title: Characterization of a mutant cell line that does not activate NF-kappaB in response to multiple stimuli.
abstract: Numerous genes required during the immune or inflammation response as well as the adhesion process are regulated by nuclear factor kappaB (NF-kappaB). Associated with its inhibitor, I kappaB, NF-kappaB resides as an inactive form in the cytoplasm. Upon stimulation by various agents, I kappaB is proteolyzed and NF-kappaB translocates to the nucleus, where it activates its target genes. The transduction pathways that lead to I kappaB inactivation remain poorly understood. In this study, we have characterized a cellular mutant, the 70/Z3-derived 1.3E2 murine pre-B cell line, that does not activate NF-kappaB in response to several stimuli. We demonstrate that upon stimulation by lipopolysaccharide, Taxol, phorbol myristate acetate, interleukin-1, or double-stranded RNA, I kappaB alpha is not degraded, as a result of an absence of induced phosphorylation on serines 32 and 36. Neither a mutation in I kappaB alpha nor a mutation in p50 or relA, the two major subunits of NF-kappaB in this cell line, accounts for this phosphorylation defect. As well as culminating in the inducible phosphorylation of I kappaB alpha on serines 32 and 36, all the stimuli that are inactive on 1.3E2 cells exhibit a sensitivity to the antioxidant pyrrolidine dithiocarbamate (PDTC). In contrast, stimuli such as hyperosmotic shock or phosphatase inhibitors, which use PDTC-insensitive pathways, induce I kappaB alpha degradation in 1.3E2. Analysis of the redox status of 1.3E2 does not reveal any difference from wild-type 70Z/3. We also report that the human T-cell leukemia virus type 1 (HTLV-1)-derived Tax trans-activator induces NF-kappaB activity in 1.3E2, suggesting that this viral protein does not operate via the defective pathway. Finally, we show that two other I kappaB molecules, I kappaB beta and the recently identified I kappaB epsilon, are not degraded in the 1.3E2 cell line following stimulation. Our results demonstrate that 1.3E2 is a cellular transduction mutant exhibiting a defect in a step that is required by several different stimuli to activate NF-kappaB. In addition, this analysis suggests a common step in the signaling pathways that trigger I kappaB alpha, I kappaB beta, and I kappaB epsilon degradation.
authors.affiliation: Unité de Biologie Moléculaire de l'Expression Génique, UMR 321 CNRS,Institut Pasteur, Paris, France.
authors.forename: G
authors.initials: G
authors.lastname: Courtois
authors.forename: S T
authors.initials: ST
authors.lastname: Whiteside
authors.forename: C H
authors.initials: CH
authors.lastname: Sibley
authors.forename: A
authors.initials: A
authors.lastname: Israel
mesh_terms: D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D001402:B-Lymphocytes / Q000502:physiology*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D016356:Gene Products, tax / Q000235:genetics / Q000502:physiology; D015368:Human T-lymphotropic virus 1; D006801:Humans; D006822:Hybrid Cells; D020672:I-kappa B Proteins; D008070:Lipopolysaccharides / Q000494:pharmacology; D051379:Mice; D009154:Mutation; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D052002:NF-kappa B p50 Subunit; D010084:Oxidation-Reduction; D010766:Phosphorylation; D011759:Pyrrolidines / Q000494:pharmacology; D012694:Serine / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology; D013859:Thiocarbamates / Q000494:pharmacology; D051996:Transcription Factor RelA; D015533:Transcriptional Activation
chemical_list: D000975:Antioxidants; D004268:DNA-Binding Proteins; D016356:Gene Products, tax; C083487:I kappa B beta protein; D020672:I-kappa B Proteins; D008070:Lipopolysaccharides; D016328:NF-kappa B; D052002:NF-kappa B p50 Subunit; C000606204:NFKBIA protein, human; C000606205:Nfkbia protein, mouse; D011759:Pyrrolidines; D013859:Thiocarbamates; D051996:Transcription Factor RelA; D000072000:NF-KappaB Inhibitor alpha; C020972:pyrrolidine dithiocarbamic acid; D012694:Serine