Qian Wenliang, Gang Xiaoxu, Zhang Tianlei, Wei Ling, Yang Xinxin, Li Zheng, Yang Yan, Song Liang, Wang Peng, Peng Jian, Cheng Daojun, Xia Qingyou
State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing 400715, China.
School of Life Science, Southwest University, Chongqing 400715, China.
J Biol Chem. 2017 Jul 28;292(30):12460-12470. doi: 10.1074/jbc.M117.775130. Epub 2017 Jun 5.
The insect-specific transcription factor Broad-Complex (BR-C) is transcriptionally activated by the steroid 20-hydroxyecdysone (20E) and regulates the expression of many target genes involved in insect growth and development. However, although the transcriptional regulation of BR-C proteins has been well studied, how BR-C is regulated at post-transcription and -translation levels is poorly understood. To this end, using liquid chromatography-tandem mass spectrometry analysis, we identified residue Ser-186 as a phosphorylation site of BR-C in silkworm. Site-directed mutagenesis and treatment with specific kinase activators and inhibitors indicated that the Ser-186 residue in silkworm BR-C is phosphorylated by protein kinase A (PKA). Immunostaining assays disclosed that PKA-mediated phosphorylation of silkworm BR-C has no effect on its nuclear import. However, luciferase reporter analysis, electrophoretic mobility shift assays, and chromatin immunoprecipitation revealed that the PKA phosphorylation event suppresses the transcriptional activation of silkworm BR-C target genes and that this inhibition was caused by repression of BR-C binding to its DNA targets. Of note, both and experiments disclosed that a continuous 20E signal inhibits the PKA-mediated BR-C phosphorylation and also the cAMP/PKA pathway, indicating that 20E's inhibitory effect on PKA-mediated phosphorylation of silkworm BR-C contributes to maintaining BR-C transcriptional activity. In conclusion, our findings indicate that PKA-mediated phosphorylation inhibits silkworm BR-C activity by interfering with its binding to DNA and that 20E signaling relieves PKA-mediated phosphorylation of BR-C, thereby maintaining its transcriptional activity.
昆虫特异性转录因子Broad-Complex（BR-C）由类固醇20-羟基蜕皮激素（20E）转录激活，并调节许多参与昆虫生长和发育的靶基因的表达。然而，尽管对BR-C蛋白的转录调控已有深入研究，但对其在转录后和翻译后水平的调控却知之甚少。为此，我们利用液相色谱-串联质谱分析，在蚕中鉴定出BR-C的第186位丝氨酸残基为磷酸化位点。定点诱变以及使用特异性激酶激活剂和抑制剂进行处理的结果表明，家蚕BR-C中的第186位丝氨酸残基被蛋白激酶A（PKA）磷酸化。免疫染色分析表明，PKA介导的家蚕BR-C磷酸化对其核输入没有影响。然而，荧光素酶报告基因分析、电泳迁移率变动分析和染色质免疫沉淀结果显示，PKA磷酸化事件抑制了家蚕BR-C靶基因的转录激活，且这种抑制是由于BR-C与其DNA靶标的结合受到抑制所致。值得注意的是，实验均表明持续的20E信号会抑制PKA介导的BR-C磷酸化以及cAMP/PKA途径，这表明20E对PKA介导的家蚕BR-C磷酸化的抑制作用有助于维持BR-C的转录活性。总之，我们的研究结果表明，PKA介导的磷酸化通过干扰BR-C与DNA的结合来抑制家蚕BR-C的活性，而20E信号传导可减轻PKA介导的BR-C磷酸化，从而维持其转录活性。
