Mechanism of processing of the NF-κB2 p100 precursor: identification of the specific polyubiquitin chain-anchoring lysine residue and analysis of the role of NEDD8-modification on the SCF^β-TrCP ubiquitin ligase

Abstract

Processing of the NF-κB2 precursor p100 to the mature p52 subunit is regulated via a unique pathway. NF-κB-inducing kinase (NIK) induces IκB kinase α (IKKα)-mediated phosphorylation of specific serine residues in the C-terminal domain of p100, leading to recruitment of the SCF^β-TrCP ubiquitin ligase. We identified a single lysine residue, K855, that serves as the ubiquitin-anchoring residue required for signal-induced processing of p100. In a reconstituted system containing purified components, p100-K855R could not be ubiquitinated. In a crude extract and cells, only residual, signal-independent ubiquitination and processing were retained. Importantly, K855 is located in a site homologous to K22 that serves as an ubiquitination site in IκBα. This suggests a common recognition mechanism for the two molecules. In contrast, p105, the p100 homologue, lacks a similar Lys residue. We also demonstrate that the NEDD8 pathway is essential for the SCF^β-TrCP activity. In a reconstituted system, efficient ubiquitination of p100 required all three components of the pathway – E1, the UBC12 E2 and NEDD8. Experiments in reconstituted systems and in cells demonstrated that SCF^β-TrCP, which contains a mutant Cul-1 that cannot be NEDDylated, cannot stimulate ubiquitination and processing. Similarly, dominant negative UBC12 inhibits, in a reversible manner, both ubiquitination and processing of p100.