DNA-Dependent Protein Kinase Phosphorylation of IκBα and IκBβ Regulates NF-κB DNA Binding Properties

ABSTRACT

Regulation of the IκBα and IκBβ proteins is critical for modulating NF-κB-directed gene expression. Both IκBα and IκBβ 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 IκBα and IκBβ. 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 IκBα and also phosphorylates threonine residue 273 in the carboxy terminus of this protein. To determine the biological relevance of DNA-PK phosphorylation of IκBα, 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 NF-κB DNA binding activity, which was not detected in extracts prepared from SCID cells complemented with the human DNA-PKcs gene. Furthermore, IκBα that was phosphorylated by DNA-PK was a more potent inhibitor of NF-κB binding than nonphosphorylated IκBα. These results suggest that DNA-PK phosphorylation of IκBα increases its interaction with NF-κB to reduce NF-κB DNA binding properties.

ACKNOWLEDGMENTS

We thank Sharon Johnson for the preparation of the manuscript; Shane Fults for the preparation of the figures; and R. Robinson, and K. Pierce for expertise in the digestion and mass spectrometry. We thank Dean Ballard for providing the IκBα and IκBβ cDNAs and Cordula Kirchgessner for providing SCID and DNA-PKcs-complemented SCID cell lines.

This work was supported by grants from the NIH, the Veterans Administration, and the Robert Welch Foundation.

Li Liu and Youn-Tae Kwak contributed equally to this work.