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