title: Stimulation of androgen-regulated transactivation by modulators of protein phosphorylation.
abstract: The effect of modulators of protein phosphorylation on the transcriptional activity of the androgen receptor (AR) was studied under transient expression conditions. Activators of protein kinase-A [8-bromo-cAMP (8-Br-cAMP)] and protein kinase-C (phorbol 12-myristate 13-acetate) or an inhibitor of protein phosphatase-1 and -2A (okadaic acid) influenced minimally pMMTV-chloramphenicol acetyl-transferase (CAT) activity in CV-1 cells cotransfected with an AR expression plasmid in the absence of androgen. In the presence of testosterone, however, all compounds enhanced AR-mediated transactivation by 2- to 4-fold. A nonsteroidal antiandrogen, Casodex, behaved as a pure antagonist; it blunted the action of testosterone and was not rendered agonistic by activators of protein kinase-A. A reporter plasmid containing two androgen response elements (AREs) in front of the thymidine kinase promoter (pARE2tk-CAT) was also used to examine promoter specificity. It was activated by 8-Br-cAMP, forskolin, or okadaic acid even without AR or androgen. However, when forskolin or okadaic acid was used together with androgen and AR, the resulting AR-dependent transactivation of pARE2tk-CAT was more than additive. Intact DNA- and ligand-binding domains, but not the N-terminal amino acid residues 40-147, of the receptor were mandatory for the synergism between protein kinase-A activators and androgen. Immunoreactive AR content in transfected COS-1 cells was not influenced by exposure to 8-Br-cAMP. Similar results were obtained by ligand binding assays. Quantitative or qualitative differences were not observed in DNA-binding characteristics between receptors extracted from cells treated with testosterone with or without protein kinase-A activator. Collectively, the synergistic stimulation of AR-dependent transactivation by androgen and protein kinase activators is not due to changes in cellular AR content or affinity of the receptor for the cognate DNA element; rather, this phenomenon seems to result from altered interaction of ligand-activated AR with other proteins in the transcription machinery.
authors.affiliation: Department of Physiology, University of Helsinki, Finland.
authors.forename: T
authors.initials: T
authors.lastname: Ikonen
authors.forename: J J
authors.initials: JJ
authors.lastname: Palvimo
authors.forename: P J
authors.initials: PJ
authors.lastname: Kallio
authors.forename: P
authors.initials: P
authors.lastname: Reinikainen
authors.forename: O A
authors.initials: OA
authors.lastname: Jänne
mesh_terms: D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology*; D000726:Androgen Antagonists / Q000494:pharmacology; D000728:Androgens / Q000494:pharmacology*; D000813:Anilides / Q000494:pharmacology; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase / Q000378:metabolism; D005576:Colforsin / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism / Q000502:physiology; D004247:DNA / Q000032:analysis / Q000235:genetics; D004789:Enzyme Activation / Q000187:drug effects; D004988:Ethers, Cyclic / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D009570:Nitriles; D019319:Okadaic Acid; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism / Q000502:physiology; D010766:Phosphorylation / Q000187:drug effects; D011493:Protein Kinase C / Q000378:metabolism / Q000502:physiology; D011944:Receptors, Androgen / Q000032:analysis / Q000235:genetics / Q000502:physiology; D013739:Testosterone / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D014105:Tosyl Compounds; D015533:Transcriptional Activation / Q000187:drug effects*
chemical_list: D000726:Androgen Antagonists; D000728:Androgens; D000813:Anilides; D004988:Ethers, Cyclic; D009570:Nitriles; D011944:Receptors, Androgen; D014105:Tosyl Compounds; D005576:Colforsin; D019319:Okadaic Acid; D015124:8-Bromo Cyclic Adenosine Monophosphate; D013739:Testosterone; D004247:DNA; C053541:bicalutamide; D015500:Chloramphenicol O-Acetyltransferase; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D010744:Phosphoric Monoester Hydrolases; D013755:Tetradecanoylphorbol Acetate