βTrCP-mediated proteolysis of NF-kB1 p105 requires phosphorylation of p105 serines 927 and 932
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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-p105758-967 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.
Lang , V , Janzen , J , Fischer , G Z , Soneji , Y , Beinke , S , Salmeron , A , Allen , H , Hay , R T , Ben-Neriah , Y & Ley , S C 2003 , ' βTrCP-mediated proteolysis of NF-kB1 p105 requires phosphorylation of p105 serines 927 and 932 ' Molecular and Cellular Biology , vol 23 , no. 1 , pp. 402-413 . DOI: 10.1128/MCB.23.1.402-413.2003
Molecular and Cellular Biology
Copyright © 2003, American Society for Microbiology. All Rights Reserved. Open Access article available from PMC
DescriptionThis 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.
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