K13 Activates NFkB Independent of TRAF6/TAK1/LUBAC Figure 7. NEMO-mediated recruitment of IKK1 and IKK2 to K13 and their activation by ‘‘T loop’’ phosphorylation. A. Co- immunoprecipitation assay showing NEMO is essential for mediating the interaction of K13 with IKK1 and IKK2. FLAG-tagged K13 was immunoprecipitated from WT and NEMO-deficient Jurkat cells using control (C) and FLAG (F) antibody beads and presence of co-immunoprecipitated IKK1, IKK2 and NEMO detected by immunoblotting. B. MEFs expressing K13-ERTAM were treated with 4OHT and phosphorylation status of IKK1, IKK2 and IkBa was determined using Pathscan phospho-IKK1 (Ser176/180), phospho-IKK2 (Ser177/181), phosphor-IkBa (Ser32) ELISA kits, respectively. doi:10.1371/journal.pone.0036601.g007 shown that NEMO is essential for K13-induced NF-kB activation [34,45]. However, the exact mechanism by which K13-NEMO interaction results in the activation of the IKK complex and the NF-kB pathway is not clear. In a recent study, we demonstrated that TRAF2, which is required for NF-kB activation by TNFa, is not required for the interaction of K13 with NEMO and for K13- induced NF-kB activation [39]. In this report, we have excluded the involvement of TRAF6, which has been implicated in NF-kB activation during signaling emanating from IL-1 and Toll like receptors [46,47], in K13-induced NF-kB activation. Taken collectively, these studies support the model that K13 bypasses TRAFs and directly interacts with the IKK complex to activate the NF-kB pathway. This model is consistent with previous studies demonstrating a direct interaction between K13 and NEMO/ IKKc in GST-pull down and yeast two-hybrid assays [18], and the recently described crystal structure of the K13-NEMO complex [63]. We next asked the question how the interaction of K13 with NEMO results in the activation of the IKK complex and the NF-kB pathway. Genetic and pharmacological studies have implicated TAK1 as the upstream kinase responsible for the activation of the IKK complex during NF-kB activation by pro-inflammatory cytokines [7,8]. A recent study also showed that TAK1 is required for NF-kB activation by KSHV-encoded viral G protein coupled receptor (vGPCR) [50]. In contrast, we found no defect in K13- induced NF-kB DNA-binding and transcriptional activities in TAK1-deficient cells. The lack of involvement of TAK1 in K13- induced NF-kB activity was further supported by our studies with 5Z-7-oxo-zeaenol, a specific inhibitor of TAK1, which failed to block K13-induced NF-kB transcriptional activity. Recent studies have implicated LUBAC-mediated linear polyubiquitination in NF-kB activation by proinflammatory cytokines [18,19,20]. Depending on their position relative to the substrate, the ubiquitin monomers in uniquitin chains can be described as Ubdistal or Ubproximal subunits [55]. A crystal structure of NEMO in complex with linear ubiquitin molecules has demonstrated that distinct residues of NEMO interact with Ubdistal and Ubproximal subunits [55]. A recent study reported that reconstitution of a NEMO-deficient clone of mouse 70Z/3 lymphoma cells, designated 1.3E2, with a murine NEMO mutant that is defective in binding to Ubdistal (NEMO-F312A) resulted in restoration of K13-induced NF-kB activation [64]. To confirm the lack of involvement of linear polyubiquitination in K13- induced NF-kB activation, we took advantage of cells that are deficient in two essential components of LUBAC, an enzyme complex required for linear polyubiquitination. Our results with HOIL-12/2 and cpdm MEFs, rule out the requirement of LUBAC- induced linear polyubiquitination in K13-induced NF-kB activa- tion. The lack of requirement of linear polyubiquitination in K13- induced NF-kB activation was further supported by our results showing that CYLD, a deubiquitylating enzyme that can cleave linear ubiquitin chains, cannot block K13-induced NF-kB activity. Taken collectively, the above results demonstrate that linear polyubiquitination is not required for K13-induced activation of the IKK complex and the NF-kB pathway. In addition to its interaction with linear ubiquitin chains, NEMO has been shown to interact with Lys63-linked ubiquitin chains [8,65] and it has been recently suggested that both types of chains cooperate for optimal activation of the NF-kB pathway by proinflammatory cytokines [66]. Interestingly, a F312A mutation in human NEMO, which corresponds to the F305A mutation in mouse NEMO tested in this study, was shown to be defective in binding to Lys63-linked ubiqutin chains [8], which suggests that K13 activates NF-kB independent of Lys63-linked chains as well. This hypothesis is further supported by our results showing that K13-induced NF-kB is not blocked by CYLD, a deubiquitylating enzyme that cleaves both linear and Lys63-linked ubiquitin chains [57,67]. Based on the lack of involvement of TRAF6, TAK1 and LUBAC in K13-induced NF-kB activation, we support a model according to which the interaction between K13 and NEMO is direct and is not mediated via the TRAF family members or the linear ubiquitin chains. We further demonstrate that binding of K13 to NEMO is required for recruitment of IKK1 and IKK2 to PLoS ONE | www.plosone.org 9 May 2012 | Volume 7 | Issue 5 | e36601