Shared pathways of IkappaB kinase-induced SCF(betaTrCP)-mediated ubiquitination and degradation for the NF-kappaB precursor p105 and IkappaBalpha.
p105 (NFKB1) acts in a dual way as a cytoplasmic IkappaB molecule and as the source of the NF-kappaB p50 subunit upon processing. p105 can form various heterodimers with other NF-kappaB subunits, including its own processing product, p50, and these complexes are signal responsive. Signaling through the IkappaB kinase (IKK) complex invokes p105 degradation and p50 homodimer formation, involving p105 phosphorylation at a C-terminal destruction box. We show here that IKKbeta 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 DSGPsi motif and is functionally most critical. The region containing both serines is homologous to the N-terminal destruction box of IkappaBalpha, -beta, and -epsilon. Upon phosphorylation by IKK, p105 attracts the SCF E3 ubiquitin ligase substrate recognition molecules betaTrCP1 and betaTrCP2, 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 IKKgamma, 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.
Heissmeyer
V
V
Max-Delbrück-Center for Molecular Medicine, 13122 Berlin, Germany.
Krappmann
D
D
Hatada
E N
EN
Scheidereit
C
C
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D017124:Conserved Sequence; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D051550:I-kappa B Kinase; D020672:I-kappa B Proteins; D066298:In Vitro Techniques; D008070:Lipopolysaccharides / Q000494:pharmacology; D008969:Molecular Sequence Data; D009154:Mutation; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D052002:NF-kappa B p50 Subunit; D010453:Peptide Synthases / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011498:Protein Precursors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D044843:SKP Cullin F-Box Protein Ligases; D014452:Ubiquitins / Q000378:metabolism
D004268:DNA-Binding Proteins; D020672:I-kappa B Proteins; D008070:Lipopolysaccharides; D016328:NF-kappa B; D052002:NF-kappa B p50 Subunit; C000606204:NFKBIA protein, human; D011498:Protein Precursors; D011994:Recombinant Proteins; D014452:Ubiquitins; D000072000:NF-KappaB Inhibitor alpha; D044843:SKP Cullin F-Box Protein Ligases; D017346:Protein Serine-Threonine Kinases; C496559:CHUK protein, human; D051550:I-kappa B Kinase; C496560:IKBKB protein, human; C496565:IKBKE protein, human; D010453:Peptide Synthases