Activation of NF-κB in vivo is regulated by multiple phosphorylations

Abstract

The activation of nuclear factor κB (NF-κB) in intact cells is mechanistically not well understood. Therefore we investigated the modifications imposed on NF-κB/IκB components following stimulation and show that the final step of NF-κB induction in vivo involves phosphorylation of several members of the NF-κB/IκB protein families. In HeLa cells as well as in B cells, TNF-α rapidly induced nuclear translocation primarily of p50-p65, but not of c-rel. Both NF-κB precursors and IκBα became strongly phosphorylated with the same kinetics. In addition to the inducible phosphorylation after stimulation, B lymphocytes containing constitutive nuclear NF-κB revealed constitutively phosphorylated p65 and IκBα. Phosphorylation was accompanied by induced processing of the precursors p100 and p105 and by degradation of IκBα. As an in vitro model we show that phosphorylation of 105 impedes its ability to interact with NF-κB, as has been shown before for IκBα. Surprisingly, even p65, but not c-rel, was phosphorylated after induction in vivo, suggesting that TNF-α selectively activates only specific NF-κB heteromers and that modifications regulate not only IκB molecules but also NF-κB molecules. In fact, cellular NF-κB activity was phosphorylation-dependent and the DNA binding activity of p65-containing NF-κB was enhanced by phosphorylation in vitro. Furthermore, we found that the induction by hydrogen peroxide of NF-κB translocation to the nucleus, which is assumed to be triggered by reactive oxygen intermediates, also coincided with incorporation of phosphate into the same subunits that were modified after stimulation by TNF-α. Thus, phosphorylation appears to be a general mechanism for activation of NF-κB in vivo.