affinity of TAB2 and TAB3 for Lys63-linked chains [7,8,17]. Recently, another novel ubiquitin ligase complex, designated LUBAC (linear ubiquitin chain assembly complex), was also shown to be part of the TNFR1 signaling complex and to play a key role in NF-kB activation by catalyzing the assembly of linear ubiquitin chains [18,19,20,21]. Activation of NF-kB by IL1b and lipopolysaccharide involve similar components. Binding of these ligands to their receptors initially results in the recruitment of MyD88 [22]. Subsequently, MyD88 recruits kinases IRAK4 and IRAK1 and E3 ligase TRAF6, which results in IRAK1 phosphorylation, release of IRAK1/TRAF6 and TRAF6 autoubiquitination [23,24,25]. Ubiquitinated TRAF6 is then believed to recruit the TAK1 complex and in concert with Ubc13, modify both TAK1 and IRAK1 with Lys63-linked chains [26,27]. The final step in this process involves recruitment of the IKKs through NEMO and their phosphorylation/activation by TAK1 [26,28]. Kaposi’s sarcoma (KS) associated herpesvirus (KSHV), also known as Human herpesvirus 8 (HHV8), is the commonest cause of malignancy among patients with Acquired Immuno- deficiency Syndrome [29,30]. Infection with KSHV has been associated with KS, primary effusion lymphoma (PEL) and multicentric Castleman’s disease (MCD) [31]. KSHV encodes a vFLIP (viral FLICE inhibitory Protein), encoded by the open reading frame K13, which is one of the few viral proteins to be expressed in cells latently-infected with KSHV [32]. Based on its structural homology to the prodomain of FLICE/caspase 8 [33], K13 was initially believed to be an inhibitor of FLICE/ caspase 8 (hence the name vFLIP) [33]. However, subsequent work by our laboratory and others have demonstrated that the main biological function of K13 is the activation of the NF-kB pathway rather than inhibition of caspase 8/FLICE, and that it utilizes this pathway to promote cellular survival, proliferation, transformation and cytokine secretion [34,35,36,37,38]. We recently demonstrated that K13 bypasses TRAF2 and TRAF3 and activates the NF-kB pathway by directly binding to the NEMO subunit of the IKK complex [39]. However, it is not clear how K13-NEMO interaction results in the activation of the IKK complex. In this study, we have investigated the role of TRAF6, TAK1 and LUBAC in K13-induced NF-kB activation. We demonstrate that K13 activates the NF-kB pathway independent of TRAF6 and TAK1. Furthermore, linear polyubiquitination of NEMO is not required for K13- induced NF-kB activation. Instead, NEMO is required for mediating the interaction between K13 and IKK1 and IKK2. Our results suggest that K13 activates the NF-kB pathway using a mechanism distinct from that utilized by proinflammatory cytokines. Materials and Methods Cell Lines and Reagents 293T, BC1, BCBL1, Jurkat and Namalwa cells were obtained from ATCC (Manassas, VA). BCBL1 and Namalwa cells expressing an empty vector and K13 have been described previously [39,40]. Wild-type and NEMO-deficient cells have also been described previously [41]. TRAF6+/+ and TRAF62/2 mouse embryonic fibroblasts (MEFs) were gifts from Tak Mak (University of Toronto, Canada) [24]. TAK1+/+ and TAK12/2 MEFs were generously provided by Dr. Stephanie S. Watowich (MD Anderson Cancer Center, Houston, Texas). HOIL-12/2 and cpdm cells were kindly provided by Dr. Kazuhiro Iwai (Osaka University, Japan). TAK1 inhibitor 5Z-7-oxo-zeaenol was purchased from Tocris Bioscience (Ellisville, MO), recombinant K13 Activates NFkB Independent of TRAF6/TAK1/LUBAC TNFa and IL-1b were from Peprotech (Rocky Hill, NJ), and 4- Hydroxytamoxifen (4OHT) was purchased from Sigma (St. Lois, MO). Plasmids Plasmids encoding K13 and 4-Hydroxytamoxifen (4OHT)- inducible K13-ERTAM, CYLD, EDAR (ectodermal dysplasia receptor) and NEMO have been described previously [36,37,39,42,43]. Retroviral constructs expressing NEMO mu- tants defective in linear ubiquitination were kindly provided by Dr. Ivan Dikic (Goethe University Medical School). Recombinant retroviruses were generated and used to generate polyclonal populations of stably transduced MEFs following selection with puromycin essentially as described previously [44]. Luciferase Reporter Assay 293T cells were transfected in a 24-well plate with various test plasmids along with an NF-kB luciferase reporter construct (75 ng/well) and a pRSV/LacZ (b-galactosidase) reporter con- struct (75 ng/well) as described previously [42]. Cells were lysed 24–36 h later, and extracts were used for the measurement of firefly luciferase and b-galactosidase activities, respectively. Luciferase activity was normalized relative to the b-galactosidase activity to control for the difference in the transfection efficiency. Transient transfection of MEFs and measurement of luciferase activity was performed essentially as described previously [45]. Briefly, MEFs were transfected in duplicate using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) in a 24-well plate with the various test plasmids along with an NF-kB/luciferase reporter construct (75 ng/well) and a Renilla luciferase reporter construct (phRG-TK, 75 ng/well, Promega, Madison, WI). The cells were lysed 48 hours later, and extracts were used for the measurement of firefly and Renilla luciferase activities as described in the Dual- LuciferaseH Reporter (DLRTM) Assay system from Promega. Firefly luciferase activity was normalized relative to the Renilla luciferase activity to control for the difference in the transfection efficiency. Western Blot Western blot analysis was performed essentially as described previously [34]. Primary antibodies used in these experiments were: NEMO, Total-IkBa, Rel B, TRAF6 (Santa Cruz Bio- technology, Santa Cruz, CA); tubulin, M2 FLAG (Sigma, St. Louis, MO), and phospho-TAK1, phospho-IkBa and A20 (Cell Signaling, Danvers, MA). A mouse monoclonal antibody against K13 (8F6) was raised in our laboratory. NF-kB DNA-binding Assays The NF-kB subunit composition of the K13-induced NF-kB complexes in wild-type and TAK12/2 MEFs was determined with an NF-kB ELISA kit (TransAM NF-kB; Active Motif, Carlsbad, CA) according to the manufacturers’ instructions. The electro- phoretic mobility shift assay was performed as described previously [34]. Pathscan ELISA Assay The PathScan Phospho-IKKa (Ser176/180), Phospho-IKKb (Ser177/181) sandwich ELISA Kits and PathScan Phospho-IkBa (Ser32) sandwich ELISA antibody pair (Cell Signaling, Danvers, MA) were used to detect endogenous levels of IKKa, IKKb and IkBa proteins when phosphorylated at Ser176/180, Ser177/181 and Ser32, respectively. PLoS ONE | www.plosone.org 2 May 2012 | Volume 7 | Issue 5 | e36601