Kaposi’s Sarcoma Associated Herpesvirus Encoded Viral FLICE Inhibitory Protein K13 Activates NF-kB Pathway Independent of TRAF6, TAK1 and LUBAC Hittu Matta, Ramakrishnan Gopalakrishnan, Ciaren Graham, Bhairavi Tolani, Akshat Khanna, Han Yi, Yulan Suo, Preet M. Chaudhary* Jane Anne Nohl Division of Hematology and Center for the Study of Blood Diseases, University of Southern California Keck School of Medicine, Los Angeles, California, United States of America Abstract Background: Kaposi’s sarcoma associated herpesvirus encoded viral FLICE inhibitory protein (vFLIP) K13 activates the NF-kB pathway by binding to the NEMO/IKKc subunit of the IkB kinase (IKK) complex. However, it has remained enigmatic how K13-NEMO interaction results in the activation of the IKK complex. Recent studies have implicated TRAF6, TAK1 and linear ubiquitin chains assembled by a linear ubiquitin chain assembly complex (LUBAC) consisting of HOIL-1, HOIP and SHARPIN in IKK activation by proinflammatory cytokines. Methodology/Principal Findings: Here we demonstrate that K13-induced NF-kB DNA binding and transcriptional activities are not impaired in cells derived from mice with targeted disruption of TRAF6, TAK1 and HOIL-1 genes and in cells derived from mice with chronic proliferative dermatitis (cpdm), which have mutation in the Sharpin gene (Sharpincpdm/cpdm). Furthermore, reconstitution of NEMO-deficient murine embryonic fibroblast cells with NEMO mutants that are incapable of binding to linear ubiquitin chains supported K13-induced NF-kB activity. K13-induced NF-kB activity was not blocked by CYLD, a deubiquitylating enzyme that can cleave linear and Lys63-linked ubiquitin chains. On the other hand, NEMO was required for interaction of K13 with IKK1/IKKa and IKK2/IKKb, which resulted in their activation by ‘‘T Loop’’ phosphorylation. Conclusions/Significance: Our results demonstrate that K13 activates the NF-kB pathway by binding to NEMO which results in the recruitment of IKK1/IKKa and IKK2/IKKb and their subsequent activation by phosphorylation. Thus, K13 activates NF- kB via a mechanism distinct from that utilized by inflammatory cytokines. These results have important implications for the development of therapeutic agents targeting K13-induced NF-kB for the treatment of KSHV-associated malignancies. Citation: Matta H, Gopalakrishnan R, Graham C, Tolani B, Khanna A, et al. (2012) Kaposi’s Sarcoma Associated Herpesvirus Encoded Viral FLICE Inhibitory Protein K13 Activates NF-kB Pathway Independent of TRAF6, TAK1 and LUBAC. PLoS ONE 7(5): e36601. doi:10.1371/journal.pone.0036601 Editor: J. Pedro Simas, Lisbon University, Portugal Received November 30, 2011; Accepted April 11, 2012; Published May 8, 2012 Copyright: 2012 Matta et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by National Institutes of Health (DE019811 and CA139119) and the Leukemia & Lymphoma Society. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: preet.chaudhary@med.usc.edu Introduction Transcription factors of the nuclear factor-k B (NF-kB) family regulate expression of hundreds of genes involved in the inflammatory and immune response [1,2,3]. The classical NF- kB complex is a heterodimer of the p65/RelA and p50 subunits and is found in most cells in association with a family of inhibitory proteins, called IkBs [4,5]. The IkB proteins are identified by the presence of multiple ankyrin repeats and retain NF-kB in the cytoplasm through the masking of the nuclear localization signal of p65. Most of the diverse signaling pathways that activate NF-kB converge on a multi-subunit IkB kinase (IKK) complex that contains two catalytic subunits, IKK1/IKKa and IKK2/IKKb, and a regulatory subunit, NEMO/IKKc [5,6]. The IKK complex induces phosphorylation of IkB proteins that leads to their ubiquitination and proteasomal-mediated degradation, allowing the NF-kB subunits to enter the nucleus and turn on the expression of their target genes [4]. A kinase complex composed of TAK1 and its associated subunits TAB1, TAB2 and TAB3 has been reported to act as the upstream kinase that activates the IKK complex by targeting two serine residues located in the conserved ‘‘T loop’’ region of the kinase domains of IKK1/IKKa and IKK2/IKKb [7,8]. Recently, various members of the E3 ligase family have recently been implicated in NF-kB activation by participating in ubiquitination processes not linked to protein degradation [8]. For example, one of the best characterized pathways that results in NF-kB activation is the Tumor Necrosis Factor Receptor 1 (TNFR1) signaling pathway, which is activated by TNFa [9,10]. Binding of TNFa to TNFR1, results in the recruitment of several adaptor proteins to the cytosolic domain of the receptor, including the adaptor protein TRADD, E3 ligase TRAF2 and cIAPs and kinases RIP, TAK1 and IKKs [10,11,12,13,14,15,16]. These macromolecular complex results in TRAF2 and cIAP1-mediated Lys63-linked polyubiquitination of RIP, which then recruits the TAK1 complex through specific PLoS ONE | www.plosone.org 1 May 2012 | Volume 7 | Issue 5 | e36601