Direct phosphorylation of NF-kappaB1 p105 by the IkappaB kinase complex on serine 927 is essential for signal-induced p105 proteolysis.
The p105 precursor protein of NF-kappaB1 acts as an NF-kappaB inhibitory protein, retaining associated Rel subunits in the cytoplasm of unstimulated cells. Tumor necrosis factor alpha (TNFalpha) and interleukin-1alpha (IL-1alpha) stimulate p105 degradation, releasing associated Rel subunits to translocate into the nucleus. By using knockout embryonic fibroblasts, it was first established that the IkappaB kinase (IKK) complex is essential for these pro-inflammatory cytokines to trigger efficiently p105 degradation. The p105 PEST domain contains a motif (Asp-Ser(927)-Gly-Val-Glu-Thr), related to the IKK target sequence in IkappaBalpha, which is conserved between human, mouse, rat, and chicken p105. Analysis of a panel of human p105 mutants in which serine/threonine residues within and adjacent to this motif were individually changed to alanine established that only serine 927 is essential for p105 proteolysis triggered by IKK2 overexpression. This residue is also required for TNFalpha and IL-1alpha to stimulate p105 degradation. By using a specific anti-phosphopeptide antibody, it was confirmed that IKK2 overexpression induces serine 927 phosphorylation of co-transfected p105 and that endogenous p105 is also rapidly phosphorylated on this residue after TNFalpha or IL-1alpha stimulation. In vitro kinase assays with purified proteins demonstrated that both IKK1 and IKK2 can directly phosphorylate p105 on serine 927. Together these experiments indicate that the IKK complex regulates the signal-induced proteolysis of NF-kappaB1 p105 by direct phosphorylation of serine 927 in its PEST domain.
Salmerón
A
A
Division of Cellular Immunology, the National Institute for Medical Research, Mill Hill, London NW7 1AA, United Kingdom.
Janzen
J
J
Soneji
Y
Y
Bump
N
N
Kamens
J
J
Allen
H
H
Ley
S C
SC
D000595:Amino Acid Sequence; D006367:HeLa Cells; D006801:Humans; D006868:Hydrolysis; D051550:I-kappa B Kinase; D008969:Molecular Sequence Data; D016328:NF-kappa B / Q000378:metabolism*; D052002:NF-kappa B p50 Subunit; D010766:Phosphorylation; D011498:Protein Precursors / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000378:metabolism*; D012694:Serine / Q000378:metabolism*; D015398:Signal Transduction
D016328:NF-kappa B; D052002:NF-kappa B p50 Subunit; D011498:Protein Precursors; D012694:Serine; D017346:Protein Serine-Threonine Kinases; C496559:CHUK protein, human; D051550:I-kappa B Kinase; C496560:IKBKB protein, human; C496565:IKBKE protein, human