title: Association of the Ku autoantigen/DNA-dependent protein kinase holoenzyme and poly(ADP-ribose) polymerase with the DNA binding domain of progesterone receptors.
abstract: Ligand-activated progesterone receptors (PR) bind to DNA at specific progesterone response elements by means of a DNA binding domain (DBD(PR)) containing two highly conserved zinc fingers. DNA-bound PRs regulate transcription via interaction with other nuclear proteins and transcription factors. We have now identified four HeLa cell nuclear proteins that copurify with a glutathionine-S-transferase-human DBD(PR )fusion protein. Microsequence and immunoblot analyses identified one of these proteins as the 113 kDa poly(ADP-ribose) polymerase. The three other proteins were identified as subunits of the DNA-dependent protein kinase (DNA-PK) holoenzyme: its DNA binding regulatory heterodimers consisting of Ku70 and Ku86, and the 460 kDa catalytic subunit, DNA-PK(CS). DNA-PK that was 'pulled-down' by DBD(PR) on the affinity resin was able to (1) autophosphorylate Ku70, Ku86, and DNA-PK(CS), (2) transphosphorylate DBD(PR), and (3) phosphorylate a DNA-PK-specific p53 peptide substrate. DNA-PK was also able to associate with the DBD of the yeast activator GAL4. However, neither a PR DBD mutant lacking a structured first zinc finger (DBD(CYS)) nor the core DBD of the estrogen receptor (DBD(ER)) copurified DNA-PK, suggesting the interaction is not non-specific for DBDs. Lastly, we found that DNA-PK copurified with full-length human PR transiently expressed in HeLa cells, suggesting that the human PR/DNA-PK complex can assemble in vivo. These data show that DNA-PK and DBD(PR) interact, that DBD(PR) is a phosphorylation substrate of DNA-PK, and suggest a potential role for DNA-PK in PR-mediated transcription.
authors.affiliation: Department of Medicine, University of Colorado Health Sciences Center, Denver, Colorado 80262, USA. Carol.Sartorius@uchsc.edu
authors.forename: C A
authors.initials: CA
authors.lastname: Sartorius
authors.forename: G S
authors.initials: GS
authors.lastname: Takimoto
authors.forename: J K
authors.initials: JK
authors.lastname: Richer
authors.forename: L
authors.initials: L
authors.lastname: Tung
authors.forename: K B
authors.initials: KB
authors.lastname: Horwitz
mesh_terms: D000595:Amino Acid Sequence; D034961:Antigens, Nuclear; D001324:Autoantigens / Q000302:isolation & purification / Q000378:metabolism; D001665:Binding Sites; D004265:DNA Helicases; D051747:DNA-Activated Protein Kinase; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D005982:Glutathione Transferase / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D000072200:Ku Autoantigen; D008024:Ligands; D008715:Methionine / Q000378:metabolism; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism*; D010766:Phosphorylation; D011065:Poly(ADP-ribose) Polymerases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D014176:Protein Biosynthesis; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D011980:Receptors, Progesterone / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors / Q000378:metabolism
chemical_list: D034961:Antigens, Nuclear; D001324:Autoantigens; D004268:DNA-Binding Proteins; D008024:Ligands; D009687:Nuclear Proteins; D011980:Receptors, Progesterone; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; C081647:high affinity DNA-binding factor, S cerevisiae; D008715:Methionine; D011065:Poly(ADP-ribose) Polymerases; D005982:Glutathione Transferase; D051747:DNA-Activated Protein Kinase; C497263:PRKDC protein, human; D017346:Protein Serine-Threonine Kinases; D004265:DNA Helicases; C106893:XRCC5 protein, human; C000606509:Xrcc6 protein, human; D000072200:Ku Autoantigen