Activation of nuclear factor kappa B 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 kappa B 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 mo lecules per cell. Stimulation of transfected fibroblasts with interleukin ( IL)-1 beta caused nuclear translocation of EGFPRELA, typically resulting in a 30-fold increase in nuclear protein at maximum induction and a concomita nt 20% decrease in cytoplasmic levels. The response of individual cells to IL-1 beta was graded, and the kinetics of nuclear translocation were depend ent on the dose of IL-1 beta 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 r educed at less than saturating doses of IL-1 beta suggesting that the pathw ay is limited by incoming signals. The response to IL-1 beta was biphasic, demonstrating a decline in nuclear import rate at expression levels above t hree to four times endogenous, This correlated with the anti-apoptotic func tion of EGFPRELA which was more prominent at low expression levels and demo nstrated successively less protection at higher levels, In comparison, tran sfection of p50 had ho effect on the level of apoptosis and demonstrated so me toxicity in combination with EGFPRELA.