Identification of the receptor component of the IkappaBalpha-ubiquitin ligase

Nature. 1998 Dec 10;396(6711):590-4. doi: 10.1038/25159.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Binding Sites
  • DNA-Binding Proteins / metabolism
  • Drosophila
  • Drosophila Proteins
  • Electrophoresis, Polyacrylamide Gel
  • GTP-Binding Proteins / isolation & purification
  • HeLa Cells
  • Humans
  • I-kappa B Kinase
  • I-kappa B Proteins*
  • Ligases / chemistry*
  • Ligases / isolation & purification
  • Ligases / metabolism
  • Mass Spectrometry
  • Mice
  • Molecular Sequence Data
  • NF-KappaB Inhibitor alpha
  • NF-kappa B / metabolism
  • Peptide Fragments / isolation & purification
  • Phosphorylation
  • Protein Binding
  • Protein-Serine-Threonine Kinases / metabolism
  • Ubiquitin-Protein Ligases
  • Ubiquitins / metabolism*
  • beta-Transducin Repeat-Containing Proteins

Substances

  • BTRC protein, human
  • Btrc protein, mouse
  • DNA-Binding Proteins
  • Drosophila Proteins
  • I-kappa B Proteins
  • NF-kappa B
  • NFKBIA protein, human
  • Nfkbia protein, mouse
  • Peptide Fragments
  • Ubiquitins
  • beta-Transducin Repeat-Containing Proteins
  • NF-KappaB Inhibitor alpha
  • Ubiquitin-Protein Ligases
  • Protein-Serine-Threonine Kinases
  • CHUK protein, human
  • Chuk protein, mouse
  • I-kappa B Kinase
  • IKBKB protein, human
  • IKBKE protein, human
  • IKKbeta protein, Drosophila
  • Ikbkb protein, mouse
  • Ikbke protein, mouse
  • GTP-Binding Proteins
  • Ligases

Associated data

  • GENBANK/AF099932
  • GENBANK/AF101784