Sequence-specific DNA Binding and Transcription Factor Phosphorylation by Ku Autoantigen/DNA-dependent Protein Kinase

doi:10.1074/jbc.272.9.5647

Sequence-specific DNA Binding and Transcription Factor Phosphorylation by Ku Autoantigen/DNA-dependent Protein Kinase

PHOSPHORYLATION OF Ser-527 OF THE RAT GLUCOCORTICOID RECEPTOR*

From the ^‡ Departments of Medicine and
^§Biochemistry, University of Ottawa, Loeb Medical Research Institute, Ottawa Civic Hospital, Ottawa, Ontario, Canada K1Y 4E9 and the
^∥Department of Cell Biology, Baylor College of Medicine, Houston, Texas 77030

** Scholar of the Medical Research Council of Canada and Cancer Research Society Inc. To whom correspondence should be addressed:
Loeb Medical Research Institute, Ottawa Civic Hospital, 1053 Carling Ave., Ottawa, Ontario, Canada K1Y 4E9.
Tel.: 613-798-5555 (ext. 6283); Fax: 613-761-5365; E-mail: hache{at}civich.ottawa.on.ca

Abstract

NRE1 is a DNA sequence element through which Ku antigen/DNA-dependent protein kinase (DNA-PK) catalytic subunit represses the induction of mouse mammary tumor virus transcription by glucocorticoids. Although Ku is an avid binder of DNA ends and has the ability to translocate along DNA, we report that direct sequence-specific Ku binding occurs with higher affinity (K_d = 0.84 ± 0.24 nM) than DNA end binding. Comparison of Ku binding to several sequences over which Ku can accumulate revealed two classes of sequence. Sequences with similarity to NRE1 competed efficiently for NRE1 binding. Conversely, sequences lacking similarity to NRE1 competed poorly for Ku and were not recognized in the absence of DNA ends. Phosphorylation of glucocorticoid receptor (GR) fusion proteins by DNA-PK reflected Ku DNA-binding preferences and demonstrated that co-localization of GR with DNA-PK on DNA in cis was critical for efficient phosphorylation. Phosphorylation of the GR fusion protein by DNA-PK mapped to a single site, Ser-527. This site occurs adjacent the GR nuclear localization sequence between the DNA and ligand binding domains of GR, and thus its phosphorylation, if confirmed, has the potential to affect receptor function in vivo.

Footnotes

↵^¶ Recipient of a studentship from the National Sciences and Engineering Research Council of Canada.
↵* This work was supported by an operating grant from the Medical Research Council of Canada (to R. J. G. H.). The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

DNA-PK

DNA-dependent protein kinase

DNA-PK_cs

DNA-dependent protein kinase catalytic subunit

NRE1

negative regulatory element 1

MMTV

mouse mammary tumor virus

LTR

long terminal repeat

GR

glucocorticoid receptor

Oct-1

octamer transcription factor 1

GST

glutathione S-transferase

EMSA

electrophoretic mobility shift assay

PAGE

polyacrylamide gel electrophoresis

HTLV

human T cell leukemia virus

IAP

intracisternal A particle

PRE

plasmacytoma repressor factor binding site

DBD

DNA binding domain

GRE

glucocorticoid-responsive element

PSE

proximal U1 promoter sequence element

HSE

heat shock response element

bp

base pair(s)

HPLC

high pressure liquid chromatography

Ab

antibody.
↵² W. Giffin and R. J. G. Haché, data not shown.
↵³ J. Kwast-Welfeld, Y. Zhang, N. Weigel, and R. J. G. Haché, unpublished observation.
↵⁴ W. Griffin and R. J. G. Haché, unpublished observation.
↵⁵ H. Torrance, W. Giffin, and R. J. G. Haché, manuscript in preparation.
↵⁶ H. Torrance, W. Giffin, and R. J. G. Haché, unpublished observation.
↵⁷ D. J. Rodda, W. Giffin, and R. J. G. Haché, unpublished observation.
- Received June 27, 1996.
- Revision received December 11, 1996.