Regulation of NF-κB RelA Phosphorylation and Transcriptional Activity by p21 and Protein Kinase Cζ in Primary Endothelial Cells

Abstract

The activity of the transcription factor NF-B is thought to be regulated mainly through cytoplasmic retention by IB molecules. Here we present evidence of a second mechanism of regulation acting on NF-B after release from IB. In endothelial cells this mechanism involves phosphorylation of the RelA subunit of NF-B through a pathway involving activation of protein kinase C (PKC) 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 PKC 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.The NF-B/Rel family of dimeric transcription factors is involved in the immediate early transcription, i.e. independent of protein synthesis, of a large array of genes induced by mitogenic or pathogen-associated stimuli. In its active form, NF-B is a nuclear homo-or heterodimeric complex of a number of different Rel family members. The canonical and most abundant form of NF-B is composed of a 50-kDa (p50, or NFB1) and a 65-kDa (p65, or RelA) subunit. Both subunits can form homodimers as well as heterodimers with other members of the Rel family i.e. c-Rel (Rel), p52 (NFB2), and RelB (1). All members of the Rel family exhibit extensive sequence similarity in their N-terminal region referred to as the Rel homology domain (RHD) 1 responsible for DNA binding and formation of Rel dimers. Only RelA, Rel, and RelB carry a transcription activating domain, and thus only dimers containing one of these proteins activate the transcription of NF-B-dependent genes efficiently. With respect to transcription activation, the RelA subunit appears to have the highest activity.In most unstimulated cells, NF-B is constitutively retained in the cytoplasm by inhibitory proteins of the IB family, namely IB␣, IB␤, IB␥, p100, p105, and IB⑀ (2). Formation of NF-B⅐IB complexes masks the nuclear localization signal sequence present in NF-B molecules and thus prevents their nuclear translocation. One of the key events in the activation of NF-B is the liberation of functional NF-B dimers from IB, which results in the translocation of NF-B to the nucleus. Cytoplasmic release of NF-B dimers involves site-specific phosphorylation of IB by kinases of the IB signalosome (3-6), ubiquitination (7), and subsequent proteolytic degradation by the 26 S proteasome pathway (8). Upon nuclear import and binding to specific decameric recognition motifs, which are reflected by the consensus GGGRNNYYCC (where R represents A or G and Y represents C or T), NF-B dimers function as transcriptional activators. IB␣ (9), IB␤, and p105 (10) have been implicated in the inhibition of DNA binding of NF-B complexes. However, there have been several reports showing that NF-B transcriptional activity can be blocked withou...

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