βTrCP-Mediated Proteolysis of NF-κB1 p105 Requires Phosphorylation of p105 Serines 927 and 932

Abstract

NF-κB1 p105 functions both as a precursor of NF-κB1 p50 and as a cytoplasmic inhibitor of NF-κB. Following the stimulation of cells with tumor necrosis factor alpha (TNF-α), the IκB kinase (IKK) complex rapidly phosphorylates NF-κB1 p105 on serine 927 in the PEST region. This phosphorylation is essential for TNF-α to trigger p105 degradation, which releases the associated Rel/NF-κB subunits to translocate into the nucleus and regulate target gene transcription. Serine 927 resides in a conserved motif (Asp-Ser⁹²⁷-Gly-Val-Glu-Thr-Ser⁹³²) homologous to the IKK target sequence in IκBα. In this study, TNF-α-induced p105 proteolysis was revealed to additionally require the phosphorylation of serine 932. Experiments with IKK1^−/− and IKK2^−/− double knockout embryonic fibroblasts demonstrate that the IKK complex is essential for TNF-α to stimulate phosphorylation on p105 serines 927 and 932. Furthermore, purified IKK1 and IKK2 can each phosphorylate a glutathione S-transferase-p105_758-967 fusion protein on both regulatory serines in vitro. IKK-mediated p105 phosphorylation generates a binding site for βTrCP, the receptor subunit of an SCF-type ubiquitin E3 ligase, and depletion of βTrCP by RNA interference blocks TNF-α-induced p105 ubiquitination and proteolysis. Phosphopeptide competition experiments indicate that βTrCP binds p105 more effectively when both serines 927 and 932 are phosphorylated. Interestingly, however, βTrCP affinity for the IKK-phosphorylated sequence on p105 is substantially lower than that on IκBα. Thus, it appears that reduced p105 recruitment of βTrCP and subsequent ubiquitination may contribute to delayed p105 proteolysis after TNF-α stimulation relative to that for IκBα.

ACKNOWLEDGMENTS

V.L. and J.J. contributed equally to this work.

We thank Nancy Bump, Alain Israel, Frank Mercurio, Keiji Tanaka, and Inder Verma for the reagents used in this study. The help of Lee Johnston, Steve Smerdon, and Steve Gamblin (National Institute for Medical Research) for suggestions on the manuscript, Sandra Watton (National Institute for Medical Research) for advice on affinity purification of antiphosphopeptide antibodies, and Lesley Thompson (University of St. Andrews) and Ada Hatzubai (Hebrew University) for helping with in vitro ubiquitination experiments is gratefully acknowledged. We are also indebted to the PhotoGraphics department (National Institute for Medical Research) for making the figures.

This work was supported by the U.K. Medical Research Council, the Arthritis Research Campaign (project grant L0536 to V.L.), and the AINP consortium, EC—5th framework.