Mechanism of processing of the NF-kappa B2 p100 precursor: identification of the specific polyubiquitin chain-anchoring lysine residue and analysis of the role of NEDD8-modification on the SCF(beta-TrCP) ubiquitin ligase

Oncogene. 2004 Apr 1;23(14):2540-7. doi: 10.1038/sj.onc.1207366.


Processing of the NF-kappa B2 precursor p100 to the mature p52 subunit is regulated via a unique pathway. NF-kappa B-inducing kinase (NIK) induces I kappa B kinase alpha (IKK alpha)-mediated phosphorylation of specific serine residues in the C-terminal domain of p100, leading to recruitment of the SCF(beta-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 I kappa B alpha. 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(beta-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(beta-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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Baculoviridae / genetics
  • Cell Cycle Proteins / metabolism
  • Cell Line
  • HeLa Cells
  • Humans
  • I-kappa B Proteins / chemistry
  • I-kappa B Proteins / metabolism
  • Ligases / metabolism*
  • Lysine / metabolism*
  • Mutagenesis, Site-Directed
  • NEDD8 Protein
  • NF-kappa B / chemistry
  • NF-kappa B / metabolism*
  • Phosphorylation
  • Point Mutation
  • Protein Precursors / chemistry
  • Protein Precursors / metabolism*
  • Protein Processing, Post-Translational*
  • Protein Structure, Tertiary
  • RNA, Small Interfering / metabolism
  • Recombinant Proteins / metabolism
  • Ubiquitins / metabolism*
  • beta-Transducin Repeat-Containing Proteins / metabolism


  • Cell Cycle Proteins
  • I-kappa B Proteins
  • NEDD8 Protein
  • NEDD8 protein, human
  • NF-kappa B
  • Protein Precursors
  • RNA, Small Interfering
  • Recombinant Proteins
  • Ubiquitins
  • beta-Transducin Repeat-Containing Proteins
  • protein p52, mammalian
  • Ligases
  • Lysine