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.
Yaron
A
A
The Lautenberg Center for Immunology, The Hebrew University-Hadassah Medical School, Jerusalem, Israel.
Hatzubai
A
A
Davis
M
M
Lavon
I
I
Amit
S
S
Manning
A M
AM
Andersen
J S
JS
Mann
M
M
Mercurio
F
F
Ben-Neriah
Y
Y
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D004268:DNA-Binding Proteins / Q000378:metabolism; D004330:Drosophila; D029721:Drosophila Proteins; D004591:Electrophoresis, Polyacrylamide Gel; D019204:GTP-Binding Proteins / Q000302:isolation & purification; D006367:HeLa Cells; D006801:Humans; D051550:I-kappa B Kinase; D020672:I-kappa B Proteins; D008025:Ligases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D013058:Mass Spectrometry; D051379:Mice; D008969:Molecular Sequence Data; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000378:metabolism; D010446:Peptide Fragments / Q000302:isolation & purification; D010766:Phosphorylation; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases; D014452:Ubiquitins / Q000378:metabolism*; D044844:beta-Transducin Repeat-Containing Proteins
C471644:BTRC protein, human; C471643:Btrc protein, mouse; D004268:DNA-Binding Proteins; D029721:Drosophila Proteins; D020672:I-kappa B Proteins; D016328:NF-kappa B; C000606204:NFKBIA protein, human; C000606205:Nfkbia protein, mouse; D010446:Peptide Fragments; D014452:Ubiquitins; D044844:beta-Transducin Repeat-Containing Proteins; D000072000:NF-KappaB Inhibitor alpha; D044767:Ubiquitin-Protein Ligases; D017346:Protein Serine-Threonine Kinases; C496559:CHUK protein, human; C496564:Chuk protein, mouse; D051550:I-kappa B Kinase; C496560:IKBKB protein, human; C496565:IKBKE protein, human; C496563:IKKbeta protein, Drosophila; C496562:Ikbkb protein, mouse; C496566:Ikbke protein, mouse; D019204:GTP-Binding Proteins; D008025:Ligases