RESUMEN
The biological ramifications of phosphorylation of the androgen receptor (AR) are largely unknown. To examine the phosphorylation of AR at serine 213, a putative substrate for Akt, a phosphorylation site-specific antibody was generated. The use of this antibody indicated that AR Ser-213 is phosphorylated in vivo and that phosphorylation is tightly regulated in a cell type-specific manner. Furthermore, Ser-213 phosphorylation took place with rapid kinetics and was inhibited by the phosphatidylinositol 3-kinase inhibitor LY294002. Phosphorylation occurred in response to R1881 and dihydrotestosterone but weakly if at all in response to testosterone. It did not occur in response to AR antagonists or growth factor stimulation in the absence of an AR agonist. Transcription assays using an AR-responsive reporter gene construct showed that activated phosphatidylinositol 3-kinase inhibited transcription mediated by wild type AR but not that of a mutant AR variant (S213A), which could not be phosphorylated at Ser-213. By immunohistochemistry, the AR Ser(P)-213 antigen was detected in prostate epithelial but not stromal cells despite the fact that an antibody recognizing both phosphorylated and non-phosphorylated forms of AR demonstrates that AR is present in both cell types as expected. In fetal tissue the AR-Ser(P)-213 antigen was present in epithelial cells of the urogenital sinus when endogenous androgen levels were high and activated Akt was prevalent, but absent at a later stage of development when endogenous androgen levels were low and Akt activation was minimal. Immunoreactivity was evident in differentiated cells lining the lumen of the urogenital sinus but not in rapidly dividing, Ki67 positive cells within the developing prostate or stromal tissue, suggesting that site-specific phosphorylation of AR Ser-213 by cellular kinases occurs in a non-proliferating cellular milieu.
Asunto(s)
Homeostasis , Próstata/citología , Proteínas Quinasas/metabolismo , Receptores Androgénicos/metabolismo , Secuencia de Aminoácidos , Especificidad de Anticuerpos , División Celular , Línea Celular , Cromonas/farmacología , Dihidrotestosterona/farmacología , Embrión de Mamíferos , Factor de Crecimiento Epidérmico/farmacología , Células Epiteliales/química , Feto/química , Técnica del Anticuerpo Fluorescente , Humanos , Immunoblotting , Inmunohistoquímica , Factor I del Crecimiento Similar a la Insulina/farmacología , Antígeno Ki-67/análisis , Riñón , Masculino , Metribolona/farmacología , Datos de Secuencia Molecular , Morfolinas/farmacología , Mutación , Fragmentos de Péptidos/química , Fragmentos de Péptidos/inmunología , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Fosforilación , Próstata/química , Próstata/embriología , Receptores Androgénicos/química , Receptores Androgénicos/genética , Serina/metabolismo , Células del Estroma/química , Relación Estructura-Actividad , Transcripción Genética , TransfecciónRESUMEN
Androgen receptor trapped clone-27 (ART-27) is a newly described transcriptional coactivator that binds to the N terminus of the androgen receptor (AR). Given the vital importance of AR signaling in prostate growth and differentiation, we investigated the role of ART-27 in these processes. Immunohistochemical studies indicate that ART-27 protein is expressed in differentiated epithelial cells of adult human prostate and breast tissue. In prostate, ART-27 is abundant in AR-positive prostate luminal epithelial cells, in contrast to the stroma, where cells express AR but not ART-27. The use of a rat model of androgen depletion/reconstitution indicates that ART-27 expression is associated with the elaboration of differentiated prostate epithelial cells. Interestingly, regulated expression of ART-27 in the androgen-sensitive LNCaP prostate cancer cell line inhibits androgen-mediated cellular proliferation and enhances androgen-mediated transcription of the prostate-specific antigen (PSA) gene. Consistent with a growth suppressive function, we show that ART-27 expression levels are negligible in human prostate cancer. Importantly, examination of ART-27 protein expression in early fetal prostate development demonstrates that ART-27 is detected only when the developing prostate gland has proceeded from a solid mass of undifferentiated cells to a stage in which differentiated luminal epithelial cells are evident. Thus, ART-27 is an AR cofactor shown to be subject to both cell type and developmental regulation in humans. Overall, the results suggest that decreased levels of ART-27 protein in prostate cancer tissue may occur as a result of de-differentiation, and indicate that ART-27 is likely to regulate a subset of AR-responsive genes important to prostate growth suppression and differentiation.