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

Ligand-activated progesterone receptors (PR) bind to DNA at specific progesterone response elements by means of a DNA binding domain (DBDPR) 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 DBDPR 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-PKCS. DNA-PK that was 'pulled-down' by DBDPR on the affinity resin was able to (1) autophosphorylate Ku70, Ku86, and DNA-PKCS, (2) transphosphorylate DBDPR, 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 (DBDCYS) nor the core DBD of the estrogen receptor (DBDER) 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 DBDPR interact, that DBDPR is a phosphorylation substrate of DNA-PK, and suggest a potential role for DNA-PK in PR-mediated transcription.