重组信号分子GRF的PH结构域抑制蛋白激酶C活性

Recombinant GRF PH domain down-regulates activity of protein kinase C

目的 鉴定表达 guanine- nucleotide- releasing factor(GRF)的 pleckstrin homology(PH)结构域与谷胱甘肽转移酶 (GST)融合蛋白的可溶性 ,并进一步检测该重组蛋白与蛋白激酶 C(PKC)的结合及对 PKC活性的影响 .方法 将表达GRF的 PH结构域与 GST融合蛋白的菌体经超声裂解 ,上清中的融合蛋白用琼脂糖珠锚定纯化 .Western blot检测融合蛋白与 PKC的结合 ,以 PKC活性检测试剂盒测定结合后PKC的活性变化 .结果 获得信号分子 GRF的 PH结构域 -GST融合蛋白的可溶性表达 .Western blot结果表明 ,GRF的 PH结构域可在体外与 PKC结合 .PKC活性实验显示 ,GRF的 PH结构域抑制 PKC活性 .结论 GRF的 PH结构域可在体外与 PKC结合并对

AIM To investigate association of guanine nucleotide releasing factor for Ras (GRF) PH domain and protein kinase C (PKC) and to examine effect of GRF PH domain on activity of protein kinase C. METHODS GRF PH domain GST fusion protein was expressed in E.coli and purified by glutathione agarose beads. The expressed fusion pro tein was harvested by lysing the E.coli with ultrasonic. After centrifugation, the fusion protein in supernatant was purified by glutathione agarose beads. Western blot was employed to confirm the binding of the PH domain with PKC in vitro . To detect change of PKC activity after the fusion protein binding to PKC, PKC activity was examined with TECT TM PKC assay system. RESULTS Association of PKC with GRF PH domain was detected out by Western blot, and the PH domain down regulated the activity of PKC. CONCLUSION Recombinant GRF PH domain binds to PKC and inhibits its kinase activity in vitro .