Gordon Vicki, Bhadel Shriti, Wunderlich Winfried, Zhang JoAnn, Ficarro Scott B, Mollah Sahana A, Shabanowitz Jeffrey, Hunt Donald F, Xenarios Ioannis, Hahn William C, Conaway Mark, Carey Michael F, Gioeli Daniel
University of Virginia, Department of Microbiology, Charlottesville, Virginia 22908, USA.
Mol Endocrinol. 2010 Dec;24(12):2267-80. doi: 10.1210/me.2010-0238. Epub 2010 Oct 27.
Previously we determined that S81 is the highest stoichiometric phosphorylation on the androgen receptor (AR) in response to hormone. To explore the role of this phosphorylation on growth, we stably expressed wild-type and S81A mutant AR in LHS and LAPC4 cells. The cells with increased wild-type AR expression grow faster compared with parental cells and S81A mutant-expressing cells, indicating that loss of S81 phosphorylation limits cell growth. To explore how S81 regulates cell growth, we tested whether S81 phosphorylation regulates AR transcriptional activity. LHS cells stably expressing wild-type and S81A mutant AR showed differences in the regulation of endogenous AR target genes, suggesting that S81 phosphorylation regulates promoter selectivity. We next sought to identify the S81 kinase using ion trap mass spectrometry to analyze AR-associated proteins in immunoprecipitates from cells. We observed cyclin-dependent kinase (CDK)9 association with the AR. CDK9 phosphorylates the AR on S81 in vitro. Phosphorylation is specific to S81 because CDK9 did not phosphorylate the AR on other serine phosphorylation sites. Overexpression of CDK9 with its cognate cyclin, Cyclin T, increased S81 phosphorylation levels in cells. Small interfering RNA knockdown of CDK9 protein levels decreased hormone-induced S81 phosphorylation. Additionally, treatment of LNCaP cells with the CDK9 inhibitors, 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole and Flavopiridol, reduced S81 phosphorylation further, suggesting that CDK9 regulates S81 phosphorylation. Pharmacological inhibition of CDK9 also resulted in decreased AR transcription in LNCaP cells. Collectively these results suggest that CDK9 phosphorylation of AR S81 is an important step in regulating AR transcriptional activity and prostate cancer cell growth.
此前我们确定S81是雄激素受体（AR）对激素应答时化学计量比最高的磷酸化位点。为探究这种磷酸化在生长过程中的作用，我们在LHS和LAPC4细胞中稳定表达野生型和S81A突变型AR。与亲代细胞和表达S81A突变体的细胞相比，野生型AR表达增加的细胞生长更快，这表明S81磷酸化的缺失会限制细胞生长。为探究S81如何调节细胞生长，我们测试了S81磷酸化是否调节AR转录活性。稳定表达野生型和S81A突变型AR的LHS细胞在内源性AR靶基因的调控上表现出差异，这表明S81磷酸化调节启动子选择性。接下来，我们试图通过离子阱质谱法鉴定S81激酶，以分析来自细胞免疫沉淀中的AR相关蛋白。我们观察到细胞周期蛋白依赖性激酶（CDK）9与AR相关联。CDK9在体外使AR的S81位点发生磷酸化。磷酸化对S81具有特异性，因为CDK9不会使AR的其他丝氨酸磷酸化位点发生磷酸化。CDK9与其同源细胞周期蛋白Cyclin T共表达会增加细胞中S81的磷酸化水平。用小干扰RNA敲低CDK9蛋白水平会降低激素诱导的S81磷酸化。此外，用CDK9抑制剂5,6 - 二氯 - 1 - β - D - 呋喃核糖基苯并咪唑和黄酮哌啶醇处理LNCaP细胞，会进一步降低S81磷酸化，这表明CDK9调节S81磷酸化。对CDK9的药理抑制也导致LNCaP细胞中AR转录减少。这些结果共同表明，AR的S81位点被CDK9磷酸化是调节AR转录活性和前列腺癌细胞生长的重要步骤。
