Abstract
In unstimulated cells, the transcription factor NF-kappaB is held in the cytoplasm in an inactive state by the inhibitor protein IkappaBalpha. Stimulation of cells results in rapid phosphorylation and degradation of IkappaBalpha, thus releasing NF-kappaB, which translocates to the nucleus and activates transcription of responsive genes. Here we demonstrate that in cells where proteasomal degradation is inhibited, signal induction by tumor necrosis factor alpha results in the rapid accumulation of higher molecular weight forms of IkappaBalpha that dissociate from NF-kappaB and are consistent with ubiquitin conjugation. Removal of the high molecular weight forms of IkappaBalpha by a recombinant ubiquitin carboxyl-terminal hydrolase and reactivity of the immunopurified material with a monoclonal antibody specific for ubiquitin indicated that IkappaBalpha was conjugated to multiple copies of ubiquitin. Western blot analysis of immunopurified IkappaBalpha from cells expressing epitope-tagged versions of IkappaBalpha and ubiquitin revealed the presence of multiple copies of covalently bound tagged ubiquitin. An S32A/S36A mutant of IkappaBalpha that is neither phosphorylated nor degraded in response to signal induction fails to undergo inducible ubiquitination in vivo. Thus signal-induced activation of NF-kappaB involves phosphorylation-dependent ubiquitination of IkappaBalpha, which targets the protein for rapid degradation by the proteasome and releases NF-kappaB for translocation to the nucleus.
Highlights
The NF-B/rel family of transcription factors are involved in the activation of a wide variety of genes, including the HIV-1 provirus, that respond to immune and inflammatory signals
Ubiquitinated IB␣ must represent a short lived intermediate in the degradative pathway, as ubiquitinated IB␣ is only readily detected when proteasomal degradation is inhibited
Phosphorylated IB␣ remains bound to NF-B but is recognized by proteins involved in ubiquitin addition
Summary
Materials—Z-LLL-H was synthesized as described previously [55], isolated by reverse-phase high performance liquid chromatography (Ͼ95% purity) and the structure confirmed by NMR spectroscopy. Plasmids—The plasmid expressing HA-tagged ubiquitin under control of the cytomegalovirus immediate early promoter [56] was obtained from D. The plasmid expressing IBctag under control of the cytomegalovirus immediate promoter was as described previously [57]. Cells were treated with Z-LLL-H or TNF␣ and Z-LLL-H or were untreated, and extracts were prepared as described below. The expression of both wild-type and S32A/S36A IBctag proteins was stabilized in HeLa cells using neomycin selection with DNA vectors driven by an immediate early cytomegalovirus promoter (pCDNAIII, InVitrogen) or an Rous sarcoma virus promoter (RcRSV, InVitrogen), respectively. After 30 min at 4 °C, the lysate was clarified by centrifugation at 14,000 rpm for 15 min at 4 °C in an Eppendorf microcentrifuge
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