Molecular Cell, Volume 40

Supplemental Information

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, and Claus Scheidereit Inventory of Supplemental Information Hinz et al.

Figure S1 and S2 are related to Figure 1 Figure S1 shows, that ATM translocation is also triggered by etoposide and is independent of the components of the PARP-1 signalosome. Figure S2 demonstrates selective calcium dependency of DNA damage induced NF-κB activation and absence of early DNA damage induced alterations of calcium concentrations. Figure S3 is related to Figures 2 and 5 Figure S3 summarizes the results of an siRNA screen for pathway components required for DNA damage induced NF-κB activation. Figure S4 is related to Figure 4 Figure S4 describes the requirement of TAK1 for DNA damage induced NF-κB activation. Moreover it shows that TAK1 activation does not depend on PARP-1 or PIASy. Figure S5 is related to Figure 5 Figure S5 shows a longer exposure of figure 5B and calcium dependency of IKKgamma monoubiquitination Figure S6 is related to Figure 5 Figure S6 shows that the association of TRAF6 with cIAP1 depends on the RING domain in TRAF6. Figure S7 is related to Figure 6 Figure S7 shows that IKKγ monoubiquitination is induced by TNFα.

Figure S1. DNA damage induced ATM translocation is independent of the PARP-1 signalosome, related to Figure 1. (A) HepG2 cells were treated with etoposide (10 µM) for 30 min and fractionated. Crude cytoplasmic (CE) and nuclear extracts (NE) were analyzed by western blotting with ATM, P-ATM, PARP-1 or tubulin antibodies, respectively. (B) HepG2 cells were transfected with control siRNA or siRNA against PARP-1 or PIASy. Cells were treated with IR for 45 min, as indicated, and fractionated. Membranes and nuclear extracts (NE) were analyzed by western blotting with ATM, P-ATM, PARP-1 or PIASy antibodies, respectively. The membrane fraction was additionally analyzed with an IKKγ antibody.

ATM

P-ATM

PARP-1

PIASy

IKKγ

- -- -- - -- + ++ ++ + ++ IREto

siContr

ol

siContr

ol

siPARP-1

siPARP-1

siPIASy

siPIASy

membranes NE

270-

270-

170-

170-

130-130-

72-

55-

55-

ATM

P-ATM

PARP-1

Tubulin

CE NE

BA

Figure S2. DNA damage- but not TNFα-induced NF-κB activation is calcium dependent, related to Figure 1. (A) HeLa cells were pre-incubated with solvent alone (DMSO) or with BAPTA for 60 min and treated with IR for the indicated time. WCE were monitored for NF-κB activation by EMSA. Free DNA probe is not shown (B) Cells were treated as in (A). Crude cytoplasmic (CE) or nuclear (NE) fractions were analyzed by western blotting with antibodies against IκBα, p65, PARP-1 or p105. (C) Cells were pre-incubated with BAPTA for 60 min and

A B

C D

DMSO DMSO

DMSODMSO

DMSO

DMSO

BAPTA BAPTA

BAPTA

BAPTA

BAPTA

BAPTA

0 0 00 0 01 1 11 1 12 2 22 2 2h post-IR h post-IR

NF- Bκ

NF- Bκ

- - -- - -+ + ++ + +TNF (15 min)α TNF (15 min)α

CE

CE

NE

NE

I Bκ α

I Bκ α

p65

p65

PARP-1

p105

p105

43-

43-

34-

34-

130-

130-

130-

72-

72-

IKKγ55-

*

*

9

11

10

12

13

min upon treatment 0 20 40 60

DMSOATP

9

11

10

12

Cal

cium

con

cent

ratio

nar

bita

ry u

nits

(x10

00)

Cal

cium

con

cent

ratio

nar

bita

ry u

nits

(x10

00)

min upon treatment 0 20 40 60

DMSOEtoE

treated with TNFα for 15 min. WCE were monitored for NF-κB activation by EMSA as in (A). (D) Cells were treated as in (C). Crude cytoplasmic (CE) or nuclear (NE) fractions were analyzed by western blotting with antibodies against IκBα, p65, p105 and IKKγ. (E) Changes in intracellular Ca2+ concentration were assessed in WT MEFs upon treatment with 100 µM etoposide (left panel) or with 1 mM ATP as a positive control (right panel). As a negative control the solvent DMSO was applied in each experiment. The fluorescence measurements were performed employing Fluo-4 NW Calcium Assay Kit (Invitrogen) and Mithras LB 940 plate reader (Berthold Technologies).

