Ku is a novel transcriptional recycling coactivator of the androgen receptor in prostate cancer cells.
The androgen receptor (AR) dynamically assembles and disassembles multicomponent receptor complexes in order to respond rapidly and reversibly to fluctuations in androgen levels. We are interested in identifying the basal factors that compose the AR aporeceptor and holoreceptor complexes and impact the transcriptional process. Using tandem mass spectroscopy analysis, we identified the trimeric DNA-dependent protein kinase (DNA-PK) complex as the major AR-interacting proteins. AR directly interacts with both Ku70 and Ku80 in vivo and in vitro, as shown by co-immunoprecipitation, glutathione S-transferase pull-down, and Sf9 cell/baculovirus expression. The interaction was localized to the androgen receptor ligand binding domain and is independent of DNA interactions. Ku interacts with AR in the cytoplasm and nucleus regardless of the presence or absence of androgen. Ku acts as a coactivator of AR activity in a luciferase reporter assay employing both Ku-defective cells and Ku small interfering RNA knock-down in a prostate cancer cell line. DNA-PK catalytic subunit (DNA-PKcs) also acts as a coactivator of androgen receptor activity in a luciferase reporter assay employing DNA-PKcs defective cells. AR nuclear translocation is not affected in Ku defective cells, implying Ku functionality may be mainly nuclear. Chromatin immunoprecipitation experiments demonstrated that both Ku70 and Ku80 interact with the prostate-specific antigen promoter in an androgen-dependant manner. Finally, in vitro transcription assays demonstrated Ku involvement in transcriptional recycling with androgen dependent promoters.
Mayeur
Greg L
GL
Department of Biological Chemistry, School of Medicine, University of California, Davis, UC Davis Cancer Center, Sacramento, California 95817, USA.
Kung
Wei-Jen
WJ
Martinez
Anthony
A
Izumiya
Chie
C
Chen
David J
DJ
Kung
Hsing-Jien
HJ
D000728:Androgens / Q000378:metabolism; D000818:Animals; D034961:Antigens, Nuclear / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D002460:Cell Line; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism; D002843:Chromatin / Q000378:metabolism; D047369:Chromatin Immunoprecipitation; D003593:Cytoplasm / Q000378:metabolism; D004247:DNA / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D004591:Electrophoresis, Polyacrylamide Gel; D017930:Genes, Reporter; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D047468:Immunoprecipitation; D007313:Insecta; D000072200:Ku Autoantigen; D008024:Ligands; D008156:Luciferases / Q000378:metabolism; D008297:Male; D013058:Mass Spectrometry; D008957:Models, Genetic; D011471:Prostatic Neoplasms / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012313:RNA / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism; D011944:Receptors, Androgen / Q000378:metabolism*; D015398:Signal Transduction; D014158:Transcription, Genetic
D000728:Androgens; D034961:Antigens, Nuclear; D002843:Chromatin; D004268:DNA-Binding Proteins; D008024:Ligands; D034741:RNA, Small Interfering; D011944:Receptors, Androgen; D012313:RNA; D004247:DNA; D008156:Luciferases; D005982:Glutathione Transferase; C000606509:Xrcc6 protein, human; D000072200:Ku Autoantigen