Date of Award
Doctor of Philosophy (PhD)
Michael J. May
NF-kappaB is activated through two signaling cascades: the classical and non-canonical pathways, which are distinguished based on the Inhibitor of kappaB Kinase (IKK) components required to activate each pathway. Whereas classical NF-kappaB requires NF-kappaB Essential Modulator (NEMO) and IKKbeta, non-canonical NF-kappaB requires IKKalpha and upstream stabilization of NF-kappaB Inducing Kinase (NIK), but not NEMO. However, we have previously shown that IKKalpha contains a functional NEMO binding domain and associates with NEMO and IKKbeta as part of the heterotrimeric IKK complex. The overarching goal of the work described in this thesis was to determine whether the interaction between NEMO and IKKalpha was required for non-canonical NF-kappaB signaling. We used a reconstitution approach in knockout cells to show that IKKalpha, independent of its association with NEMO, is sufficient to control the non-canonical NF-kappaB pathway. Thus we provide biochemical evidence to support the tenet that IKKalpha alone regulates non-canonical NF-kappaB signaling. Importantly, while pursuing these results we made the surprising finding that NEMO, and furthermore, IKKbeta, play a compulsory role in the constraint of basal non-canonical NF-kappaB activity. These studies reveal that the previous model depicting a NEMO-independent non-canonical NF-kappaB signaling cascade is incomplete. We show that an intact and catalytically competent classical IKK complex constrains basal NIK accumulation. This novel mechanism of non-canonical NF-kappaB regulation requires classical NF-kappaB transcription driven by the prototypic NF-kappaB family member p65. Thus classical NF-kappaB activity is essential to maintain the quiescent state of the non-canonical NF-kappaB signaling pathway. Upon activation, the duration of non-canonical signaling is controlled by a recently described negative feedback mechanism that promotes NIK turnover. We used a combination of translation and proteasome inhibitors to show that turnover of active NIK is proteasome-dependent. Furthermore, we utilized a Smac mimetic compound to show that degradation of active NIK does not require cellular inhibitor of apoptosis (cIAP)1/2, which distinguishes this feedback mechanism from the cIAP1/2-dependent mechanism of basal NIK turnover. Collectively, these studies advance our understanding of a recently described mechanism of non-canonical NF-kappaB down-regulation and reveal a novel interplay between classical and non-canonical NF-kappaB activity, which revises the current model of non-canonical NF-kappaB signaling.
Gray, Carolyn Margaret, "Mechanisms of Non-canonical NF-kappaB Regulation" (2014). Publicly Accessible Penn Dissertations. 1293.