CDK11p58激酶活性及SGT/PIAS1相互作用与凋亡的研究

【作者】 恽小婧；

【导师】 顾建新；

【作者基本信息】 复旦大学 ， 生物化学与分子生物学， 2009， 博士

【摘要】 第一部分CDK11p58激酶活性与凋亡的研究CDK11p58是一种在G2/M期特异性表达的蛋白激酶,在许多细胞中被证明与细胞凋亡相关,但机制仍不明确。我们实验室之前的研究证明CDK11p58能够促进放线菌酮(cycloheximide,CHX)诱导的SMMC-7721肝癌细胞的凋亡,然而其机制尚不明确。本文首次发现在SMMC-7721细胞中外转表达CDK11p58能够在CHX诱导凋亡过程中下调抗凋亡蛋白Bcl-2的蛋白水平,也下调Bcl-2的Ser70和Ser87的磷酸化水平。同时,我们也发现过表达Bcl-2能够抑制CDK11p58的促凋亡活性。进一步研究表明,CDK11p58的激酶活性在下调Bcl-2蛋白水平以及促凋亡过程中发挥关键作用。综上所述,CDK11p58在促凋亡过程中下调Bcl-2及其Ser70和Ser87磷酸化水平,并且该过程依赖于CDK11p58的蛋白激酶活性。第二部分:SGT与PIS1相互作用及其在凋亡中的作用Small glutamine-rich tetratricopeptide repeat-containing protein(SGT)在细胞中发挥分子伴侣蛋白的辅分子的作用,与一些重要的伴侣蛋白相互作用,例如热休克蛋白70和热休克蛋白90。我们实验室之前的研究表明SGT具有促凋亡的作用,但是其生物学机制并不清楚。为了进一步研究SGT的功能,我们采用酵母双杂交的方法,以SGT全长为诱饵筛人胎肝eDNA文库,发现了与其相互作用的蛋白PIAS1(protein inhibitor ofactivated STAT 1),体外GST-pull down实验也验证了它们之间的相互作用。由于SGT与PIAS1在细胞内的定位不同,PIAS1定位于胞核,而SGT一般定位于胞浆中,SGT在格尔德酶素(Geldanamycin,GA,一种热休克蛋白90的抑制剂)或凋亡信号的刺激下向核内转运,在体内试验中我们发现SGT在转运至核内后才与PIAS1发生相互作用。进一步的研究表明,采用RNA干扰的方式抑制PIAS1的表达之后,影响了SGT的促凋亡活性。通过本研究我们得出结论:SGT在一些凋亡信号的刺激下,向核内转运,通过与PIAS1相互作用促进细胞凋亡,PIAS1在SGT促凋亡的过程中发挥重要作用。更多还原

【Abstract】 PARTⅠ:The research of CDK11p58 protein kinase activity in apoptosisCDK11p58,a G2/M-specific protein kinase,has been shown to be associated with apoptosis in many cell lines,with largely unknown mechanisms.Our previous study proved that CDK11p58 enhanced cycloheximide(CHX)-induced apoptosis in SMMC-7721 hepatocarcinoma cells.Here we reported for the first time that ectopic expression of CDK11p58 down-regulated the expression of Bcl-2 and its Ser70,Ser87 phosphorylation in CHX-induced apoptosis in SMMC-7721 cells.Overexpression of Bcl-2 counteracted the pro-apoptotic activity of CDK11p58.Furthermore,we confirmed that the kinase activity of CDK11p58 was essential to the down-regulation of Bcl-2 as well as apoptosis.Taken together,these results demonstrated that CDK11p58 down-regulated Bcl-2 in pro-apoptosis pathway depending on its kinase activity,which elicited survival signal in hepatocarcinoma cells. PARTⅡ:The research of the interaction of SGT with PIAS1 and its function in apoptosisSmall glutamine-rich tetratricopeptide repeat-containing protein(SGT) acts as a co-chaperone,interacting with several essential chaperones,such as Hsp70 and Hsp90. Our previous study showed the pro-apoptosis activity of SGT.However,the biological mechanism remains unclear.To further investigate its functions,we used the yeast two-hybrid system to screen a human fetal liver cDNA library and identified PIAS1(protein inhibitor ofαctivated STAT 1) as an interacting partner of SGT.The association of SGT with PIAS1 was further confirmed by GST-pull down in vitro. Based on the different subcellular location of this two molecule and the fact that SGT translocates into nucleus after Geldanamycin(GA,an Hsp90 inhibitor) or other apoptosis inducer treatment,we revealed SGT interacted with PIAS1 in vivo after it translocated into the nucleus.Further studies showed that inhibition of PIAS1 expression by siRNA infected the pro-apoptosis activity of SGT.Collectively we concluded that SGT transloacted into the nucleus and induced apoptosis upon stress stimuli.SGT interacted with PIAS1 in the nucleus,and PIAS1 played a role in the pro-apoptosis process of SGT.更多还原