Shared Pathways of IκB Kinase-Induced SCF^βTrCP-Mediated Ubiquitination and Degradation for the NF-κB Precursor p105 and IκBα

Abstract

p105 (NFKB1) acts in a dual way as a cytoplasmic IκB molecule and as the source of the NF-κB p50 subunit upon processing. p105 can form various heterodimers with other NF-κB subunits, including its own processing product, p50, and these complexes are signal responsive. Signaling through the IκB kinase (IKK) complex invokes p105 degradation and p50 homodimer formation, involving p105 phosphorylation at a C-terminal destruction box. We show here that IKKβ phosphorylation of p105 is direct and does not require kinases downstream of IKK. p105 contains an IKK docking site located in a death domain, which is separate from the substrate site. The substrate residues were identified as serines 923 and 927, the latter of which was previously assumed to be a threonine. S927 is part of a conserved DSGΨ motif and is functionally most critical. The region containing both serines is homologous to the N-terminal destruction box of IκBα, -β, and -ɛ. Upon phosphorylation by IKK, p105 attracts the SCF E3 ubiquitin ligase substrate recognition molecules βTrCP1 and βTrCP2, resulting in polyubiquitination and complete degradation by the proteasome. However, processing of p105 is independent of IKK signaling. In line with this and as a physiologically relevant model, lipopolysaccharide (LPS) induced degradation of endogenous p105 and p50 homodimer formation, but not processing in pre-B cells. In mutant pre-B cells lacking IKKγ, processing was unaffected, but LPS-induced p105 degradation was abolished. Thus, a functional endogenous IKK complex is required for signal-induced p105 degradation but not for processing.

ACKNOWLEDGMENTS

We thank Michael Karin for the gift of IKKα and -β expression vectors and baculovirus constructs, Yinon Ben-Neriah for the βTrCP1 expression vector, Serge Fuchs and Ze'ev Ronai for the HOS/βTrCP2 plasmid, and Ulf Rapp for Cot constructs. 1.3E2 cells were provided by Carol Sibley. We thank Erika Scharschmidt for expert technical assistance and Rudolf Dettmer for purification of IKKs.