DNA-dependent protein kinase phosphorylation of IκBβ and IκBβ regulates NF-κB DNA binding properties

Regulation of the I kappa B alpha and I kappa B beta proteins is critical for modulating NF-kappa B-directed gene expression. Both I kappa B alpha and I kappa B beta are substrates for cellular kinases that phosphorylate the amino and carboxy termini of these proteins and regulate their function. In this study, we utilized a biochemical fractionation scheme to purify a kinase activity which phosphorylates residues in the amino and carboxy termini of both I kappa B alpha and I kappa B beta. Peptide microsequence analysis by capillary high-performance liquid chromatography ion trap mass spectroscopy revealed that this kinase was the DNA-dependent protein kinase catalytic subunit (DNA-PKcs). DNA-PK phosphorylates serine residue 36 but not serine residue 32 in the amino terminus of I kappa B alpha and also phosphorylates threonine residue 273 in the carboxy terminus of this protein. To determine the biological relevance of DNA-PK phosphorylation of I kappa B alpha murine severe combined immunodeficiency (SCID) cell lines which lack the DNA-PKcs gene were analyzed. Gel retardation analysis using extract prepared from these cells demonstrated constitutive nuclear NP-kappa B DNA binding activity, which was not detected in extracts prepared from SCID cells complemented with the human DNA-PKcs gene. Furthermore, I kappa B alpha that was phosphorylated by DNA-PK was a more potent inhibitor of NF-kappa B binding than nonphosphorylated I kappa B alpha. These results suggest that DNA-PK phosphorylation of I kappa B alpha increases its interaction with NF-kappa B to reduce NF-kappa B DNA binding properties.