Phosphorylation of serine 468 by GSK-30 negatively regulates basal p65 NF-κB activity

Abstract

The activity of NF-κB is controlled at several levels including the phosphorylation of the strongly transactivating p65 (RelA) subunit. However, the overall number of phosphorylation sites, the signaling pathways and protein kinases that target p65 NF-κB and the functional role of these phosphorylations are still being uncovered. Using a combination of peptide arrays with in vitro kinase assays we identify serine 468 as a novel phosphorylation site of p65 NF-κB. Serine 468 lies within a GSK-3β consensus site, and recombinant GSK-3β specifically phosphorylates a GST-p65-(354-551) fusion protein at Ser468 in vitro. In intact cells, phosphorylation of endogenous Ser468 of p65 is induced by the PP1/PP2A phosphatase inhibitor calyculin A and this effect is inhibited by the GSK-3β inhibitor LiCl. Reconstitution of p65-deficient cells with a p65 protein where serine 468 was mutated to alanine revealed a negative regulatory role of serine 468 for NF-κB activation. Collectively our results suggest that a GSK-3β-PP1-dependent mechanism regulates phosphorylation of p65 NF-κB at Ser468 in unstimulated cells and thereby controls the basal activity of NF-κB.