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目的:明确信号传导与转录激活因子3(signal transducer and activator of transcription 3,STAT3)中Y705与S727在磷酸化过程中的相互作用。方法:通过构建STAT3 Y705F和S727A两个磷酸化位点突变的质粒,将其分别转染至TSC2-/-MEF细胞中,蛋白质印迹法检测STAT3 Y705F对STAT3 S727磷酸化水平和STAT3 S727A对STAT3 Y705磷酸化水平的影响。结果:基因测序结果显示编码丝氨酸的TCC突变为GCC,编码酪氨酸的TAC突变为TTC;蛋白质印迹检测结果显示转染STAT3 Y705F组中STAT3 Y705的磷酸化水平显著低于转染HA-STAT3组,且STAT3 Y705F下调STAT3 S727磷酸化水平;转染STAT3 S727A组中STAT3 S727的磷酸化水平显著低于转染HA-STAT3组,且STAT3 S727A下调Y705磷酸化水平。结论:STAT3中Y705与S727两个磷酸化位点具有协同作用。
Objective: To clarify the interaction between Y705 and S727 in phosphorylation of signal transducers and activator of transcription 3 (STAT3). Methods: Plasmids containing two phosphorylation sites, STAT3 Y705F and S727A, were constructed and transfected into TSC2 - / - MEF cells respectively. Western blotting was used to detect the phosphorylation of STAT3 S727 and the effect of STAT3 S727A on STAT3 Y705 Effect of phosphorylation level. Results: The results of sequencing showed that the TCC mutation of serine was GCC, and the TAC mutation of tyrosine was TTC. Western blot results showed that the phosphorylation of STAT3 Y705 in STAT3 Y705F group was significantly lower than that in HA-STAT3 transfected group , And STAT3 Y705F downregulated the phosphorylation level of STAT3 S727. The phosphorylation level of STAT3 S727 in STAT3 S727A group was significantly lower than that of the transfected HA-STAT3 group, and STAT3 S727A down-regulated the phosphorylation level of Y705. Conclusion: There are synergistic effects between Y705 and S727 phosphorylation sites in STAT3.