Blok L J, de Ruiter P E, Brinkmann A O
Department of Endocrinology & Reproduction, Faculty of Medicine and Health Sciences, Erasmus University Rotterdam, The Netherlands.
Biochemistry. 1998 Mar 17;37(11):3850-7. doi: 10.1021/bi9724422.
When androgen receptor containing cells are cultured in the presence of the PKA stimulator forskolin, a rapid dephosphorylation of the androgen receptor occurs resulting in a decrease in the amount of 112 kDa androgen receptor isoform and an increase in 110 kDa androgen receptor isoform on SDS-PAGE. To establish which amino acid residues in the androgen receptor were phosphorylated in control and forskolin-treated cells, trypsin-digested androgen receptors were subjected to RP-HPLC analysis and subsequently to Edman degradation. It was observed that serine residues 506, 641, and 653 were potentially phosphorylated in control cells, while after forskolin treatment strong evidence was obtained that phosphorylation of serines 641 and 653 was significantly reduced. When the dephosphorylated androgen receptor was analyzed for its transcription activation capacity, it was observed that androgen-induced transcriptional regulation of two endogenous genes (PSA) and beta 1-subunit of Na,K-ATPase), in cells cultured in the presence of forskolin, was inhibited as compared to the control situation. The observation that the dephosphorylated androgen receptor was transcriptionally less active was further strengthened by the finding that the dephosphorylated androgen receptor was markedly impaired in ligand binding (Bmax was found to be reduced by approximately 40%). The current investigations show for the first time a clear function for the rapid phosphorylation which occurs directly after synthesis of the androgen receptor, namely, effective ligand binding.
当含有雄激素受体的细胞在蛋白激酶A（PKA）刺激剂福斯高林存在的情况下进行培养时，雄激素受体发生快速去磷酸化，导致在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）上112 kDa雄激素受体异构体的量减少，而110 kDa雄激素受体异构体的量增加。为了确定在对照细胞和经福斯高林处理的细胞中雄激素受体的哪些氨基酸残基被磷酸化，对经胰蛋白酶消化的雄激素受体进行反相高效液相色谱（RP-HPLC）分析，随后进行埃德曼降解。观察到丝氨酸残基506、641和653在对照细胞中可能被磷酸化，而在福斯高林处理后，有强有力的证据表明丝氨酸641和653的磷酸化显著减少。当分析去磷酸化的雄激素受体的转录激活能力时，观察到与对照情况相比，在福斯高林存在的情况下培养的细胞中，雄激素诱导的两个内源性基因（前列腺特异性抗原（PSA）和钠钾-ATP酶β1亚基）的转录调控受到抑制。去磷酸化的雄激素受体转录活性较低这一观察结果，因去磷酸化的雄激素受体在配体结合方面明显受损（发现最大结合容量（Bmax）降低约40%）这一发现而进一步得到加强。目前的研究首次表明了雄激素受体合成后直接发生的快速磷酸化的明确功能，即有效的配体结合。
