NF-κB RelA Phosphorylation Regulates RelA Acetylation

Abstract

The nuclear functions of NF-κB p50/RelA heterodimers are regulated in part by posttranslational modifications of its RelA subunit, including phosphorylation and acetylation. Acetylation at lysines 218, 221, and 310 differentially regulates RelA's DNA binding activity, assembly with IκBα, and transcriptional activity. However, it remains unclear whether the acetylation is regulated or simply due to stimulus-coupled nuclear translocation of NF-κB. Using anti-acetylated lysine 310 RelA antibodies, we detected p300-mediated acetylation of RelA in vitro and in vivo after stimulation of cells with tumor necrosis factor alpha (TNF-α). Coexpression of catalytically inactive mutants of the catalytic subunit of protein kinase A/mitogen- and stress-activated kinase 1 or IKK1/IKK2, which phosphorylate RelA on serine 276 or serine 536, respectively, sharply inhibited RelA acetylation on lysine 310. Furthermore, phosphorylation of RelA on serine 276 or serine 536 increased assembly of phospho-RelA with p300, which enhanced acetylation on lysine 310. Reconstitution of RelA-deficient murine embryonic fibroblasts with RelA S276A or RelA S536A decreased TNF-α-induced acetylation of lysine 310 and expression of the endogenous NF-κB-responsive E-selectin gene. These findings indicate that the acetylation of RelA at lysine 310 is importantly regulated by prior phosphorylation of serines 276 and 536. Such phosphorylated and acetylated forms of RelA display enhanced transcriptional activity.

SUPPLEMENTAL MATERIAL

Supplemental material for this article may be found at http://mcb.asm.org/.

ACKNOWLEDGMENTS

We thank A. Beg, Y. Shi, G. Haegeman, J. S. Arthur, and Q.-H. Zhang for the gift of reagents and Z.-Q. Zhang (Massachusetts General Hospital) for advice on the purification of recombinant RelA. We also thank J. Carroll and C. Goodfellow for assistance in the preparation of figures and S. Ordway and G. Howard for editorial assistance.

L.-F.C. is the recipient of an Arthritis Foundation Investigator Award. This work was supported in part by a National Institutes of Health training grant (AI07305) to L.-F.C., by a National Institutes of Health grant (RO1 CA89001-02) to W.C.G., and by funds from the J. David Gladstone Institutes and Pfizer, Inc., and benefited from core facilities provided through the UCSF-GIVI Center for AIDS Research (National Institutes of Health grant P30 MH59037).