Baratchian, M; (2015) Mechanism of IKK activation by the Kaposi's sarcoma-associated herpesvirus protein vFLIP and its cellular homologues. Doctoral thesis , UCL (University College London).

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Abstract

Activation of the NF-κB pathway is linked to cancer development and progression. Kaposi’s sarcoma-associated herpesvirus (KSHV/HHV8) encodes vFLIP which binds to the NEMO/IKKγ subunit of IKK and constitutively activates NF-κB, leading to tumourigenesis. Cellular FLIPs, which share sequence homology with KSHV vFLIP and induce NF-κB activation, are upregulated in a variety of malignancies and are therefore promising targets for anti-cancer therapies. The cFLIP family consists of three splice variant isoforms (cFLIPL, cFLIPS and cFLIPR) and two proteolytic fragments (p43-FLIP and p22-FLIP). Much is known about how cFLIPs regulate apoptosis but the mechanisms by which they activate NF-κB are not well understood. Potential similarities to vFLIP-induced activation have been suggested but not investigated. Here we show that, unlike KSHV vFLIP, cFLIP variants are not found in stable complexes with NEMO and all require upstream events to mediate signalling to IKK. By mutational analysis on NEMO and protein expression knockdowns, we demonstrate that all cFLIP isoforms require the ubiquitin binding domain (UBD) of NEMO while it is redundant for vFLIP’s function. Similarly, our data reveals that TAK1 is essential for induction of IKK by cFLIP isoforms but not vFLIP. We further show that different cFLIP isoforms have different requirements for IKK activation. While cFLIPL needs LUBAC to activate NF-κB, cFLIPS and p22-FLIP require FADD and RIP1. Contrary to existing reports, our results suggest that processing of cFLIPL to p22-FLIP or p43-FLIP fragments by caspase-8 is not necessary for its IKK activation. Finally, we propose that vFLIP-mediated activation of IKK is most likely to occur through induction of multimerisation and re-orientation of the IKK complexes within higher order IKK assemblies that lead to autophosphorylation of the enzymatic subunits, IKKα and β. In conclusion, the work in this thesis provides evidence that vFLIP, cFLIPL, cFLIPS and p22-FLIP have specific and different mechanisms of inducing IKK activation. This has implications for the design of therapeutics to block pathological NF-κB activation in viral and non-viral tumours.

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