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1 Figure S1. Silver staining and immunoblotting of the purified TAK1 kinase complex. The TAK1 kinase complex was purified through tandem affinity methods (Protein A and FLAG), and aliquots of the purified complex were analyzed by silver staining and immunoblotting with the indicated antibodies. Asterisk (*) denotes a truncated TAK1 fragment. 1

2 Figure S2. TRAF6 does not undergo auto-polyubiquitination in the presence of Ubc13/Uev1A. Ubiquitination reactions were carried out in the presence of E1, Ubc13/Uev1A, TRAF6 and varying concentrations of ubiquitin as indicated. Aliquots of the reaction products were analyzed by immunoblotting with an antibody against His 6, ubiquitin, or TRAF6. 2

3 Figure S3. TAK1-stimulatory polyub chains are not conjugated to any detectable target protein. a, Mono S fraction 15 as shown in Fig. 2d was incubated with IsoT, CYLD or CYLD(C601S), then immunoblotted with an antibody against ubiquitin or His 6. In the bottom panel, His 6 -tagged TRAF6 (lane 5: 1.2 ng; lane 6: 2.5 ng; lane 7: 5 ng), Ubc13/Uev1A (10 ng; lane 8) and E1 (10 ng; lane 9) were loaded as controls for immunoblotting. b, Mono S fractions 14, 15 and 16 (as shown in Fig. 2d) were incubated with IsoT, CYLD or CYLD(C601S), then detected by silver staining or immunoblotting. The asterisks (* and **) indicate contaminants in the CYLD and IsoT preparations. 3

4 Figure S4. NEMO binds to unanchored polyubiquitin chains following IL-1β stimulation. HEK293/IL-1R cells were stimulated with 10 ng/ml of IL-1β for 2 minutes, and the lysate was subjected to immunoprecipitation using NEMO antibody. Precipitated proteins were treated with or without Isopeptidase T (10 μg/ml) for 1 hour at 30 C, and then analyzed by immunoblotting with an antibody against ubiquitin or NEMO. N.S. non-specific. 4

5 Figure S5. IKK activation by TRAF6 in vitro does not require IRAK1 or ubiquitination of TRAF6. a, TRAF6 and ATP were added to cytosolic extracts from the wild type or IRAK1-deficient HEK293T/IL-1R cells. Aliquots of the reactions were analyzed by immunoblotting. b, HEK293T/IL-1R cells lacking IRAK1 (I1a) and the parental cells (WT) were stimulated with IL-1β, then aliquots of the cell lysates were analyzed by immunoblotting. c, Wild-type or mutant (7KR) TRAF6 was added to HeLa S100 in the presence or absence of ATP. Aliquots of the reactions were heated in 1% SDS, then immunoprecipitated with a TRAF6 antibody. Lane 6 in the bottom panel was loaded with polyub chains synthesized by Ubc13/Uev1A and TRAF6. 5

6 Figure S6. Differential activation of TAK1 and IKK by distinct polyub chains. a. Ubiquitination reactions were carried out in the presence of TRAF6 and Ubc13/Uev1A or UbcH5C, then NEM was added to inactivate E1 and E2 before the reaction mixtures were analyzed by immunoblotting. b, The ubiquitination products from (a) were tested for activation of TAK1 (top) and IKK (bottom). c, PolyUb chains synthesized in the presence of TRAF6 and Ubc13/Uev1A (lane ii in a) were pre-incubated with isopeptidase T or its mutant C335A, then incubated with the TAK1 complex and MKK6(K82A) to measure TAK1 activation. d, Immunoblotting of isopeptidase T and its C335A mutant used in (c). 6

7 Figure S7. Binding of polyub chains to NEMO is required for IKK activation in vitro. a, Ubiquitination reactions containing E1, Ub, TRAF6, and UbcH5C were carried out in the presence or absence of ATP. Aliquots of the reactions were analyzed by immunoblotting with an antibody against ubiquitin. b. Reaction mixtures containing ubiquitin (-ATP) or polyub chains (+ATP) as shown in (a) were incubated with the IKK complex isolated from NEMO-deficient cells. NEMO or its Ub-binding defective mutant Y308S was added to the reactions as indicated, and the activation of IKK was measured by immunoblotting with an antibody against IKKα/β, phospho-ikkα/β, NEMO or IκBα. 7

8 Figure S8. Unconventional polyub chains synthesized by TRAF6 and UbcH5C activate IKK. a, Diagram of ubiquitin mutants used in the assays. b, Polyubiquitin chain synthesis was carried out in the presence of E1, UbcH5C (E2), TRAF6 and wild type (WT) or mutant ubiquitin as indicated. E1 and E2 were inactivated with NEM, then the reaction mixtures were incubated with IKK complex and its substrate IκBα in the presence of ATP. Activation of IKK was analyzed by immunoblotting. The top panel was deliberately overexposed to reveal likely multiple monoubiquitination of TRAF6 in the presence of ubiquitin mutants (lanes 3, 5 & 7). 8

