Regulation of NF-kappa B RelA phosphorylation and transcriptional activityby p21(ras) and protein kinase C zeta in primary endothelial cells

The activity of the transcription factor NF-kappa B is thought to be regula ted mainly through cytoplasmic retention by I kappa B molecules. Here we pr esent evidence of a second mechanism of regulation acting on NF-kappa B aft er release from I kappa B. In endothelial cells this mechanism involves pho sphorylation of the RelA subunit of NF-kappa B through a pathway involving activation of protein kinase C zeta (PKC zeta) and p21(ras). We show that t ranscriptional activity of RelA is dependent on phosphorylation of the N-te rminal Rel homology domain but not the C-terminal transactivation domain. I nhibition of phosphorylation by dominant negative mutants of PKC zeta or p2 1(ras) results in loss of RelA transcriptional activity without interfering with DNA binding. Raf/MEK, small GTPases, phosphatidylinositol 3-kinase, a nd stress-activated protein kinase pathways are not involved in this mechan ism of regulation.