibodies. CE and NE were immunoblotted with PARP-1, tubulin, or IKKγ antibodies.
(D) HeLa cells were transfected with HA-IKKγ-WT or HA-IKKγ-DK (K277A/K309A). Forty-five minutes after IR, lysates were prepared and immunoprecipitated with anti-HA beads. IP extracts were analyzed for IKKγ ubiquitination with IKKγ antibodies (upper panel). In addition, IP extracts and lysates were immunoblotted with cIAP1, TRAF6, and HA antibodies. Images separated by a black line are from the same gel. Asterisks indicate modified IKKγ (A–D) or PIASy (B).
(E) Diagram illustrating a model for DNA damage-induced NF-κB activation. Genotoxic stress activates PARP-1 and ATM. PARP-1 synthesizes poly(ADP-ribose), resulting in assembly of IKKγ, PIASy, and ATM. Signalosome formation promotes IKKγ SUMOylation and likely IKKγ phosphorylation by ATM. Modified IKKγ translocates to the cytoplasm and integrates into IKK complexes, which are then primed for activation. In parallel, ATM translocates to cytosol and plasma membrane and induces a TRAF6-Ubc13-mediated K63-linked polyubiquitin-dependent cascade, involving TAB2, TAK1, and cIAP1. Both signaling branches cooperate to catalyze IKKγ monoubiquitination, which is required for IKKβ phosphorylation and enzymatic IKK activation.
Molecular Cell  , 63-74DOI: ( /j.molcel )
Copyright © 2010 Elsevier Inc. Terms and Conditions