Lipopolysaccharide stimulates both nuclear localization of the nuclear factor kappa B 50-kDa subunit and loss of the 105-kDa precursor in RAW264 macrophage-like cells.
Nuclear factor kappa B (NF-kappa B) is an important regulator of gene expression in cells of the immune system. One such gene, tumor necrosis factor, is induced by bacterial lipopolysaccharide (LPS) in macrophages, and this induction has been shown to be mediated in part by NF-kappa B activation in murine macrophages. In this study, immunochemical analysis was used to follow LPS activation of the NF-kappa B 50-kDa subunit in the RAW264 macrophage-like cell line. The recombinant NF-kappa B 50-kDa subunit was used as an immunogen to produce a rabbit antiserum, which was then affinity-purified using a portion of the NF-kappa B 50-kDa subunit that does not have homology to other members of the c-rel gene family. Untreated macrophages had little NF-kappa B in the nucleus as detected by Western immunoblotting. The protein was predominantly localized in the cytoplasmic fraction. Interestingly, NF-kappa B was found as the 50-kDa mature protein and 105-kDa precursor. After LPS treatment, there was a rapid nuclear translocation of NF-kappa B as detected by immunoblot analysis. There was also a rapid decrease in the amount of the cytoplasmic 105-kDa protein. This may indicate that the 105-kDa protein is a reservoir for the 50-kDa protein and that one of the actions of LPS is to increase the rate of 105-kDa precursor processing.
Zheng
S
S
Department of Biochemistry and Molecular Biology, State University of New York, Health Science Center, Syracuse 13210.
Brown
M C
MC
Taffet
S M
SM
D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D015153:Blotting, Western; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008264:Macrophages / Q000378:metabolism* / Q000648:ultrastructure; D051379:Mice; D016328:NF-kappa B / Q000378:metabolism*; D011498:Protein Precursors / Q000378:metabolism*
D008070:Lipopolysaccharides; D016328:NF-kappa B; D011498:Protein Precursors