TY - JOUR
T1 - Identification of the receptor component of the IκBα-ubiquitin ligase
AU - Yaron, Avraham
AU - Hatzubai, Ada
AU - Davis, Matti
AU - Lavon, Iris
AU - Amit, Sharon
AU - Manning, Anthony M.
AU - Andersen, Jens S.
AU - Mann, Matthias
AU - Mercurio, Frank
AU - Ben-Neriah, Yinon
PY - 1998/12/10
Y1 - 1998/12/10
N2 - NF-κB, 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, IκBs. 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 pIκ̄α-ubiquitin ligase (pIκBα-E3) that attaches ubiquitin, a small protein which marks other proteins for degradation by the proteasome system, to the phosphorylated NF-κB inhibitor pIκBα. Taking advantage of its high affinity to pIκBα, 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 that pIκBα degradation motif as an F-box/WD-domain protein belonging to a recently distinguished family of β-TrCP/Slimb proteins. This component, which we denote E3RS(IκB) (pIκBα-E3 receptor subunit), binds specifically to pIκBα 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(IκB), which tightly binds pIκBα but does not support its ubiquitination, acts in vivo as a dominant-negative molecule, inhibiting the degradation of pIκBα and consequently NF-κB activation. E3RS(IκB) 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.
AB - NF-κB, 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, IκBs. 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 pIκ̄α-ubiquitin ligase (pIκBα-E3) that attaches ubiquitin, a small protein which marks other proteins for degradation by the proteasome system, to the phosphorylated NF-κB inhibitor pIκBα. Taking advantage of its high affinity to pIκBα, 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 that pIκBα degradation motif as an F-box/WD-domain protein belonging to a recently distinguished family of β-TrCP/Slimb proteins. This component, which we denote E3RS(IκB) (pIκBα-E3 receptor subunit), binds specifically to pIκBα 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(IκB), which tightly binds pIκBα but does not support its ubiquitination, acts in vivo as a dominant-negative molecule, inhibiting the degradation of pIκBα and consequently NF-κB activation. E3RS(IκB) 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.
UR - http://www.scopus.com/inward/record.url?scp=17944401842&partnerID=8YFLogxK
U2 - 10.1038/25159
DO - 10.1038/25159
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C2 - 9859996
AN - SCOPUS:17944401842
SN - 0028-0836
VL - 396
SP - 590
EP - 594
JO - Nature
JF - Nature
IS - 6711
ER -