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Mol Cell Biol, July 1998, p. 4221-4234, Vol. 18, No. 7
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

DNA-Dependent Protein Kinase Phosphorylation of Ikappa Balpha and Ikappa Bbeta Regulates NF-kappa B DNA Binding Properties

Li Liu,1 Youn-Tae Kwak,1 Françoise Bex,1 León F. García-Martínez,1 Xiao-Hua Li,1 Katheryn Meek,2 William S. Lane,3 and Richard B. Gaynor1 *

Divisions of Molecular Virology and Hematology-Oncology1 and Departments of Medicine and Microbiology,2 University of Texas Southwestern Medical Center, Dallas, Texas 75235, and Harvard Microchemistry Facility, Cambridge, Massachusetts 021383

Received 27 October 1997/Returned for modification 18 December 1997/Accepted 28 April 1998

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

* Corresponding author. Mailing address: Division of Hematology-Oncology, Department of Internal Medicine, University of Texas Southwestern Medical School, 5323 Harry Hines Blvd., Dallas, Texas 75235-8594. Phone: (214) 648-7570. Fax: (214) 648-8862. E-mail: Gaynor{at}UTSW.SWMed.edu.

Mol Cell Biol, July 1998, p. 4221-4234, Vol. 18, No. 7
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


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