9 Figure S9. Short ubiquitin chains inhibit TAK1 and IKK activation by long polyubiquitin chains. a, Coomassie blue staining of short, defined length, ubiquitin chains used in the assays. b, Linear or K63-linked ubiquitin chains were incubated with the TAK1 complex and its substrate MKK6(K82A) in the presence or absence of polyub chains synthesized by TRAF6 and Ubc13/Uev1A. Activation of TAK1 was determined by immunoblotting with an antibody against phospho-mkk6. c, Linear or K63-linked ubiquitin chains were incubated with the IKK complex in the presence or absence of polyub chains synthesized by TRAF6 and UbcH5C. Activation of IKK was determined by immunoblotting with an antibody against phospho-ikkα/β. 9

10 Figure S10. Linear polyub chains do not activate TAK1 or IKK. a, FLAG-tagged TRAF6, RNF31 and RBCK1 proteins were incubated with HeLa S100 supplemented with ATP. Phosphorylation of endogenous IκBα was analyzed by immunoblotting. b, Ubiquitination reactions were carried out using E1, UbcH5C, RNF31-RBCK1 and the Ub mutants as indicated, and the polyub chains were detected with a ubiquitin antibody. c, PolyUb chains synthesized using different combinations of E2s (UbcH5C and Ubc13/Uev1A) and E3s (TRAF6 and RNF31-RBCK1) as indicated were incubated with IKK and IκBα (middle two panels) or TAK1 and MKK6(K82A) (bottom two panels) in the presence of ATP, and the reaction products were analyzed by immunoblotting with the indicated antibodies. 10

11 Figure S11. CYLD preferentially cleaves unanchored K63 polyubiquitin chains. a, K63 polyubiquitination of RIG-I(N) [Ub n -RIG-I(N)] was performed by incubating MBP- Ub-RIG-I(N) with E1, Ub and TRIM25 in the presence or absence of Ubc13/Uev1A. Aliquots of the reaction mixtures were analyzed by immunoblotting with an antibody specific for RIG-I or K63-polyUb. b. Ub n -RIG-I(N) synthesized as described in (a) was incubated with CYLD, IsoT or viral OTU. The reaction products were analyzed by immunoblotting. The ubiquitin antibody does not detect Ub-RIG-I(N) at low concentrations because mono-ubiquitin does not react strongly with the antibody. c. Coomassie blue staining of viral OTU. 11

12 Figure S12. Inhibition of NF-κB by Isopeptidase T and CYLD. Expression vectors encoding CYLD, IsoT or IsoT mutants were transfected into HEK293T/IL-1R cells, together with an NF-κB-luciferase reporter. To activate NF-κB, cells were co-transfected with expression vectors for TRAF6 (0.3 μg) or NIK (0.1 μg) or stimulated with IL-1β (20 ng/ml). Luciferase reporter assays were carried out 48 hours after transfection. C335A mutation disrupts catalysis by IsoT, whereas R221A mutation disrupts binding of ZnF- UBP to ubiquitin. The error bars represent variation ranges from duplicate experiments. 12

13 Supplementary Notes In vitro reconstitution of TAK1 activation with purified proteins His 6 -E1 and His 6 -TRAF6 were purified from baculovirus-infected insect cells, His 6 -Ubc13, His 6 -Uev1A, ubiquitin and its mutants were purified from E. coli, and the TAK1 complex was purified from HEK293 cells stably expressing TAK1, which contains an N-terminal tandem affinity tag (Protein A-Tev protease - Calmodulin-binding peptide) and a C-terminal FLAG tag to facilitate purification. The purified TAK1 kinase complex contains TAB1 and TAB2 in addition to TAK1, as verified by immunoblotting (Supplementary Fig. S1) and silver staining (Fig.1a). These proteins were incubated together with an ATP buffer at 30 o C for 1 hour, and the activation of TAK1 was analyzed by immunoblotting with an antibody specific for TAK1 or phospho-tak1 phosphorylated at Thr-187. Ubiquitination of IRAK1 and TRAF6 is dispensable for IKK activation Previous studies have suggested that polyubiquitination of IRAK1 and TRAF6 is important for IKK activation in the IL-1R/TLR pathways 1-4. However, we found that TRAF6 was still capable of activating IKK in cell lysates derived from an IRAK1- deficient 293 cell line (Supplementary Fig. S5a). When the IRAK1-deficent cells were stimulated with IL-1β, IκBα degradation was blocked (Supplementary Fig. S5b), suggesting that IRAK1 functions upstream of TRAF6, consistent with a previous report 5. Interestingly, while both IκBα and IRAK1 were degraded in cells stimulated with IL-1β, only IRAK1 but not IκBα was degraded in TRAF6-supplemented cytosolic extracts (S100). However, we found that IRAK1 degradation was dependent on ATP, but not 13

