Sequence-specific DNA binding and transcription factor phosphorylation by Ku Autoantigen/DNA-dependent protein kinase. Phosphorylation of Ser-527 of the rat glucocorticoid receptor.
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 (Kd = 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.
Giffin
W
W
Department of Medicine, University of Ottawa, Loeb Medical Research Institute, Ottawa Civic Hospital, Ottawa, Ontario, Canada K1Y 4E9.
Kwast-Welfeld
J
J
Rodda
D J
DJ
Préfontaine
G G
GG
Traykova-Andonova
M
M
Zhang
Y
Y
Weigel
N L
NL
Lefebvre
Y A
YA
Haché
R J
RJ
D000818:Animals; D034961:Antigens, Nuclear; D001667:Binding, Competitive; D004247:DNA / Q000378:metabolism*; D004265:DNA Helicases; D004260:DNA Repair; D051747:DNA-Activated Protein Kinase; D004268:DNA-Binding Proteins / Q000378:metabolism*; D000072200:Ku Autoantigen; D051379:Mice; D009687:Nuclear Proteins / Q000378:metabolism*; D010449:Peptide Mapping; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D051381:Rats; D011965:Receptors, Glucocorticoid / Q000378:metabolism*; D012694:Serine; D013329:Structure-Activity Relationship; D014157:Transcription Factors / Q000378:metabolism*
D034961:Antigens, Nuclear; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D011965:Receptors, Glucocorticoid; D014157:Transcription Factors; D012694:Serine; D004247:DNA; D051747:DNA-Activated Protein Kinase; D017346:Protein Serine-Threonine Kinases; D004265:DNA Helicases; C106893:XRCC5 protein, human; C000606509:Xrcc6 protein, human; C000606510:Xrcc6 protein, mouse; C000606511:Xrcc6 protein, rat; D000072200:Ku Autoantigen