Isolation and identification of L-dopa decarboxylase as a protein that binds to and enhances transcriptional activity of the androgen receptor using the repressed transactivator yeast two-hybrid system.
The AR (androgen receptor) is a ligand-regulated transcription factor, which belongs to the steroid receptor family and plays an essential role in growth and development of the prostate. Transcriptional activity of steroid receptors is modulated by interaction with co-regulator proteins and yeast two-hybrid analysis is commonly used to identify these steroid receptor-interacting proteins. However, a limitation of conventional two-hybrid systems for detecting AR protein partners has been that they only allow for analysis of the ligand- and DNA-binding domains of the receptor, as its NTD (N-terminal domain) possesses intrinsic transactivation activity. To identify AR N-terminus-interacting proteins, its NTD was used in the RTA (repressed transactivator) system, which is specifically designed for transactivator bait proteins and was shown to be suitable for two-hybrid analysis with the AR NTD. DDC (L-dopa decarboxylase) was detected multiple times as a novel AR-interacting protein, which was subsequently confirmed in vitro and in vivo. Furthermore, transient transfection of DDC in prostate cancer cells strongly enhanced ligand-dependent AR transcriptional activity, an effect that was antagonized using high concentrations of the anti-androgen bicalutamide. Glucocorticoid receptor activity was also strongly enhanced with DDC co-transfection, while oestrogen receptor activity was only mildly affected. Together, our data demonstrate that DDC interacts with AR to enhance steroid receptor transactivation, which may have important implications in prostate cancer progression.
Wafa
Latif A
LA
Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, BC, V6T 2B5, Canada.
Cheng
Helen
H
Rao
Mira A
MA
Nelson
Colleen C
CC
Cox
Michael
M
Hirst
Martin
M
Sadowski
Ivan
I
Rennie
Paul S
PS
D000595:Amino Acid Sequence; D000813:Anilides / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D015153:Blotting, Western; D045744:Cell Line, Tumor; D004296:Dopa Decarboxylase / Q000235:genetics / Q000378:metabolism*; D047628:Estrogen Receptor alpha; D015971:Gene Expression Regulation, Enzymologic; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008297:Male; D008969:Molecular Sequence Data; D009570:Nitriles; D011485:Protein Binding; D051381:Rats; D011944:Receptors, Androgen / Q000235:genetics / Q000378:metabolism*; D011960:Receptors, Estrogen / Q000235:genetics / Q000378:metabolism; D011965:Receptors, Glucocorticoid / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D014105:Tosyl Compounds; D015533:Transcriptional Activation / Q000187:drug effects; D020798:Two-Hybrid System Techniques
D000813:Anilides; D047628:Estrogen Receptor alpha; D009570:Nitriles; D011944:Receptors, Androgen; D011960:Receptors, Estrogen; D011965:Receptors, Glucocorticoid; D011993:Recombinant Fusion Proteins; D014105:Tosyl Compounds; C053541:bicalutamide; D008156:Luciferases; D004296:Dopa Decarboxylase