14 TRAF6 or proteasome (Pineda G., and Chen, Z., unpublished). It is not clear why IκBα was phosphorylated, but not degraded in the in vitro system (Fig. S5a). One possibility is that some factor(s) required for ubiquitination or degradation of IκBα was absent in the cytosolic extracts. Like IRAK1, TRAF6 ubiquitination was detectable following IL-1β stimulation, and a recent report showed that mutation of a specific lysine (K124) of TRAF6 prevented its ubiquitination and IKK activation by IL-1β 3. However, we found that simultaneous mutation of 7 lysines in the RING domain of TRAF6, including K124, did not affect the ability of TRAF6 to activate IKK (Fig. S5c). These results, together with a recent report 6, suggest that polyubiquitination of TRAF6 is dispensable for IKK activation. Long polyubiquitin chains synthesized by TRAF6 activate TAK1 and IKK A recent study shows that a ubiquitin ligase complex composed of two RING domain proteins, RNF31 (also known as HOIP) and RBCK1 (also known as HOIL-1L), and UbcH5C catalyze the synthesis of linear Ub chains 7. These linear Ub chains can bind to NEMO 8, and are believed to be important for IKK, but not TAK1 activation 7. We tested whether short, defined-length, linear or K63-linked ubiquitin chains could directly activate or inhibit TAK1 or IKK (Supplementary Figure S9). None of the linear or K63- linked ubiquitin polymers (Ub2-Ub4) were able to directly activate TAK1 or IKK. Instead, these ubiquitin polymers inhibited IKK activation by UbcH5C-synthesized polyub chains, with the longer polymers exhibiting a stronger inhibitory effect (Fig. S9c). The K63-linked Ub3 and Ub4, and to a lesser extent, linear Ub3, also inhibited TAK1 activation (Fig. S9b). These results suggest that only long polyub chains 14

15 synthesized by TRAF6 could activate TAK1 and IKK. Consistent with this notion, TRAF6, but not the RNF31-RBCK1 E3 complex, was able to trigger IKK activation in HeLa cytosolic extracts (S100; Supplementary Fig. S10a). As reported 7, RNF31-RBCK1 synthesized linear polyub chains (Fig. S10b); however, these chains did not activate TAK1 or IKK (Fig. S10c). CYLD cleaves unanchored K63 polyubiquitin chains but not the chains conjugated to RIG-I To test if CYLD could remove K63 polyub chains from a target protein, we carried out in vitro ubiquitination of RIG-I, a viral RNA sensor protein known to be conjugated by K63 polyub chains through the E3 ligase TRIM25 9. In the presence of Ubc13/Uev1A, TRIM25 catalyzes K63 polyubiquitination of a RIG-I fragment containing the N-terminal CARD domains (Supplementary Fig. S11a). Both CYLD and IsoT cleaved unanchored K63 polyub chains, but not the chains conjugated to RIG-I (Fig. S11b). In contrast, the viral OTU from CCHFV-L cleaved both K63 chains on RIG- I and unanchored K63 chains. It is interesting to note that the viral OTU, which has been shown to be a potent inhibitor of NF-κB 10, cleaves unanchored K63-linked, but not linear polyub chains, further suggesting that unanchored linear polyub chains may not be involved in NF-κB activation. The viral OTU may specifically cleave an isopeptide bond, but not a conventional peptide bond in a linear Ub linkage, providing a potential explanation for why it removes the linear polyub chains from NEMO, but does not cleave linear polyub chains (Fig. 4e; lane 6, lower panel) or Ub-RIG-I (N), in which ubiquitin is fused to the N-terminus of RIG-I (Fig. S11b; lane 6, upper panel). 15

