betaTrCP-mediated proteolysis of NF-kappaB1 p105 requires phosphorylation of p105 serines 927 and 932.
NF-kappaB1 p105 functions both as a precursor of NF-kappaB1 p50 and as a cytoplasmic inhibitor of NF-kappaB. Following the stimulation of cells with tumor necrosis factor alpha (TNF-alpha), the IkappaB kinase (IKK) complex rapidly phosphorylates NF-kappaB1 p105 on serine 927 in the PEST region. This phosphorylation is essential for TNF-alpha to trigger p105 degradation, which releases the associated Rel/NF-kappaB subunits to translocate into the nucleus and regulate target gene transcription. Serine 927 resides in a conserved motif (Asp-Ser(927)-Gly-Val-Glu-Thr-Ser(932)) homologous to the IKK target sequence in IkappaBalpha. In this study, TNF-alpha-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-alpha 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 betaTrCP, the receptor subunit of an SCF-type ubiquitin E3 ligase, and depletion of betaTrCP by RNA interference blocks TNF-alpha-induced p105 ubiquitination and proteolysis. Phosphopeptide competition experiments indicate that betaTrCP binds p105 more effectively when both serines 927 and 932 are phosphorylated. Interestingly, however, betaTrCP affinity for the IKK-phosphorylated sequence on p105 is substantially lower than that on IkappaBalpha. Thus, it appears that reduced p105 recruitment of betaTrCP and subsequent ubiquitination may contribute to delayed p105 proteolysis after TNF-alpha stimulation relative to that for IkappaBalpha.
Lang
Valerie
V
Division of Immune Cell Biology, National Institute for Medical Research, London NW7 1AA, United Kingdom.
Janzen
Julia
J
Fischer
Gregory Zvi
GZ
Soneji
Yasmina
Y
Beinke
Sören
S
Salmeron
Andres
A
Allen
Hamish
H
Hay
Ronald T
RT
Ben-Neriah
Yinon
Y
Ley
Steven C
SC
D000595:Amino Acid Sequence; D000818:Animals; D001667:Binding, Competitive; D002478:Cells, Cultured; D005347:Fibroblasts; D019204:GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D051550:I-kappa B Kinase; D051379:Mice; D018345:Mice, Knockout; D008969:Molecular Sequence Data; D016328:NF-kappa B / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D052002:NF-kappa B p50 Subunit; D010766:Phosphorylation; D011498:Protein Precursors / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012694:Serine / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D025801:Ubiquitin / Q000378:metabolism; D044844:beta-Transducin Repeat-Containing Proteins
C471644:BTRC protein, human; C471643:Btrc protein, mouse; D016328:NF-kappa B; D052002:NF-kappa B p50 Subunit; D011498:Protein Precursors; D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha; D025801:Ubiquitin; D044844:beta-Transducin Repeat-Containing Proteins; D012694:Serine; D017346:Protein Serine-Threonine Kinases; C496559:CHUK protein, human; C496564:Chuk protein, mouse; D051550:I-kappa B Kinase; C496560:IKBKB protein, human; C496565:IKBKE protein, human; C496562:Ikbkb protein, mouse; C496566:Ikbke protein, mouse; D019204:GTP-Binding Proteins