K13 Activates NFkB Independent of TRAF6/TAK1/LUBAC Figure 5. The ability of K13 to activate NF-kB pathway does not require linear polyubiquitination of NEMO. A–B. MEF NEMO2/Y cells were stably transduced with wild-type NEMO and two NEMO mutants defective in linear ubiquitination of NEMO (NEMO-Y293A/Y301A/K302A and NEMO-F305A) and their expression was confirmed by immunoblotting using NEMO antibody. These cells were further transduced with either an empty retroviral vector or a retroviral vector expressing FLAG-tagged K13 and their expression was determined by immunoblotting with FLAG antibody. The blot was re-probed with a tubulin antibody to show equal loading of protein. C. Upon expression of K13, these cells were able to activate NF-kB p65 DNA binding as determined by ELISA. The values shown are averages (mean6SE) of one representative experiment out of three in which the level of p65 DNA binding was measured in triplicate. Asterisks (*) indicate significance at levels of p#0.05. doi:10.1371/journal.pone.0036601.g005 Figure 7A, while interaction of K13 with NEMO, IKK1, and IKK2 was readily detected in samples immunoprecipitated with Flag antibody from wild-type Jurkat cells, no significant interaction between K13 and IKK1 or IKK2 was observed in the NEMO- deficient Jurkat cells, Stimulation of cells with TNFa or IL-1 induces recruitment of IKK1 and IKK2 to NEMO and their subsequent phosphorylation and activation [61,62]. The relevant phosphorylation sites in IKK1 and IKK2 have been mapped to regions in their catalytic domain that shares strong homology with ‘‘T loop’’ regulatory sequences found in the members of the mitogen activated protein kinase kinase (MAP2K) family of enzymes [61,62]. These sites include Ser176 and Ser180 for IKK1 and Ser177 and Ser181 for IKK2. We next examined if recruitment of IKK1 and IKK2 to the K13-NEMO complex is also associated with ‘‘T Loop’’ phosphorylation of IKK1, IKK2 or both. The K13-ERTAM expressing MEFs were treated with 4OHT and phosphorylation status of IKK1 and IKK2 was determined using Pathscan phospho-IKK1(Ser176/180) and phospho-IKK2 (Ser177/181) ELISA kits, respectively. As shown in Figure 7C, 4OHT treatment of K13-ERTAM cells resulted in a significant increase in ‘‘T loop’’ phosphorylation of IKK1, IKK2 as compared to the untreated cells. The K13-induced increased ‘‘T loop’’ phosphorylation of IKK1 and IKK2 was also associated with increased phosphorylation of IkBa. Thus, recruitment of IKK1 and IKK2 to the K13-NEMO complex results in their activation by ‘‘T loop’’ phosphorylation. Discussion K13 is known to activate the NF-kB pathway by binding to activating the ,700 kDa IKK complex consisting of IKK1/ IKKa, IKK2/IKKb and NEMO/IKKc [34]. We have previously Figure 6. Deubiquitinating enzyme CYLD, a negative regulator of NF-kB pathway does not block K13-induced NF-kB activity. A–D. The 293T cells were co-transfected with a control vector or vectors encoding FLAG-K13, TNFR1, CD40 and EDAR (250 ng/ml) and a CYLD plasmid construct (750 ng/well) along with NF-kB-Luc reporter construct (75 ng/well) and a pRSV/LacZ (b-galactosidase) reporter construct (75 ng/well), and the reporter assay performed as described in ‘‘Materials and Methods’’ The values shown are averages (mean6SE) of one representative experiment out of three in which each transfection was performed in duplicate. doi:10.1371/journal.pone.0036601.g006 PLoS ONE | www.plosone.org 8 May 2012 | Volume 7 | Issue 5 | e36601