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作 者:李晓燕[1] 路程[1] 吴宁华[1] 沈珝琲[1]
机构地区:[1]中国医学科学院中国协和医科大学基础医学研究所医学分子生物学国家重点实验室,北京100005
出 处:《中国医学科学院学报》2002年第3期264-268,共5页Acta Academiae Medicinae Sinicae
基 金:国家自然科学基金(39930050)资助
摘 要:目的探讨Rac-MEKK-JNK(Rac-丝裂原活化的蛋白激酶的激酶的激酶-C-junN端蛋白激酶)信号通路对热诱导hsp90β基因表达的调控作用以及Hsp90蛋白对Rac信号通路传递的调节过程。方法将载体PcDNA3、显性负突变Rac(DN-Rac),显性负突变MEKK(DN-MEKK)和显性负突变JNK(DN-JNK)表达质粒分别与hsp90β基因转录调控片段介导的氯霉素乙酰基转移酶(CAT)报告基因质粒β3.1共转染Jurkat和LETPa-2细胞,采用以竞争性RT-PCR为基础的系统检测CAT报告基因转录水平。用Western印迹分析检测转染了DN-Rac、DN-MEKK和DN-JNK的细胞在热刺激前后c-Jun的表达水平和磷酸化状态。用体外激酶活性分析法检测geldanamycin(GA)对热诱导的JNK活性的影响。结果转染DN-Rac、DN-MEKK及DN-JNK均能抑制42℃1h诱导的β3.1报告基因的表达,Jurkat细胞中CAT的热诱导表达分别为对照组的(72.8±5)%、(60±13.2)%及(47.7±12.1)%;LETPa-2细胞中CAT的热诱导表达分别为对照组的(16.17±5.1)%、(50.2±8.7)%及(47.5±10)%。转录因子c-Jun的表达和磷酸化水平也在一定程度上被抑制。GA处理细胞可明显抑制热诱导激活的JNK激酶活性和c-Jun的磷酸化水平,但不影响JNK和c-Jun的蛋白表达水平。结论Rac-MEKK-JNK通路参与hsp90β基因的热诱导表达,hsp90可能对热激活化?Objective To study the effect of Rac-MEKK-JNK(Rac-mitogen activated protein kinase kinase kinase-C-jun N-terminal protein kinase)signal pathway on heat shock-induced hsp90βgene expression and the impact of Hsp90on the regulation of the pathway.Methods DN-Rac,DN-MEKK or DN-JNK were cotransfected with hsp90βCAT reporter plasmidβ3.1into Jurkat or LETPa-2cells individually,the CAT mRNA expression was then determined quantitatively by competitive RT-PCR based system.Western blot was carried out to detect the expression level and phosphorylation of c-Jun in Jurkat and LETPa-2cells that were transfected with DN-Rac,DN-MEKK or DN-JNK.By in vitro kinase activity assay and Western blot,the effect of geldnamycin(GA)on heat induced JNK activity were evaluated.Results In Jurkat cell transfected with DN-Rac,DN-MEKK or DN-JNK,heat shock induced relative CAT mRNA expression level was decreased to(72.8±5)%,(60±13.2)%and(47.7±12.1)%of the control respectively;while in LETPa-2cell hsp90β3.1reporter gene expression was accordingly suppressed to(16.17±5.1)%,(50.2±8.7)%and(47.5±10)%of control.C-Jun expression and phosphorylation were inhibited by the transfection of either one of DN-Rac,DN-MEKK or DN-JNK.With GA treatment,heat shock induced JNK activity was repressed,while the expression level of JNK or c-Jun was not obviously changed.Conclusions Rac-MEKK-JNK pathway promotes heat shock induced hsp90βgene expression and hsp90may participate in the regulation of heat shock activated Rac-MEKK-JNK signal pathway in both Jurkat and LETPa-2cells.
关 键 词:Rac-MEKK-JNK信号通路 Rac蛋白 丝裂原活化的蛋白激酶的激酶的激酶 C-junN端蛋白激酶 HSP90Β基因 基因表达调控
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