H-Ras is well known as one of the essential components of Ras/Raf/MEK/ERK cascade, which is a critical prosurvival signaling mechanism in most eukaryotic cells. Ras targets Raf/MEK/ERK cascade by integrating and trans...H-Ras is well known as one of the essential components of Ras/Raf/MEK/ERK cascade, which is a critical prosurvival signaling mechanism in most eukaryotic cells. Ras targets Raf/MEK/ERK cascade by integrating and transmitting extracellular signals from growth factor receptors to Raf, leading to the propagation of signals to modulate a serious of cellular survival events. Apoptosis signal-regulating kinasel (ASK1) serves as a general mediator of cell death because it is responsive to a variety of death signals. In this study, we found that H-Ras interacted with ASK1 to cause the inhibition of both ASK1 activity and ASK1-induced apoptosis in vivo, which was reversed only partially by addition of RafS621A, an antagonist of Raf, whereas MEK inhibitor, PD98059, and PI3K inhibitor, LY294002, did not disturb the inhibitory effect of H-Ras on ASK-1-induced apoptosis. Furthermore, by means of immunoprecipitate and kinase assays, we demonstrated that the interaction between H-Ras and ASK1 as well as the inhibition of ASK1 activity were dependent on the binding activity of H-Ras. These results suggest that a novel mechanism may be involved in H-Rasmediated cell survival in addition to the well established MEK/ERK and PI3K/Akt kinase-dependent enhancement of cell survival.展开更多
[目的]构建人ASK1基因原核表达载体并进行生物信息学分析。[方法]以pCDNA3.1-hASK1为模板扩增hASK1基因,构建pGEX-KG-hASK1重组质粒,菌落PCR筛选阳性克隆并进行测序验证,最后对hASK1蛋白进行生物信息学分析。[结果]成功构建原核表达载体...[目的]构建人ASK1基因原核表达载体并进行生物信息学分析。[方法]以pCDNA3.1-hASK1为模板扩增hASK1基因,构建pGEX-KG-hASK1重组质粒,菌落PCR筛选阳性克隆并进行测序验证,最后对hASK1蛋白进行生物信息学分析。[结果]成功构建原核表达载体pGEX-KG-hASK1。生物信息学分析显示,hASK1蛋白由1 374个氨基酸残基组成,等电点5.52,相对分子质量154.5 k Da。该蛋白无跨膜区结构,二级结构由α-螺旋、折叠延伸链、无规卷曲构成。[结论]hASK1蛋白是含1 374个氨基酸残基的亲水蛋白,含79个磷酸化位点,能与多种信号蛋白相互作用。展开更多
文摘H-Ras is well known as one of the essential components of Ras/Raf/MEK/ERK cascade, which is a critical prosurvival signaling mechanism in most eukaryotic cells. Ras targets Raf/MEK/ERK cascade by integrating and transmitting extracellular signals from growth factor receptors to Raf, leading to the propagation of signals to modulate a serious of cellular survival events. Apoptosis signal-regulating kinasel (ASK1) serves as a general mediator of cell death because it is responsive to a variety of death signals. In this study, we found that H-Ras interacted with ASK1 to cause the inhibition of both ASK1 activity and ASK1-induced apoptosis in vivo, which was reversed only partially by addition of RafS621A, an antagonist of Raf, whereas MEK inhibitor, PD98059, and PI3K inhibitor, LY294002, did not disturb the inhibitory effect of H-Ras on ASK-1-induced apoptosis. Furthermore, by means of immunoprecipitate and kinase assays, we demonstrated that the interaction between H-Ras and ASK1 as well as the inhibition of ASK1 activity were dependent on the binding activity of H-Ras. These results suggest that a novel mechanism may be involved in H-Rasmediated cell survival in addition to the well established MEK/ERK and PI3K/Akt kinase-dependent enhancement of cell survival.
文摘[目的]构建人ASK1基因原核表达载体并进行生物信息学分析。[方法]以pCDNA3.1-hASK1为模板扩增hASK1基因,构建pGEX-KG-hASK1重组质粒,菌落PCR筛选阳性克隆并进行测序验证,最后对hASK1蛋白进行生物信息学分析。[结果]成功构建原核表达载体pGEX-KG-hASK1。生物信息学分析显示,hASK1蛋白由1 374个氨基酸残基组成,等电点5.52,相对分子质量154.5 k Da。该蛋白无跨膜区结构,二级结构由α-螺旋、折叠延伸链、无规卷曲构成。[结论]hASK1蛋白是含1 374个氨基酸残基的亲水蛋白,含79个磷酸化位点,能与多种信号蛋白相互作用。
