Nuclear translocation of green fluorescent protein-nuclear factor kappaB with a distinct lag time in living cells.
A highly fluorescent mutant form of the green fluorescent protein (GFP) has been fused to the human nuclear factor kappaB (NF-kappaB) p50 and p105 (p50/IkappaB gamma), a precursor protein of NF-kappaB p50. GFP-p50 and GFP-p105 were expressed in monkey COS-7 cells and human HeLa cells. Translocation of these chimeric proteins was observed by confocal laser scanning microscopy. GFP-p50 (without IkappaB gamma) in the transfected cells resided in the nucleus. On the other hand, GFP-p105 (GFP-p50 with IkappaB gamma) localized only in the cytoplasm before stimulation and translocated to the nucleus with stimulant specificity similar to that of native NF-kappaB/IkappaB. In addition, the translocation of NF-kappaB to the nucleus had a distinct lag time (a quiescent time) in the target cells. The lag time lasted 10-20 min after stimulation with hydrogen peroxide or tumor necrosis factor alpha. It was suggested that this might be due to the existence of a limiting step where NF-kappaB is released from NF-kappaB/IkappaB by the proteasome.
Faculty of Pharmaceutical Sciences, Nagoya City University, Nagoya, Japan.
K
K
Tenjinbaru
T
T
Furuno
N
N
Hirashima
M
M
Nakanishi
D019943:Amino Acid Substitution; D000818:Animals; D015153:Blotting, Western; D019556:COS Cells; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism*; D003593:Cytoplasm / Q000187:drug effects / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D020672:I-kappa B Proteins; D008164:Luminescent Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D018613:Microscopy, Confocal; D016328:NF-kappa B / Q000096:biosynthesis / Q000378:metabolism*; D052002:NF-kappa B p50 Subunit; D011498:Protein Precursors / Q000096:biosynthesis / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000378:metabolism; D037401:Scyphozoa; D013997:Time Factors; D014157:Transcription Factors / Q000096:biosynthesis / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D004268:DNA-Binding Proteins; D020672:I-kappa B Proteins; D008164:Luminescent Proteins; D016328:NF-kappa B; D052002:NF-kappa B p50 Subunit; C489548:NFKB1 protein, human; D011498:Protein Precursors; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D014409:Tumor Necrosis Factor-alpha; D049452:Green Fluorescent Proteins; D006861:Hydrogen Peroxide