Phosphorylation of serine 468 by GSK-3beta negatively regulates basal p65 NF-kappaB activity.
The activity of NF-kappaB 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-kappaB 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-kappaB. Serine 468 lies within a GSK-3beta consensus site, and recombinant GSK-3beta specifically phosphorylates a GST-p65-(354-551) fusion protein at Ser(468) in vitro. In intact cells, phosphorylation of endogenous Ser(468) of p65 is induced by the PP1/PP2A phosphatase inhibitor calyculin A and this effect is inhibited by the GSK-3beta 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-kappaB activation. Collectively our results suggest that a GSK-3beta-PP1-dependent mechanism regulates phosphorylation of p65 NF-kappaB at Ser(468) in unstimulated cells and thereby controls the basal activity of NF-kappaB.
Institute of Pharmacology, Medical School Hannover, Carl-Neuberg Strasse 1, D-30625 Hannover, Germany.
Holger
H
Buss
Anneke
A
Dörrie
M Lienhard
ML
Schmitz
Ronald
R
Frank
Mark
M
Livingstone
Klaus
K
Resch
Michael
M
Kracht
D015536:Down-Regulation; D038362:Glycogen Synthase Kinase 3 / Q000378:metabolism*; D000071679:Glycogen Synthase Kinase 3 beta; D006367:HeLa Cells; D006801:Humans; D007375:Interleukin-1 / Q000378:metabolism*; D009154:Mutation; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D012694:Serine / Q000235:genetics / Q000378:metabolism*; D051996:Transcription Factor RelA
D007375:Interleukin-1; D016328:NF-kappa B; D051996:Transcription Factor RelA; D012694:Serine; C000605506:GSK3B protein, human; D000071679:Glycogen Synthase Kinase 3 beta; D038362:Glycogen Synthase Kinase 3