A Ubiquitin Replacement Strategy in Human Cells Reveals Distinct Mechanisms of IKK Activation by TNFα and IL-1β  Ming Xu, Brian Skaug, Wenwen Zeng, Zhijian J. Chen  Molecular Cell  Volume 36, Issue 2, Pages 302-314 (October 2009) DOI: 10.1016/j.molcel.2009.10.002 Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 1 Tetracycline-Induced Replacement of Endogenous Ubiquitin with Ub(K63R) (A) A diagram of human ubiquitin genes and their chromosomal locations. Also shown is the strategy to express wild-type (WT) or K63R ubiquitin genes fused to ribosomal subunits (L40 and S27a) in the U2OS-shUb cells. Ubr indicates that silent mutations have been introduced to ubiquitin genes to render them resistant to RNAi. Both ubiquitin expression cassettes are under the control of a tetracycline promoter. An HA epitope is fused to the second ubiquitin gene, whose translation is controlled by an internal ribosomal entry site (IRES). (B) U2OS-shUb-Ub(WT) (lanes 1–4) or U2OS-shUb-Ub(K63R) (lanes 5–8) cells were treated with tetracycline (1 μg/ml) for 4 days, and then total RNA was isolated for RT-PCR analyses of endogenous Ub genes or exogenous Ub transgene (Ubr) expression. GAPDH RNA expression was used as a control. RT, reverse transcriptase. (C) Quantification of wild-type or K63R ubiquitin by mass spectrometry. U2OS-shUb-Ub(K63R) cells were treated with tetracycline (1 μg/ml) for 0, 3, 5, and 7 days, and then ubiquitin was isolated for trypsin digestion and mass spectrometry. The ratio of peptides containing K63, K63R, or K48 to a reference ubiquitin peptide (E64-R72) as a function of tetracycline induction time is presented. (D) U2OS-shUb-Ub(WT) and U2OS-shUb-Ub(K63R) cells were treated with tetracycline (1 μg/ml) for 4 days before cell lysates were prepared for immunoprecipitation with an HA antibody. The precipitated proteins were analyzed by immunoblotting with an antibody specific for the K63 linkage of ubiquitin chains or with the HA antibody to detect total ubiquitin. Molecular Cell 2009 36, 302-314DOI: (10.1016/j.molcel.2009.10.002) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 2 K63-Linked Polyubiquitination Is Required for IKK Activation by IL-1β, but Not TNFα (A–C) U2OS-shUb (A), U2OS-shUb-Ub(WT), and U2OS-shUb-Ub(K63R) cells (B and C) were treated with or without tetracycline (Tet) (1 μg/ml) for 3 (A) or 4 days (B and C) and then stimulated with IL-1β or GST-TNFα as indicated. Cell lysates were prepared for immunoblotting with an antibody against IκBα, ubiquitin, tubulin (Tub), or HA. For IKK activity assay, the IKK complex was immunoprecipitated with a NEMO antibody and incubated with GST-IκBα (NT) and γ-32P-ATP. The amount of IKKβ in the NEMO immunoprecipitates was analyzed by immunoblotting. Molecular Cell 2009 36, 302-314DOI: (10.1016/j.molcel.2009.10.002) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 3 Ubc13 Is Required for IKK Activation by IL-1β, but Not TNFα (A) Tetracycline (Tet) was used to induce the expression of shRNA against Ubc13 in U2OS cells. After stimulation with IL-1β (left) or GST-TNFα (right) for the indicated time, phosphorylation and degradation of IκBα were analyzed by immunoblotting with an IκBα antibody. IKK assay was also carried out after immunoprecipitation with a NEMO antibody. The efficiency of Ubc13 RNAi was verified by immunoblotting. (B and C) Tetracycline (Tet) was used to induce the expression of Ubc13 shRNA and an RNAi-resistant Flag-Ubc13 or Flag-Ubc13 (C87A) in U2OS cells simultaneously. After stimulation with IL-1β (B) or GST-TNFα (C) for the indicated time, cell lysates were analyzed by immunoblotting with an antibody against IκBα, Ubc13, or tubulin (Tub). Molecular Cell 2009 36, 302-314DOI: (10.1016/j.molcel.2009.10.002) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 4 UbcH5 Is Required