Activation of nuclear factor kappaB in single living cells. Dependence of nuclear translocation and anti-apoptotic function on EGFPRELA concentration.
We have studied the dynamics of nuclear translocation during nuclear factor kappaB activation by using a p65(RELA)-enhanced green fluorescent protein (EGFP) fusion construct. Quantitation of expression levels indicates that EGFPRELA can be detected at physiological concentrations of about 60,000 molecules per cell. Stimulation of transfected fibroblasts with interleukin (IL)-1beta caused nuclear translocation of EGFPRELA, typically resulting in a 30-fold increase in nuclear protein at maximum induction and a concomitant 20% decrease in cytoplasmic levels. The response of individual cells to IL-1beta was graded, and the kinetics of nuclear translocation were dependent on the dose of IL-1beta and the level of EGFPRELA expression. The rate of nuclear uptake was saturable, and the time lag for uptake increased at higher EGFPRELA expression levels. Furthermore, nuclear translocation was reduced at less than saturating doses of IL-1beta suggesting that the pathway is limited by incoming signals. The response to IL-1beta was biphasic, demonstrating a decline in nuclear import rate at expression levels above three to four times endogenous. This correlated with the anti-apoptotic function of EGFPRELA which was more prominent at low expression levels and demonstrated successively less protection at higher levels. In comparison, transfection of p50 had no effect on the level of apoptosis and demonstrated some toxicity in combination with EGFPRELA.
Carlotti
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F
Division of Molecular Medicine, The Medical School, University of Sheffield, Sheffield S10 2JF, United Kingdom.
Chapman
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R
Dower
S K
SK
Qwarnstrom
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EE
D017209:Apoptosis / Q000187:drug effects; D001483:Base Sequence; D001692:Biological Transport; D002467:Cell Nucleus / Q000378:metabolism*; D002478:Cells, Cultured; D003513:Cycloheximide / Q000494:pharmacology; D017931:DNA Primers; D005455:Fluorescent Antibody Technique; D049452:Green Fluorescent Proteins; D006801:Humans; D007700:Kinetics; D008025:Ligases / Q000378:metabolism*; D008164:Luminescent Proteins / Q000378:metabolism*; D016328:NF-kappa B / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D017931:DNA Primers; D008164:Luminescent Proteins; D016328:NF-kappa B; D011993:Recombinant Fusion Proteins; D014409:Tumor Necrosis Factor-alpha; D049452:Green Fluorescent Proteins; D003513:Cycloheximide; D008025:Ligases; C028812:guanosine 3',5'-polyphosphate synthetases