Figure S3. RNAi based NF-κB pathway analysis, related to Figures 2 and 5. (A) Cells were transfected with siRNAs against NF-κB signaling proteins as indicated. WCE were monitored for NF-κB activation by EMSA. “+” transfection of two different siRNAs significantly reduced DNA damage induced NF-κB activation, “-“ no reduction, “n.d.” not determined. (B) HepG2 cells were transfected with siRNAs against RIP-1. Cells were treated with IR for 2 h. WCE were monitored for NF-κB activation (EMSA) and immunoblotted for RIP-1 and PARP-1, respectively. (C) HepG2 cells were transfected with control siRNA or siRNA against cIAP1 and treated with IR for 2 h. WCE were monitored for NF-κB activation (EMSA) and cIAP1 expression (WB).

RNAi based NF- B pathway analysis*κ

HeLa 293HepG2

* readout: EMSA

cIAP1 + n.d.+

Ubc13 ++ +

TAK1 ++ +

TAB2 ++ +

TRAF6 + + +

MALT1 n.d. - -

BCL10 n.d. - -

TRAF2 - - -

RIP1 - - -

TAB1 - - -

- + ++UT UT

IRsiR

IP1_

1

siRIP

1-2

NF- Bκ

72-

EMSA

WB

130-

RIP1

PARP-1

A

B

EMSA

WB

72-

55-

55-XIAP

cIAP-1

NF- Bκ

IR

sicIAP1-1

siContr

ol

- + +

C

Figure S4. TAK1 requirement for DNA damage induced NF-κB activation, related to Figure 4. (A) HepG2 cells were transfected with control siRNAs or siRNAs against TAK1. Cells were treated with IR (10 Gy) for 1 h. Cells were divided and WCE were monitored for NF-κB activation (EMSA), while cytoplasmic extracts were immunoblotted for P-IKK, IKKβ, P-TAK1 and TAK1. (B) HeLa cells were transfected with control siRNAs or siRNAs against TAK1. Cells were treated with IR (10 Gy) for 1 h. Lysates were immunoprecipitated with IKKγ and analyzed for IKK kinase activity. Lysates were immunoblotted for TAK1 and IKKγ. (C) and (D) HepG2 cells were transfected with control siRNA or siRNAs against PARP-1 (C) or PIASy (D) and processed as in (A). Immunoblotting was performed with anti-P-TAK1, anti-TAK1, anti PARP-1 (C) or anti-PIASy (D). (E) HepG2 cells were treated with IR and fractionated. Membranes and cytosol were analyzed by western blotting as indicated.

A

B

C

D

P-TAK1

P-TAK1TAK1

TAK1

TAK1

72-

72-

72-

72-

72-

72-

72-

72-

UT UT

UT

siTAK1_

1

siTAK1_

1

siPARP-1-

1

siTAK1_

2

siPARP-1-

2

siPIASy

siContr

ol

siRIP

1

siContr

ol

siContr

ol

siContr

ol

- -

-

--

+ + +

+

+

+ +

-

-

+ +

+

++

P-IKK

PARP-1

PIASy

IKKβ

P-TAK1

TAK1

EMSA

EMSA

EMSA

WB

WB

WB

WB

IR IR

IR

IR

95-

95-

130-

*

*

*

GST-I Bas1-53)

κ α(

IKKγ

KA

55-

membranes cytosol

0 0 min post-IR

72- TAK1

10 1045 45

E

55-

43-Tubulin

55-

72-TNFR

72- P-TAK1

TAB272-

55- TAB1

NF- Bκ

n.s.

NF- Bκ

n.s.

NF- Bκ

n.s.

Figure S5. DNA damage induced IKKγ monoubiquitination takes place in the cytoplasm, related to Figure 5. (A) Long exposure of the experiment in figure 5B. (B) HepG2 cells were pre-incubated with solvent alone (DMSO) or with BAPTA for 60 min, treated with IR for 45 min, lysed and immunoprecipitated with anti-IKKγ. Lysates and IP extracts were immunoblotted with IKKγ antibodies.

- -+ + IR

*55-

DMSO BAPTA

55-

minpost-IR00 2020 4040 6060

IP: IKKWB: IKK

γγ

IP: IKKWB: IKK

γγ

A BCE NE

*

Figure S6. DNA damage induces an interaction between TRAF6 and cIAP1, related to Figure 5. 293 cells, transfected with FLAG-TRAF6-WT or FLAG-TRAF6Δ, a deletion mutant lacking the RING domain, were either left untreated or exposed to IR for 40 min. Cytoplasmic extracts were immunoprecipitated with anti-FLAG. Lysate and IP extracts were immunoblotted with cIAP1 and TRAF6 antibodies.

