van Laar J H, Berrevoets C A, Trapman J, Zegers N D, Brinkmann A O
Department of Biochemistry II, Medical Faculty, Erasmus University Rotterdam, The Netherlands.
J Biol Chem. 1991 Feb 25;266(6):3734-8.
Phosphorylation of the androgen receptor was investigated in the absence of hormone as well as during and after transformation of the receptor to the tight nuclear binding form. Human prostate tumor cells (LNCaP) were labeled for 4 h with [32P]orthophosphate in the presence or absence of steroid. Subsequently, androgen receptors were immunoprecipitated either from total cell lysates or from nuclear extracts using a specific monoclonal antibody. The immunoprecipitated receptor preparations were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blotting, using a polyclonal antiserum, and autoradiography. It was observed that the androgen receptor is already phosphorylated in the absence of hormone, but undergoes a hormone-induced additional phosphorylation. After administration of 10 nM R1881, a 1.8-fold increase in phosphorylation over nonstimulated control cells was reached. Moreover, the amount of nuclear extractable androgen receptor was increased; the acquisition of tight nuclear binding capacity was accompanied by hormone-induced receptor phosphorylation.
在缺乏激素的情况下以及受体转变为紧密核结合形式的过程中和之后，对雄激素受体的磷酸化进行了研究。在存在或不存在类固醇的情况下，用人前列腺肿瘤细胞（LNCaP）与[32P]正磷酸盐标记4小时。随后，使用特异性单克隆抗体从总细胞裂解物或核提取物中免疫沉淀雄激素受体。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳、使用多克隆抗血清的蛋白质印迹法和放射自显影对免疫沉淀的受体制剂进行分析。观察到雄激素受体在缺乏激素的情况下已经被磷酸化，但会经历激素诱导的额外磷酸化。给予10 nM R1881后，磷酸化水平比未刺激的对照细胞增加了1.8倍。此外，核可提取雄激素受体的量增加；紧密核结合能力的获得伴随着激素诱导的受体磷酸化。
