Regulation of NF-κB RelA phosphorylation and transcriptional activity by p21(ras) and protein kinase Cζ in primary endothelial cells

Abstract

The activity of the transcription factor NF-κB is thought to be regulated mainly through cytoplasmic retention by IκB molecules. Here we present evidence of a second mechanism of regulation acting on NF-κB after release from IκB. In endothelial cells this mechanism involves phosphorylation of the RelA subunit of NF-κB through a pathway involving activation of protein kinase Cζ (PKCζ) and p21(ras). We show that transcriptional activity of RelA is dependent on phosphorylation of the N- terminal Rel homology domain but not the C-terminal transactivation domain. Inhibition of phosphorylation by dominant negative mutants of PKCζ or p21(ras) results in loss of RelA transcriptional activity without interfering with DNA binding. Raf/MEK, small GTPases, phosphatidylinositol 3-kinase, and stress-activated protein kinase pathways are not involved in this mechanism of regulation.