0 00 040 40 40 40 time upon IR40 40

IP FLAG

IP FLAG

TRAF6

72 -

26 -

43 -

72 -

cont

rol

cont

rol

55 -

34 -

55 -

cIAP1

T6-WT T6-WTT6-Δ T6-Δ

Input

*

Figure S7. TNFα signaling induces IKKγ ubiquitination, related to Figure 6. HepG2 cells were treated with TNFα for the indicated time. Lysates were immunoprecipitated with IKKγ antibody and immunoblotted with antibodies against IKKγ and Ubiquitin (Ub).

0 3 5 10 15 0 3 5 10 15

Input IP: IKKγmin

TNFα

55-

55-

IKKγ

Ub

*

*

Supplemental Experimental Procedures

Cell culture

MEFs were maintained in Hepes-buffered Dulbecco's modified Eagle's medium-Glutamax-I

(Invitrogen) containing 4.5 g/l glucose, 10% fetal calf serum (PAA Gold) and supplemented with

100 U/ml penicillin/streptomycin. For plasmid transfection Transfectin (Biorad) was used.

HepG2 and 1.3E2 cells were grown in RPMI 1640, HeLa and 293 cells in DMEM, all

supplemented with 10 % fetal calf serum, 2 mM L-glutamine and 100 U/ml

penicillin/streptomycin. For 1.3E2 cells, 50 µM β-ME was added. For irradiation, cells were

exposed to 10 Gy (HeLa, 293, 1.3E2), 30 Gy (HepG2), 80 Gy (MEF) using an OB29 Irradiator

(STS Braunschweig).

Reagents

Human recombinant TNFα and BAPTA/AM were purchased from Alexis Corporation, ATM

inhibitor Ku55933 and camptothecine from Calbiochem and etoposide from Biomol. Western

blots and immunoprecipitations were performed with antibodies directed against ATM (Bethyl

Laboratories), phospho-ATM (4526, Cell Signal Technology or 600-401-398, Rockland), CDK4

(sc-601, Santa-Cruz Biotechnology), cIAP1 (AF8181, R&D Systems) or (Proteintech), Flag

(M2), GFP and HA (all Sigma), HA affinity matrix (Roche) IκBα (sc-371, Santa-Cruz

Biotechnology), Phospho-IκBα (9241, New England Biolabs), IKKα (556532, BD

Pharmingen), IKKβ (2684, Cell Signal Technology), IKKγ (BD Transduction Laboratories) or

(sc-8330, sc-8256, Santa-Cruz Biotechnology), P-IKK (2697, Cell Signal Technology), p105/p50

(sc-7178, Santa-Cruz Biotechnology), p65 (sc-109, Santa-Cruz Biotechnology), PARP-1 (sc-

8007, Santa-Cruz Biotechnology), PIASy (sc-30875, Santa-Cruz Biotechnology), RIP1 (551041,

BD Pharmingen), SUMO-1 (Zymed), TAB1 (sc-6053, Santa-Cruz Biotechnology), TAB2 (sc-

11850 or sc-11851, Santa-Cruz Biotechnology), TAK1 (sc-7162 or sc-7967, Santa-Cruz

Biotechnology), Phospo-TAK1, (4508, Cell Signal Technology), TRAF6 (sc-7221 and sc-33897,

Santa-Cruz Biotechnology), TNF-R (sc-7895, Santa-Cruz Biotechnology), Tubulin (2-28-33,

Sigma), Ubc13 (37-1100 Invitrogen) and Ubiquitin (clone FK2, Assay Designs or sc-8017 Santa-

Cruz Biotechnology). Mouse monoclonal (CII-10) and rabbit polyclonal (288) antibodies against

PARP-1 were described previously (Mortusewicz et al., 2007).

Plasmids

Plasmids pRK5TRAF6Δ (289–522), pcDNA3-Flag-IKKγ-WT and pEF-MALT1 were described

previously (Oeckinghaus et al., 2007; Tegethoff et al., 2003). Human IKKγ-WT was subcloned

into pcDNA3-HA vector. Murine IKKγ-WT was purchased from RZPD and cloned into pTracer.

QuickChange® Site-Directed Mutagenesis Kit (Stratagene) was used to generate mutant IKKγ

constructs, TRAF6-C70A and pEF-MALT1-2EA (E642/795A). TRAF6-C70A mutant protein is

defective in E3 ligase activity (Deng et al., 2000). MALT1-2EA mutant protein is defective in

TRAF6 binding and activation (Sun et al., 2004)). pEFUBC13-C87A (catalytic inactive mutant)

has been described (Deng et al., 2000). For construction of GFP-ATM 9 (2141-2428) and GFP-

ATM 9ΔT6 (2158-2428) DNA fragments were generated by PCR from the corresponding

bacterial expression construct, and cloned into pEGFP-C3 (Clontech Labatories Inc.).

