Statistical Analyses Two-tailed paired Student’s t test was used to test for differences between two groups. Differences with a p#0.05 were considered as statistically significant. All experiments were repeated a mini- mum of three times with duplicate/triplicate samples. Results TRAF6 is not Required for K13-induced NF-kB Activation Different members of the TRAF family are required for NF-kB activation by distinct stimuli. Thus, while TRAF2 is known to be required for NF-kB activation by TNFa, TRAF6 has been implicated in the activation of this pathway signaling via interleukin 1 and Toll like receptors [46,47]. We have recently demonstrated that TRAF2 is not involved in K13-induced NF-kB activation [39]. To rule out the involvement of TRAF6 in K13- induced NF-kB activity, we transiently transfected TRAF6+/+ and TRAF62/2 MEFs with an empty vector or a K13 expression construct and examined the activation of a cotransfected NF-kB- Luc reporter construct. As shown in Figure 1A, we observed near equivalent K13-induced NF-kB-Luc activity in the TRAF6+/+ and TRAF62/2 MEFs. Essentially similar results were obtained when the experiment was repeated using the K13-ERTAM construct followed by treatment with 4OHT (Fig. 1B). Finally, we generated stable populations of TRAF6+/+ and TRAF62/2 MEFs expressing an empty vector or the K13-ERTAM construct. The mutated estrogen receptor (ERTAM) in the K13-ERTAM construct does not bind to its physiological ligand estrogen but binds with very high affinity to the synthetic ligand 4OHT (4-hydroxytamoxifen) and allows the control of K13 activity in a 4OHT-dependent fashion [48]. We treated the resulted cells with 4OHT to activate K13 activity and assessed the activation of the NF-kB pathway by measuring the upregulation of A20, a protein known to be induced by K13-induced NF-kB activity [49]. As shown in Fig. 1C, treatment with 4OHT resulted in equivalent upregulation of A20 in the TRAF6+/+ and TRAF62/2, which argues against the involvement of TRAF6 in K13-induced NF-kB activation. TAK1 is not Required for K13-induced NF-kB Activation TRAF6 is believed to activate NF-kB by activating TAK1, which in turn phosphorylates and activates the IKK complex resulting in NF-kB activation [7,8]. TAK1 was also shown to be required for NF-kB activation by KSHV-encoded viral G protein coupled receptor (vGPCR) [50]. To study the role of TAK1 in K13-induced NF-kB activation, we took advantage of murine embryonic fibroblast (MEFs) cells deficient in this protein. We used retroviral-mediated gene transfer to generate stable pools of wild-type and TAK1-deficient MEFs expressing an empty vector and FLAG-tagged K13. An immunoblot analysis confirmed equivalent expression of the K13 in the wild-type (WT) and TAK12/2 MEFs (Fig. 2A). The expression of K13 in the wild-type (WT) and TAK12/2 MEFs, however, was lower than its expression in the KSHV-infected BC1 cell line (Fig. 2A). We subsequently used an electrophoretic mobility shift assay (EMSA) to compare the ability of K13 to activate the NF-kB pathway in the two cell lines. Consistent with the known ability of K13 to activate the NF- kB pathway, ectopic expression of K13 in the TAK+/+ MEFs resulted in increased nuclear NF-kB DNA binding activity (Fig. 2B). However, an equivalent increase in NF-kB DNA binding activity was observed upon ectopic expression of K13 in the TAK12/2 cells (Fig. 2B). To confirm these results, we generated stable clones of WT and TAK12/2 MEFs expressing the K13-ERTAM fusion protein and used an ELISA-based DNA- binding assay to study the subunit composition of NF-kB K13 Activates NFkB Independent of TRAF6/TAK1/LUBAC complexes induced following treatment with 4OHT in the two cell lines. This assay revealed that p65 and p50 were the major NF-kB subunits induced by 4OHT in both the WT and TAK12/2 K13-ERTAM expressing MEFs, with modest induction of p52, RelB and cRel subunits (Fig. 2C). Thus, TAK1 has no major effect on either the amount or subunit composition of NF-kB complexes induced by K13. We next examined the effect of TAK1 on K13- induced NF-kB transcriptional activity. For this purpose, we transiently transfected an empty vector or a K13-ERTAM-fusion construct in wild-type and TAK12/2 MEFs and measured NF-kB transcriptional activation using an NF-kB luciferase reporter assay. As shown in Figure 2D, transfection of TAK+/+ MEFs with K13- ERTAM followed by treatment with 4OHT resulted in a significant increase in NF-kB-Luc reporter activity. However, 4OHT treatment of K13-ERTAM-transfected TAK2/2 MEFs also led to an equivalent increase in NF-kB-Luc activity, thereby demon- strating that TAK1 is not required for K13-induced NF-kB transcriptional activation. K13 does not Induce TAK1 Phosphorylation Human T-cell Leukemia virus 1 (HTLV-1) encoded Tax protein resembles K13 in activating the NF-kB pathway by binding to NEMO [51]. It was recently shown that TAK1 is constitutively activated by Thr-187 phosphorylation in Tax- positive HTLV-1-transformed T cells [52]. Therefore, to examine whether K13 would similarly induce TAK1 Thr-187 phosphor- ylation, we used retroviral-mediated gene transfer and generated polyclonal populations of two lymphoma cell lines, BCBL1 and Namalwa, expressing an empty vector and FLAG-K13. We chose these cell lines as they possess low to none constitutive NF-kB activity endogenously (Fig. 2E). Ectopic expression of K13 in both BCBL1 and Namalwa cells failed to induce phosphorylation of TAK1 as determined by Western blotting with a phospho- Thr184/Thr187 TAK1 antibody, whereas treatment with TNFa successfully did so (Fig. 2F). Thus, in contrast to HTLV-1 oncoprotein Tax, K13 does not induce TAK1 phosphorylation. K13-induced NF-kB is not Blocked by a Specific Inhibitor of TAK1 5Z-7-Oxozeaenol, a resorcyclic lactone of fungal origin, is a potent and selective inhibitor of TAK1 [53]. To confirm the lack of involvement of TAK1 in K13-induced NF-kB activity, we studied the effect of increasing doses of 5Z-7-Oxozeaenol treatment on 4OHT induced NF-kB luciferase activity in 293NF-kB-Luc- K13- ERTAM cells which stably express an NF-kB-driven luciferase reporter construct and the K13-ERTAM fusion protein. As shown in Figure 2G, treatment with up to 1 mM 5Z-7-Oxozeaenol had no significant inhibitory effect on 4OHT-induced NF-kB reporter activity. In comparison, 0.5 mM 5Z-7-Oxozeaenol significantly inhibited TNFa and IL-1b-induced NF-kB activity in the 293NF- kB-Luc reporter cells (Fig. 2H). Taken collectively with previous studies, the above results confirm that TAK1 is not required for K13-induced NF-kB activation. HOIL-1 is not Essential for K13-induced NF-kB Activation Recently, a novel ubiquitin ligase complex, designated LUBAC (linear ubiquitin chain assembly complex), which is composed of two RING finger proteins, HOIL-1 (also known as RBCK1) and HOIP, and SHARPIN (SHANK-associated RH domain interact- ing protein in postsynaptic density) was shown to play a key role in the activation of the NF-kB pathway by binding to NEMO and conjugating linear polyubiquitin chains onto its specific Lys residues [18,19,20]. We used HOIL-1-null MEFs to test the PLoS ONE | www.plosone.org 3 May 2012 | Volume 7 | Issue 5 | e36601