for IKK Activation by TNFα, but Not IL-1β (A) Tetracycline (Tet) was used to induce the expression of shRNA against both UbcH5b and UbcH5c. After stimulation with GST-TNFα or IL-1β for the indicated time, the activation of IKK was analyzed by immunoblotting with an IκBα antibody or by IKK activity assay. (B) U2OS cells expressing UbcH5 shRNA and RNAi-resistant Flag-UbcH5c (left) or Flag-UbcH5c (C85A) (right) were treated with tetracycline for 7 days. The cells were stimulated with GST-TNFα for the indicated time, and then IKK activation was measured. IKK activity was quantified using PhosphoImager, and the fold activation as compared to unstimulated cells is shown. Molecular Cell 2009 36, 302-314DOI: (10.1016/j.molcel.2009.10.002) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 5 TNFα-Induced RIP1 Polyubiquitination Depends on UbcH5, but Not Ubc13 (A–C) U2OS-shUbcH5 cells were treated with tetracycline (Tet) to knock down endogenous UbcH5. In (C), the cells also contain a tetracycline-inducible expression cassette for Flag-UbcH5c or Flag-UbcH5c (C85A). These cells were stimulated with GST-TNFα (A and C) or IL-1β (B) for the indicated time, and then a NEMO antibody was used to coimmunoprecipitate polyubiquitinated RIP1 (A and C) or IRAK1 (B). (D and E) Similar to (A) and (B), except that the cells contain shRNA for Ubc13 instead of UbcH5. Molecular Cell 2009 36, 302-314DOI: (10.1016/j.molcel.2009.10.002) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 6 TNFα-Induced RIP1 Polyubiquitination Is Not Restricted to K63 of Ubiquitin (A–D) U2OS-shUb (A and B) and U2OS-shUb-Ub(K63R) (C and D) were treated with tetracycline (Tet) for 3 (A and B) or 4 (C and D) days and then stimulated with GST-TNFα (A and C) or IL-1β (B and D) for the indicated time. A NEMO antibody was used to coimmunoprecipitate polyubiquitinated RIP1 (A and C) or IRAK1 (B and D). (E) U2OS cells and those expressing wild-type or K63R ubiquitin were induced with tetracycline for 4 days and then stimulated with GST-TNFα for 10 min before a NEMO antibody was used to immunoprecipitate NEMO-associated proteins. These proteins were eluted with 1% SDS, and then ubiquitinated proteins were immunoprecipitated with an HA antibody, followed by immunoblotting with a RIP1 antibody. (F) U2OS-shUb-Ub(K63R) cells (± Tet) were treated with GST-TNFα for the indicated time. Cells were lysed in 6 M urea and diluted 20-fold before immunoprecipitation with a K63-Ub antibody. RIP1 in the immunoprecipitates as well as in cell lysates was detected by immunoblotting. Molecular Cell 2009 36, 302-314DOI: (10.1016/j.molcel.2009.10.002) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 7 Polyubiquitination of RIP1 by UbcH5 and cIAP1 (A) U2OS cells were treated with SMAC mimetic (500 nM) for 10 min to induce degradation of endogenous cIAPs. The cells were stimulated with GST-TNFα for the indicated time, and then a NEMO antibody was used to immunoprecipitate the IKK complex as well as polyubiquitinated RIP1. The IKK activity was measured, and the amounts of RIP1, cIAP1, and IκBα in the cell lysates were determined by immunoblotting. (B) A catalytically inactive mutant of RIP1 (D138N) was expressed and purified from Sf9 cells and used as a substrate in ubiquitination reactions containing E1, ubiquitin, UbcH5c, or its C85A mutant, cIAP1, and ATP as indicated. After incubation at 30°C for 1 hr, ubiquitination of RIP1 was analyzed by immunoblotting. (C–E) Similar to (B), except that Ubc13/Uev1A, TRAF2, TRAF6, or ubiquitin lysine mutant as indicated was also tested for RIP1 ubiquitination. KO, no lysine in ubiquitin; K48 only or K63 only, containing only one lysine at position 48 or 63 of ubiquitin. Molecular Cell 2009 36, 302-314DOI: (10.1016/j.molcel.2009.10.002) Copyright © 2009 Elsevier Inc. Terms and Conditions