Direct phosphorylation of NF-κB1 p105 by the IκB kinase complex on serine 927 is essential for signal-induced p105 proteolysis

The p105 precursor protein of NF-kappa B1 acts as an NF-KB inhibitory protein, retaining associated Rel subunits in the cytoplasm of unstimulated cells. Tumor necrosis factor alpha (TNF alpha) and interleukin-1 alpha (IL-1 alpha) stimulate p105 degradation, releasing associated Rel subunits to translocate into the nucleus. By using knockout embryonic fibroblasts, it was first established that the I kappaB 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 I kappaB alpha, 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 TNF alpha and IL-1 alpha 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 TNF alpha or IL-1 alpha stimulation. lie 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-kappa B1 p105 by direct phosphorylation of serine 927 in its PEST domain.