Association of the Ku autoantigen/DNA-dependent protein kinase holoenzyme and poly(ADP-ribose) polymerase with the DNA binding domain of progesterone receptors

J Mol Endocrinol. 2000 Apr;24(2):165-82. doi: 10.1677/jme.0.0240165.


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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Antigens, Nuclear*
  • Autoantigens / isolation & purification
  • Autoantigens / metabolism
  • Binding Sites
  • DNA Helicases*
  • DNA-Activated Protein Kinase
  • DNA-Binding Proteins / chemistry
  • DNA-Binding Proteins / isolation & purification
  • DNA-Binding Proteins / metabolism*
  • Glutathione Transferase / genetics
  • HeLa Cells
  • Humans
  • Ku Autoantigen
  • Ligands
  • Methionine / metabolism
  • Molecular Sequence Data
  • Nuclear Proteins / metabolism*
  • Phosphorylation
  • Poly(ADP-ribose) Polymerases / chemistry
  • Poly(ADP-ribose) Polymerases / isolation & purification
  • Poly(ADP-ribose) Polymerases / metabolism*
  • Protein Biosynthesis
  • Protein Serine-Threonine Kinases / chemistry
  • Protein Serine-Threonine Kinases / isolation & purification
  • Protein Serine-Threonine Kinases / metabolism*
  • Receptors, Progesterone / chemistry
  • Receptors, Progesterone / isolation & purification
  • Receptors, Progesterone / metabolism*
  • Recombinant Fusion Proteins / metabolism
  • Saccharomyces cerevisiae Proteins*
  • Transcription Factors / metabolism


  • Antigens, Nuclear
  • Autoantigens
  • DNA-Binding Proteins
  • Ligands
  • Nuclear Proteins
  • Receptors, Progesterone
  • Recombinant Fusion Proteins
  • Saccharomyces cerevisiae Proteins
  • Transcription Factors
  • high affinity DNA-binding factor, S cerevisiae
  • Methionine
  • Poly(ADP-ribose) Polymerases
  • Glutathione Transferase
  • DNA-Activated Protein Kinase
  • PRKDC protein, human
  • Protein Serine-Threonine Kinases
  • DNA Helicases
  • XRCC5 protein, human
  • Xrcc6 protein, human
  • Ku Autoantigen