Direct phosphorylation of NF-kappa B1 p105 by the I kappa B kinase complexon serine 927 is essential for signal-induced p105 proteolysis

The p105 precursor protein of NF-kappa B1 acts as an NF-KB inhibitory prote in, retaining associated Rel subunits in the cytoplasm of unstimulated cell s. Tumor necrosis factor alpha (TNF alpha) and interleukin-1 alpha (IL-1 al pha) stimulate p105 degradation, releasing associated Rel subunits to trans locate into the nucleus. By using knockout embryonic fibroblasts, it was fi rst established that the I kappaB kinase (IKK) complex is essential for the se pro-inflammatory cytokines to trigger efficiently p105 degradation. The p105 PEST domain contains a motif (Asp-Ser(927) Gly-Val Glu-Thr), related t o the IKK target sequence in I kappaB alpha, which is conserved between hum an, 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 essenti al for p105 proteolysis triggered by IKK2 overexpression. This residue is a lso 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 an d 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 p 105 on serine 927. Together these experiments indicate that the IKK complex regulates the signal-induced proteolysis of NF-kappa B1 p105 by direct pho sphorylation of serine 927 in its PEST domain.