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.
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.
Amir
Ruthie E
RE
Department of Biochemistry and the Rappaport Family Institute for Research in the Medical Sciences, the Bruce Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Haifa 31096, Israel.
Haecker
Hans
H
Karin
Michael
M
Ciechanover
Aaron
A
D016367:Baculoviridae / Q000235:genetics; D018797:Cell Cycle Proteins / Q000378:metabolism; D002460:Cell Line; D006367:HeLa Cells; D006801:Humans; D020672:I-kappa B Proteins / Q000737:chemistry / Q000378:metabolism; D008025:Ligases / Q000378:metabolism*; D008239:Lysine / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D000075687:NEDD8 Protein; D016328:NF-kappa B / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D017354:Point Mutation; D011498:Protein Precursors / Q000737:chemistry / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D034741:RNA, Small Interfering / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D014452:Ubiquitins / Q000378:metabolism*; D044844:beta-Transducin Repeat-Containing Proteins / Q000378:metabolism
D018797:Cell Cycle Proteins; D020672:I-kappa B Proteins; D000075687:NEDD8 Protein; C082860:NEDD8 protein, human; D016328:NF-kappa B; D011498:Protein Precursors; D034741:RNA, Small Interfering; D011994:Recombinant Proteins; D014452:Ubiquitins; D044844:beta-Transducin Repeat-Containing Proteins; C111664:protein p52, mammalian; D008025:Ligases; D008239:Lysine