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(ADPribose) 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 'pulleddown' 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.

Simultaneous Suppression of Epidermal Growth Factor Receptor and c-erbB-2 Reverses Aneuploidy and Malignant Phenotype of a Human Ovarian Carcinoma Cell Line

“…3B). The significant increase in PR expression and induction of apoptosis by simultaneous inhibition of EGFR and c-erbB-2 are consistent with the results of the study showing a direct interaction of PARP with the DNA binding domain of human PR (16). This provides an insight into the cellular mechanisms of regulation of receptor tyrosine-kinase signaling pathway.…”

CDK2-dependent activation of PARP-1 is required for hormonal gene regulation in breast cancer cells

“…PARP-1 activation is the result of phosphorylation by the hormoneactivated cyclin-dependent kinase CDK2 at two consecutive serine residues, leading to enhanced PARylation capabilities. As PARP-1 is required for ligand-induced transcriptional activation following 17b estradiol and retinoic acid treatment (Parvi et al 2005;) and interacts with progesterone receptor (PR) (Sartorius et al 2000), we demonstrate the mechanism of PARP-1 activation in breast cancer cells. PARP-1 activation contributes to displacement of histone H1, an essential step for regulation of the majority of hormone-responsive genes and for the effect of progestins on cell cycle progression.…”

The Ku Protein Complex Interacts with YY1, Is Up-Regulated in Human Heart Failure, and Represses α Myosin Heavy-Chain Gene Expression

“…Ku70 interacts with YY1 in vitro. Ku has been shown to interact with various transcription factors (15,30,34,35,45), and we hypothesized that one or more of its interactions might facilitate the interaction of Ku with the ␣MyHC promoter. Recently, Ku has been shown to be important for the transcription reinitiation process (46) and to bind directly to RNA polymerase II (42).…”

“…In addition to its repressive effect on the ␣MyHC promoter, YY1 has also been shown to interact with the basic transcription machinery (41) and, interestingly, recent reports have implicated YY1 as being involved in DNA repair through interaction with poly(ADP-ribose) (PARP) (26,27). Since Ku is part of the DNA repair complex and also interacts with PARP (29,30), we tested the hypothesis that Ku and YY1 interact by performing coimmunoprecipitation experiments in HeLa cells. As seen in Fig.…”