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

Ligand-activated progesterone receptors (PR) bind to DNA at specific proges terone response elements by means of a DNA binding domain (DBDPR) containin g two highly conserved zinc fingers. DNA-bound PRs regulate transcription v ia interaction with other nuclear proteins and transcription factors. We ha ve now identified four HeLa cell nuclear proteins that copurify with a glut athionine-S-transferase-human DBDPR fusion protein. Microsequence and immun oblot analyses identified one of these proteins as the 113 kDa poly(ADP-rib ose) polymerase. The three other proteins were identified as subunits of th e DNA-dependent protein kinase (DNA-PK) holoenzyme: its DNA binding regulat ory heterodimers consisting of Ku70 and Ku86, and the 460 kDa catalytic sub unit, DNA-PKCS. DNA-PK that was 'pulled-down' by DBDPR on the affinity resi n was able to (1) autophosphorylate Ku70, Ku86, and DNA-PKCS, (2) transphos phorylate DBDPR, and (3) phosphorylate a DNA-PK-specific p53 peptide substr ate. 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 fing er (DBDCYS) nor the core DBD of the estrogen receptor (DBDER) copurified DN A-PK, suggesting the interaction is not non-specific for DBDs. Lastly, we f ound 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 phos phorylation substrate of DNA-PK, and suggest a potential role for DNA-PK in PR-mediated transcription.