Proteomic identification of 14-3-3 sigma as a common component of the androgen receptor and the epidermal growth factor receptor signaling pathways of the human prostate epithelial cell line M12.

Abstract

The epidermal growth factor receptor and androgen receptor (AR) both play major roles in the control of prostate growth. Our hypothesis is that shared downstream components of these two signaling pathways are significant participants in androgen-independent growth. Our first objective was to identify proteins whose activation and/or expression in AR-positive prostate epithelial cells are induced by both epidermal growth factor (EGF) and dihydrotestosterone (DHT). AR expression was induced in a tumorigenic, metastatic subline of the SV40 large T-antigen immortalized human prostate epithelial subline M12 by stable transfection with human wild-type AR cDNA. These M12AR (+) cells with functional AR were treated in parallel with EGF (10 ng/ml) or DHT (10(-8) M) for 24 h before 2D gel electrophoresis and Western immunoblotting with antiphosphotyrosine monoclonal antibody. Coomassie blue-stained spots on a 2D gel run in parallel were aligned with the phosphoproteins on the Western immunoblot, and identified by matrix-assisted laser desorption ionization/time-of-flight mass spectroscopy. The most interesting of the seven proteins that appeared to be phosphorylated by these criteria was 14-3-3 protein sigma. Protein extracted after either EGF or DHT treatment, immunoprecipitated with antiphosphotyrosine monoclonal antibody, and immunoblotted by anti-14-3-3 sigma confirmed phosphorylation of 14-3-3 sigma. Addition of either DHT or EGF to the M12AR(+) cells induced subcellular migration of 14-3-3 sigma and activated a 14-3-3 sigma reporter construct. Immunohistochemical analysis revealed nuclear localization of 14-3-3 sigma in higher Gleason grade prostate cancers relative to benign glands. These findings implicate 14-3-3 sigma in the development of human prostate cancer cells and could provide a new target for intervention in prostate cancer.