MST1的酵母双杂交及与Salvador体外结合的研究

利用酵母双杂交系统,以鼠M ST 1(M amm alian STE 20-1 ike 1)为诱饵蛋白,在鼠胚胎库中筛选到22个Salvador的片段。通过E.coli表达系统纯化了6个组氨酸(6H is)融合的M ST 1和谷胱甘肽硫转移酶(GST)融合的Salvador,并做体外蛋白质结合实验,进一步证实了两蛋白质相互结合。通过体外激酶活性分析,发现M ST 1并不直接磷酸化Salvador,但是Salvador能够较强地抑制M ST 1对M BP(磷酸丁酯)的磷酸化,提示有可能Salvador通过影响M ST 1的激酶活性来参与了M ST 1介导的细胞凋亡途径。

Yeast Two Hybrid of MST1 and Its Association with Salvador

Mammalian STE20-like 1 is a Ser/Thr kinase,which can be activated by caspase 3,leading to DNA fragmentation,membrane blebbing and resulting in chromatin condensation by phosphoralating H2B.Salvador can lead to cell apoptosis and cell cycle exit in Drosophila system,but the function of apoptosis and its signal pathway are not found in mammalian cells.In this work,22 fragments of(Salvador) were obtained from mouse embryo library using MST1 as a bait in yeast two-hybrid system.By (using) the purified 6His-MST1 and GST-Salvador expressed by E.coli,it was further demonstrated that these two proteins can associate with each other in vitro.In addition,MST1 cannot phosphorylate Salvador in vitro,but Salvador can strongly inhibit the phosphorylation of monobuthlphosphate(MBP) catalysed by MST1,suggesting Salvador is likely to participate MST1 induced apoptosis process by influencing MST1 kinase activity.