Association of the Ku autoantigen/DNA-dependent protein kinase holoenzyme and poly(ADP-ribose) polymerase with the DNA binding domain of progesteronereceptors
Ca. Sartorius et al., Association of the Ku autoantigen/DNA-dependent protein kinase holoenzyme and poly(ADP-ribose) polymerase with the DNA binding domain of progesteronereceptors, J MOL ENDOC, 24(2), 2000, pp. 165-182
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.