重组GST-Crosstide蛋白的表达纯化及其在AGC型蛋白激酶活性检测中的应用

Expression and purification of GST-Crosstide and its application in activity analysis of AGC protein kinases

AGC型蛋白激酶在信号通路中占据着极为重要的地位,通过对各种信号通路的调控来影响细胞的生长、增殖和凋亡进程.为方便检测AGC型蛋白激酶的活性,设计并构建了新型AGC型蛋白激酶特异底物GST-Crosstide.通过同位素标记体外磷酸化的方法研究了酵母表达的Pkh1、Ypk1和大肠杆菌表达的Akt1三种AGC型蛋白激酶磷酸化GSTCrosstide的能力.结果显示,酵母表达的有活性的Pkh1和Ypk1均可有效磷酸化GST-Crosstide,并且由于Pkh1对Ypk1的进一步激活,二者共同作用可显著增加GST-Crosstide磷酸化.而仅采用大肠杆菌表达的没有被活化的Akt1则无法磷酸化GST-Crosstide,但Akt1可以被Pkh1或Ypk1激活,从而获得磷酸化GST-Crosstide的能力.研究表明,GST-Crosstide可以作为AGC蛋白激酶活性检测的有效工具,并将有效促进真核生物蛋白激酶调控的细胞信号转导通路的研究.

AGC protein kinases play important roles in cellular signal pathways which affect cell growth, proliferation and apoptosis. The purpose of this study was to advance the researches towards elucidating signal pathways regulated by AGC kinases by providing a new protein substrate. We designed and constructed GST-Crosstide, a new specific protein substrate of AGC kinases. Radiolabeled phosphorylation assays were performed in vitro using AGC kinases including Pkhl, Ypkl and Aktl to phosphorylate GST-Crosstide. The results demonstrated that GST-Crosstide was phosphorylated effectively by active Pkhl and Ypk1 which were expressed in yeast. The interaction between Pkhl and Ypkl enhanced the phosphorylation of GST- Crosstide. The inactive form of Aktl expressed in E. coli was unable to phosphorylate GST-Crosstide until it was activated by Pkhl or Ypkl. Our study indicated that GST-Crosstide could be used as an efficient tool to analyze AGC protein kinase activity. GST-Crosstide may benefit future studies on cellular signal transduction pathways regulated by AGC protein kinases.