Distinct Activation Mechanisms of NF-κB Regulator Inhibitor of NF-κB Kinase (IKK) by Isoforms of the Cell Death Regulator Cellular FLICE-like Inhibitory Protein (cFLIP)

The viral FLICE-like inhibitory protein (FLIP) protein from Kaposi sarcoma-associated herpesvirus activates the NF-κB pathway by forming a stable complex with a central region (amino acids 150–272) of the inhibitor of NF-κB kinase (IKK) γ subunits, thereby activating IKK. Cellular FLIP (cFLIP) forms are also known to activate the NF-κB pathway via IKK activation. Here we demonstrate that cFLIP(L), cFLIP(S), and their proteolytic product p22-FLIP all require the C-terminal region of NEMO/IKKγ (amino acids 272–419) and its ubiquitin binding function for activation of the IKK kinase (or kinase complex), but none form a stable complex with IKKγ. Our results further reveal that cFLIP(L) requires the linear ubiquitin chain assembly complex and the kinase TAK1 for activation of the IKK kinase. Similarly, cFLIP(S) and p22-FLIP also require TAK1 but do not require LUBAC. In contrast, these isoforms are both components of complexes that incorporate Fas-associated death domain and RIP1, which appear essential for kinase activation. This conservation of IKK activation among the cFLIP family using different mechanisms suggests that the mechanism plays a critical role in their function.