Calcium measurements

Detection of changes in intracellular calcium concentration was performed using Fluo-4 NW

Calcium Assay Kit (Invitrogen) in WT MEFs according to manufacturer's instructions. Briefly,

3x104 MEF cells were plated per a well of a black 96-well plate (Nalge Nunc International) 24

hrs before the experiment. Cells were washed once with HBSS (20 mM Hepes) and loaded with

100 µl Fluo-4 AM solution for 20 min at 37°C. Subsequently the staining solution was removed

and after washing with HBSS, cells were tempered to 30°C for 10 min. For application of ATP

(1mM) or etoposide (100 µM) solutions and for the fluorescence measurement Mithras LB 940

plate reader (Berthold Technologies) was used. Fluorescence was measured for 1 sec with 485

nm excitation and 535 emission filter every 15 sec for a period of one hr after application of the

test solution.

siRNA sequences

The following target sequences (5’ to 3’) were used:

siATM-1 CCATGGAAGTGATGAGAAA

siATM-2 GGTAGAAGATTGTGTCAAA

siATM-3 GCAGAAACGUGCUUAGAAA

siBCL10-1 GTGCUGAAACUUAGAAAUA

siBCL10-2 GGACUAAAAUGUAGCAGUU

sicIAP1-1 GGCUUGAGGUGUUGGGAAU

sicIAP1-2 GGAUCCACCUCUAAGAAUA

siIKKγ: GGAAGAGCCAACUGUGUGA

siMALT1-1 GGAAGUGAAUGUUGGGAAA

siMALT1-2 GGAUGAAGUUGCAGAAGAU

siRIP1_1: CCACUAGUCUGACGGAUA

siRIP1_2: GCAAAGACCUUACGAGAAU

siPARP-1-1 AGCCUCCGCUCCUGAACAA

siPARP-1-2 GAUAGAGCGUGAAGGCGAA

siPIASy CCGAAUUAGUCCCACAGAA

siTAK1-1 GCCACAAAUGAUACUAUUA

siTAK1-2 UGGCUUAUCUUACACUGGA

siTAB1-1 CCACAGAGAACGAGGAUGA

siTAB1-2 GGGAUUACAAGGUUAAAUA

siTAB2-1 CAGCAUUAGUGAUGGACAA

siTAB2-2 GGUGCAUGUUACAGAAUAA

siTRAF2-1 AUACGAGAGCUGCCACGAA

siTRAF2-2 GGCCAGUCAACGACAUGAA

siTRAF6-1 CCAGCUCCUGUAGCGCUGUAACAAA

siTRAF6-2 CCACGAAGAGAUAAUGGAU

siUbc13-1 CUAGGCUAUAUGCCAUGAA

siUbc13-2 CAGUUCUGCUAUCGAUCCA

Supplemental References Deng, L., Wang, C., Spencer, E., Yang, L., Braun, A., You, J., Slaughter, C., Pickart, C., and Chen, Z. J. (2000). Activation of the IkappaB kinase complex by TRAF6 requires a dimeric ubiquitin-conjugating enzyme complex and a unique polyubiquitin chain. Cell 103, 351-361. Mortusewicz, O., Ame, J. C., Schreiber, V., and Leonhardt, H. (2007). Feedback-regulated poly(ADP-ribosyl)ation by PARP-1 is required for rapid response to DNA damage in living cells. Nucleic Acids Res 35, 7665-7675. Oeckinghaus, A., Wegener, E., Welteke, V., Ferch, U., Arslan, S. C., Ruland, J., Scheidereit, C., and Krappmann, D. (2007). Malt1 ubiquitination triggers NF-kappaB signaling upon T-cell activation. Embo J 26, 4634-4645. Sun, L., Deng, L., Ea, C. K., Xia, Z. P., and Chen, Z. J. (2004). The TRAF6 ubiquitin ligase and TAK1 kinase mediate IKK activation by BCL10 and MALT1 in T lymphocytes. Mol Cell 14, 289-301. Tegethoff, S., Behlke, J., and Scheidereit, C. (2003). Tetrameric oligomerization of IkappaB kinase gamma (IKKgamma) is obligatory for IKK complex activity and NF-kappaB activation. Mol Cell Biol 23, 2029-2041.