CHARACTERIZATION OF A MUTANT-CELL LINE THAT DOES NOT ACTIVATE NF-KAPPA-B IN RESPONSE TO MULTIPLE STIMULI

Numerous genes required during the immune or inflammation response as well as the adhesion process are regulated by nuclear factor kappa B ( NF-kappa B). Associated with its inhibitor, I kappa B, NF-kappa B resi des as an inactive form in the cytoplasm. Upon stimulation by various agents, I kappa B is proteolyzed and NF-kappa B translocates to the nu cleus, where it activates its target genes. The transduction pathways that lead to I kappa B inactivation remain poorly understood. In this study, we have characterized a cellular mutant, the 70/Z3-derived 1.3E 2 murine pre-B cell line, that does not activate NF-kappa B in respons e to several stimuli. We demonstrate that upon stimulation by lipopoly saccharide, Taxol, phorbol myristate acetate, interleukin-l, or double -stranded RNA, I kappa B alpha is not degraded, as a result of an abse nce of induced phosphorylation on serines 32 and 36. Neither a mutatio n in I kappa B alpha nor a mutation in p50 or relA, the two major subu nits of NF-kappa B in this cell line, accounts for this phosphorylatio n defect. As well as culminating in the inducible phosphorylation of I kappa B alpha on serines 32 and 36, all the stimuli that are inactive on 1.3E2 cells exhibit a sensitivity to the antioxidant pyrrolidine d ithiocarbamate (PDTC). In contrast, stimuli such as hyperosmotic shock or phosphatase inhibitors, which use PDTC-insensitive pathways, induc e I kappa B alpha degradation in 1.3E2. Analysis of the redox status o f 1.3E2 does not reveal any difference from wild-type 70Z/3. We also r eport that the human T-cell leukemia virus type 1 (HTLV-1)-derived Tax trans-activator induces NF-kappa B activity in 1.3E2, suggesting that this viral protein does not operate via the defective pathway. Finall y, we show that two other I kappa B molecules, I kappa B beta and the recently identified I kappa B epsilon, are not degraded in the 1.3E2 c ell line following stimulation. Our results demonstrate that 1.3E2 is a cellular transduction mutant exhibiting a defect in a step that is r equired by several different stimuli to activate NF-KB. In addition, t his analysis suggests a common step in the signaling pathways that tri gger I kappa B alpha, I kappa B beta, and I kappa B epsilon degradatio n.