16 Effects of Isopeptidase T and CYLD on NF-κB activation To determine if specific removal of unanchored polyub chains in cells prevents NF-κB activation, we tested whether overexpression of IsoT inhibits NF-κB activation. IsoT strongly inhibited NF-κB activation by IL-1β and TRAF6; however, it also inhibited NF-κB activation by NIK, a kinase known to activate IKK through direct phosphorylation 11 (Supplementary Fig. S12). Furthermore, various IsoT mutants, including those lacking the catalytic activity, also inhibited NF-κB activation by all stimuli tested, suggesting that IsoT has complex, perhaps nonspecific, inhibitory effects on gene expression in cells. In contrast to IsoT, CYLD specifically inhibits NF-κB activation by IL-1β and TRAF6, but not NIK (Fig. S12). TNFα and IL-1R/TLR may employ distinct signaling mechanisms Previous studies have shown that TNFα induces rapid polyubiquitination of RIP1 at a specific lysine and that this site-specific ubiquitination is important for the activation of TAK1 and IKK 12,13. Unlike the IL-1R/TLR pathways, which signal through TRAF6, TNF receptor recruits TRAF2, TRAF5, ciap1 and ciap2, all of which are RING-domain ubiquitin ligases. In particular, ciaps have been shown to be involved in RIP1 ubiquitination 14. Among these E3s, only TRAF6 has been shown to activate TAK1 and IKK in cytosolic extracts. Therefore, TNFα and IL-1β pathways likely employ different ubiquitin-dependent mechanisms to activate downstream signaling cascades, and it is possible that only TRAF6-dependent pathways employ unanchored polyub chains to activate protein kinases. In this regard, it is interesting to note that in the Drosophila IMD 16

17 pathway, which is analogous to the mammalian TNF pathway, diap2 rather than a TRAF functions as an E3 to activate a signaling pathway that is quite distinct from the Toll pathway 15. References for Supplementary Information 1. Windheim, M., Stafford, M., Peggie, M. & Cohen, P. Interleukin-1 (IL-1) induces the Lys63-linked polyubiquitination of IL-1 receptor-associated kinase 1 to facilitate NEMO binding and the activation of IkappaBalpha kinase. Mol Cell Biol 28, (2008). 2. Conze, D.B., Wu, C.J., Thomas, J.A., Landstrom, A. & Ashwell, J.D. Lys63- linked polyubiquitination of IRAK-1 is required for interleukin-1 receptor- and toll-like receptor-mediated NF-kappaB activation. Mol Cell Biol 28, (2008). 3. Lamothe, B., et al. Site-specific Lys-63-linked Tumor Necrosis Factor Receptorassociated Factor 6 Auto-ubiquitination Is a Critical Determinant of I{kappa}B Kinase Activation. J Biol Chem 282, (2007). 4. Wang, C., et al. TAK1 is a ubiquitin-dependent kinase of MKK and IKK. Nature 412, (2001). 5. Li, X., et al. Mutant cells that do not respond to interleukin-1 (IL-1) reveal a novel role for IL-1 receptor-associated kinase. Mol Cell Biol 19, (1999). 6. Walsh, M.C., Kim, G.K., Maurizio, P.L., Molnar, E.E. & Choi, Y. TRAF6 autoubiquitination-independent activation of the NFkappaB and MAPK pathways in response to IL-1 and RANKL. PLoS ONE 3, e4064 (2008). 7. Tokunaga, F., et al. Involvement of linear polyubiquitylation of NEMO in NFkappaB activation. Nat Cell Biol 11, (2009). 8. Wu, C.J., Conze, D.B., Li, T., Srinivasula, S.M. & Ashwell, J.D. Sensing of Lys 63-linked polyubiquitination by NEMO is a key event in NF-kappaB activation [corrected]. Nat Cell Biol 8, (2006). 9. Gack, M.U., et al. TRIM25 RING-finger E3 ubiquitin ligase is essential for RIG- I-mediated antiviral activity. Nature 446, (2007). 10. Frias-Staheli, N., et al. Ovarian tumor domain-containing viral proteases evade ubiquitin- and ISG15-dependent innate immune responses. Cell Host Microbe 2, (2007). 11. Ling, L., Cao, Z. & Goeddel, D.V. NF-kappaB-inducing kinase activates IKKalpha by phosphorylation of Ser-176. Proc Natl Acad Sci U S A 95, (1998). 17

18 12. Ea, C.K., Deng, L., Xia, Z.P., Pineda, G. & Chen, Z.J. Activation of IKK by TNFalpha requires site-specific ubiquitination of RIP1 and polyubiquitin binding by NEMO. Mol Cell 22, (2006). 13. Li, H., Kobayashi, M., Blonska, M., You, Y. & Lin, X. Ubiquitination of RIP is required for tumor necrosis factor alpha-induced NF-kappaB activation. J Biol Chem 281, (2006). 14. Bertrand, M.J., et al. ciap1 and ciap2 facilitate cancer cell survival by functioning as E3 ligases that promote RIP1 ubiquitination. Mol Cell 30, (2008). 15. Gesellchen, V., Kuttenkeuler, D., Steckel, M., Pelte, N. & Boutros, M. An RNA interference screen identifies Inhibitor of Apoptosis Protein 2 as a regulator of innate immune signalling in Drosophila. EMBO Rep (2005). 18