Abstract
Proteasome-mediated processing of the nfkappab2 gene product p100 is a regulated event that generates the NF-kappaB subunit p52. This event can be induced through p100 phosphorylation by a signaling pathway involving the nuclear factor-kappaB-inducing kinase (NIK). The C-terminal region of p100, which contains its phosphorylation site and a death domain, plays a pivotal role in regulating the processing of p100. To understand the biochemical mechanism of p100 processing, we searched for cellular factors interacting with the C-terminal regulatory region of p100 using the yeast two-hybrid system. This led to the identification of S9, a non-ATPase subunit of the 19 S proteasome with no known functions. Interestingly, the S9/p100 interaction could be induced by NIK but not by a catalytically inactive NIK mutant. This inducible molecular interaction required p100 ubiquitination and was dependent on the intact death domain. We further demonstrated that the death domain is essential for NIK-induced post-translational processing of p100, thus providing a functional link between the S9 binding and the processing of p100. Finally, we provide genetic evidence for the essential role of S9 in the inducible processing of p100.
Highlights
The NF-B1 family of transcription factors plays a pivotal role in diverse biological processes, including innate and adaptive immune responses, cell growth, differentiation, and survival [1,2,3]
We further demonstrated that the death domain is essential for nuclear factor-B-inducing kinase (NIK)-induced post-translational processing of p100, providing a functional link between the S9 binding and the processing of p100
We examined the effect of NIK on the S9/p100 interaction, because this kinase serves as a potent inducer of p100 processing [8]
Summary
The NF-B1 family of transcription factors plays a pivotal role in diverse biological processes, including innate and adaptive immune responses, cell growth, differentiation, and survival [1,2,3]. Upon activation by upstream signals, IKK phosphorylates the IBs at specific serines, which targets these inhibitors for ubiquitination and proteasome-mediated degradation. In addition to this so-called canonical pathway of activation, NF-B is subjected to regulation by other mechanisms. The first is to recognize and position ubiquitinated protein substrates, and the second is to unfold the protein substrates so that they can be inserted to the proteolytic chamber of the 20 S catalytic particle How these events occur in the 19 S particle is unclear, and how the individual subunits participate in the regulatory functions remains unknown. These results provide an important insight into the biochemical mechanisms of p100 processing in the proteasome
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