Phosphorylation of p105 PEST sequences via a redox-insensitive pathway up-regulates processing to p50 NF-κB

Abstract

The p 105 Rel protein has dual functions; it is the precursor of the p50 subunit of NF-κB, and it acts as an IκB-like inhibitor to retain other Rel subunits in the cytoplasm. We have investigated the posttranslational regulation of p105 following activation of Jurkat T cells and find that a rapid and sustained phosphorylation of p105 is induced. The inducible phosphorylation occurs on multiple serines in the C-terminal-most 150 amino acids of the molecule, a region rich in Pro, Glu, Ser, and Thr residues. Phosphorylation of p105 in Jurkat cells treated with phorbol 12-myristate 13-acetate/ionomycin or with okadaic acid, another activator of NF-κB, is correlated with an increase in proteolytic processing to p50. Intact PEST sequences are required for the phorbol 12-myristate 13-acetate/ionomycin-induced p105 processing, as a 68-amino acid C-termmal deletion abolishes the response to stimulation. When compounds that block IκBαα phosphorylation and degradation were tested, the serine protease inhibitors L-1-tosylamido-2-phenylethyl chloromethyl ketone and 1-ehloro-3-tosylamido-7-amino-2-heptanone blocked inducible p105 phosphorylation, but the antioxidants pyrrolidine dithiocarbamate and butylated hydroxyanisol did not. Thus, while regulation of the p105 IκB resembles that of IκBα, involving inducible serine phosphorylation and proteolysis of the inhibitory ankyrin repeat domain, it depends on a different, redox-insensitive, signaling pathway.