K13 Activates NFkB Independent of TRAF6/TAK1/LUBAC Figure 1. TRAF6 is not required for K13-induced NF-kB activation. A. TRAF6+/+ and TRAF62/2 MEFs were transfected with a control vector or vector encoding K13 along with an NF-kB-Luc construct (75 ng/well) and a Renilla reporter construct (75 ng/well, normalization control) using Lipofectamine 2000-mediated transaction. The luciferase reporter assay was performed 48 hours post transfection essentially as described in ‘‘Materials and Methods’’ section. Asterisks (*) indicate significance at levels of p#0.05. B. TRAF6+/+ and TRAF62/2 MEFs were transfected with a control vector or a vector encoding K13-ERTAM along with an NF-kB-Luc construct and a Renilla reporter construct. The transfected cells were subsequently treated with 4OHT (20 nM) for 48 hours and the reporter assays performed as described above. The values shown are averages (mean6SE) of one representative experiment out of three in which each transfection was performed in duplicate. Asterisks (*) indicate significance at levels of p#0.05. C. TRAF6+/+ and TRAF62/2 MEF cells expressing an empty vector or the FLAG-tagged K13-ERTAM treated with 4OHT were examined by immunoblot analysis for upregulation of A20. Tubulin was used as a loading control. doi:10.1371/journal.pone.0036601.g001 involvement of LUBAC-induced linear polyubiquitination in K13- induced NF-kB activation. For this purpose, we generated stable populations of wild-type and HOIL-1-null MEFs expressing and empty vector and FLAG-tagged K13-ERTAM construct. A Western blot analysis confirmed expression of the K13-ERTAM- fusion protein in both wild-type and HOIL-12/2 MEFs, with slightly higher expression in the latter (Fig. 3A). We measured NF- kB transcriptional activation in the resulting cells with and without 4OHT treatment using an NF-kB luciferase reporter assay. As shown in Figure 3B, we observed robust activation of NF-kB-Luc activity upon 4OHT treatment in both the WT-K13-ERTAM and HOIL-12/2 K13-ERTAM cells, arguing against the possibility that HOIL-1 is required for K13-induced NF-kB activity. Indeed, the HOIL-12/2 K13-ERTAM cells showed a greater increase in NF- kB-Luc activity as compared to the WT-K13-ERTAM cells upon 4OHT treatment, which was probably due to the higher level expression of the K13-ERTAM protein in the former. Consistent with the lack of requirement of HOIL-1 in K13-induced NF-kB activity, we observed equivalent increase in nuclear p65 DNA- binding activity in the wild-type and HOIL-12/2 MEFs upon ectopic K13 expression (Fig. 3D–E). In contrast, TNFa-induced NF-kB-Luc and p65 DNA binding activities were markedly reduced in HOIL-12/2 MEFs (Fig. 3C, 3F). Finally, to confirm the lack of involvement of HOIL-1 in K13-induced NF-kB activity, we used Western blot analysis to compare the phosphorylation of IkBa and expression of A20 and RelB, two proteins that are known to be upregulated by the NF-kB pathway, in the wild-type and HOIL-12/2 MEFs stably expressing K13. As shown in Figure 3G, we observed increased phosphorylation of IkBa and upregulation of A20 and RelB in both wild-type and HOIL-12/2 MEFs upon ectopic K13 expression, which argues against an essential role of HOIL-1 in K13-induced NF-kB activity. Indeed, K13-expressing HOIL-12/2 MEFs showed slightly more IkBa phosphorylation and A20 and RelB expression as compared to the K13-expressing wild-type MEFs. This was probably due to slightly higher level of ectopic K13 expression in the latter (Fig. 3G). Sharpin is not Essential for K13-induced NF-kB Activation Mutation in Sharpin (SHANK-associated RH domain interact- ing protein in postsynaptic density) gene are responsible for cpdm (chronic proliferative dermatitis) phenotype in mice that resembles the phenotype of patients with X-linked hyper-IgM syndrome and hypohydrotic ectodermal dysplasia (XHMED), which is caused by mutations in NEMO [54]. SHARPIN shows significant similarity to HOIL-1 and was recently shown to be a component of LUBAC and to be required for linear ubiquitination of NEMO and NF-kB activation [19,20,21]. To examine whether SHARPIN is required for K13-induced NF-kB activity, we examined the ability of K13 to activate an NF-kB-Luc reporter construct in cpdm MEFs. As shown in Figure 4A, transient transfection of K13 strongly activated the NF-kB-Luc reporter in cpdm MEFs, whereas treatment with TNFa failed to do so. We also generated stable populations of wild-type and cpdm MEFs expressing K13 and observed equivalent increase in the nuclear p65/RelA DNA- binding activity upon ectopic K13 expression (Fig. 4B). Finally, ectopic K13 expression also resulted in an increase in IkBa phosphorylation and enhanced expression of the NF-kB target proteins A20 and RelB in the cpdm MEFs (Fig. 4C). Taken collectively, the above results demonstrate that SHARPIN is not essential for K13-induced NF-kB activity. NEMO Mutants Defective in Binding to Linear Polyubiquitin Chains Support K13-Induced NF-kB NEMO was recently shown to selectively bind linear ubiquitin chains and NEMO mutants V293A/Y301A/K302A and F305A that are defective in binding linear ubiquitin chains were shown to be incapable of supporting NF-kB activation by TNFa and other agonists [55]. Therefore, as an independent test of the involvement PLoS ONE | www.plosone.org 4 May 2012 | Volume 7 | Issue 5 | e36601