Identification of the receptor component of the IkappaBalpha-ubiquitin ligase.
NF-kappaB, a ubiquitous, inducible transcription factor involved in immune, inflammatory, stress and developmental processes, is retained in a latent form in the cytoplasm of non-stimulated cells by inhibitory molecules, IkappaBs. Its activation is a paradigm for a signal-transduction cascade that integrates an inducible kinase and the ubiquitin-proteasome system to eliminate ... inhibitory regulators. Here we isolate the pIkappaBalpha-ubiquitin ligase (pIkappaBalpha-E3) that attaches ubiquitin, a small protein which marks other proteins for degradation by the proteasome system, to the phosphorylated NF-kappaB inhibitor pIkappaBalpha. Taking advantage of its high affinity to pIkappaBalpha, we isolate this ligase from HeLa cells by single-step immunoaffinity purification. Using nanoelectrospray mass spectrometry, we identify the specific component of the ligase that recognizes the pIkappaBalpha degradation motif as an F-box/WD-domain protein belonging to a recently distinguished family of beta-TrCP/Slimb proteins. This component, which we denote E3RSIkappaB (pIkappaBalpha-E3 receptor subunit), binds specifically to pIkappaBalpha and promotes its in vitro ubiquitination in the presence of two other ubiquitin-system enzymes, E1 and UBC5C, one of many known E2 enzymes. An F-box-deletion mutant of E3RS(IkappaB), which tightly binds pIkappaBalpha but does not support its ubiquitination, acts in vivo as a dominant-negative molecule, inhibiting the degradation of pIkappaBalpha and consequently NF-kappaB activation. E3RS(IkappaB) represents a family of receptor proteins that are core components of a class of ubiquitin ligases. When these receptor components recognize their specific ligand, which is a conserved, phosphorylation-based sequence motif, they target regulatory proteins containing this motif for proteasomal degradation.
Mesh Terms:
Amino Acid Sequence, Animals, Binding Sites, DNA-Binding Proteins, Drosophila, Drosophila Proteins, Electrophoresis, Polyacrylamide Gel, GTP-Binding Proteins, Hela Cells, Humans, I-kappa B Kinase, I-kappa B Proteins, Ligases, Mass Spectrometry, Mice, Molecular Sequence Data, NF-kappa B, Peptide Fragments, Phosphorylation, Protein Binding, Protein-Serine-Threonine Kinases, Ubiquitin-Protein Ligases, Ubiquitins, beta-Transducin Repeat-Containing Proteins
Amino Acid Sequence, Animals, Binding Sites, DNA-Binding Proteins, Drosophila, Drosophila Proteins, Electrophoresis, Polyacrylamide Gel, GTP-Binding Proteins, Hela Cells, Humans, I-kappa B Kinase, I-kappa B Proteins, Ligases, Mass Spectrometry, Mice, Molecular Sequence Data, NF-kappa B, Peptide Fragments, Phosphorylation, Protein Binding, Protein-Serine-Threonine Kinases, Ubiquitin-Protein Ligases, Ubiquitins, beta-Transducin Repeat-Containing Proteins
Nature
Date: Dec. 10, 1998
PubMed ID: 9859996
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