SCF<sup>β-TrCP</sup> ubiquitin ligase-mediated processing of NF-κB p105 requires phosphorylation of its C-terminus by IκB kinase
Abstract
Processing of the p105 precursor to form the active subunit p50 of the NF-κB transcription factor is a unique case in which the ubiquitin system is involved in limited processing rather than in complete destruction of the target substrate. A glycine-rich region along with a downstream acidic domain have been demonstrated to be essential for processing. Here we demonstrate that following IκB kinase (IκK)-mediated phosphorylation, the C-terminal domain of p105 (residues 918–934) serves as a recognition motif for the SCF ubiquitin ligase. Expression of IκKβ dramatically increases processing of wild-type p105, but not of p105-Δ918–934. Dominant-negative β-TrCP inhibits IκK-dependent processing. Furthermore, the ligase and wild-type p105 but not p105-Δ918–934 associate physically following phosphorylation. In vitro, SCF specifically conjugates and promotes processing of phosphorylated p105. Importantly, the TrCP recognition motif in p105 is different from that described for IκBs, β-catenin and human immunodeficiency virus type 1 Vpu. Since p105-Δ918–934 is also conjugated and processed, it appears that p105 can be recognized under different physiological conditions by two different ligases, targeting two distinct recognition motifs.
Acknowledgements
The authors thank Dr Arnim Pause (Boehringer Ingelheim, Canada) for the baculovirus cullin-1 expression vector, Dr Michele Pagano from NYU Medical Center for the ΔF-box Skp2 mammalian cell expression vector, and Drs Eyal Bengal (Faculty of Medicine, Technion, Haifa, Israel) and Chaim Kahana (Weizmann Institute, Rehovot, Israel) for helpful discussions. This research was supported by grants from the Israel Science Foundation founded by the Israeli Academy of Sciences and Humanities—Centers of Excellence Program, the German–Israeli Foundation for Scientific Research and Development (GIF), the Israel Cancer Society, the German–Israeli Project Cooperation (DIP), the Foundation for Promotion of Research at the Technion, a research grant administered by the Vice President of the Technion for Research (to A.C.), a grant from the National Institutes of Health (NIH; to A.L.S.), a grant from the US–Israel Binational Science Foundation (to A.C. and A.L.S.) and a TMR grant from the European Community (to A.I. and A.C.). Partial support was also obtained from Signal Pharmaceuticals, Inc., San Diego, CA. A.O. was supported by a Fellowship from the Clore Foundation. Purchasing of the ABI 310 autosequencer was supported partially by a grant from the Israel Science Foundation founded by the Israeli Academy of Sciences and Humanities.