β-TrCP binding and processing of NF-κB2/p100 involve its phosphorylation at serines 866 and 870

Processing of the NF-κB2 precursor protein p100 is a major step in noncanonical NF-κB signaling. This signaling step requires the NF-κB inducing kinase (NIK) and its downstream kinase, IκB kinase alpha (IKKα). We show here that p100 undergoes phosphorylation at serines 866, 870, and possibly 872, in cells stimulated with noncanonical NF-κB stimuli or transfected with NIK and IKKα. Phosphorylation of this serine cluster creates a binding site for β-TrCP, the receptor subunit of the β-TrCP^SCF ubiquitin ligase. Mutation of either serine 866 or serine 870 abolishes the β-TrCP recruitment and ubiquitination of p100. The functional significance of p100 phosphorylation is further supported by the finding that this molecular event occurs in a NIK- and IKKα-dependent manner. Additionally, induction of p100 phosphorylation can be blocked by a protein synthesis inhibitor, suggesting the requirement of de novo protein synthesis. These data suggest that p100 processing involves its phosphorylation at specific terminal serines, which form a binding site for β-TrCP thereby regulating p100 ubiquitination.