摘要
BclGs属于仅含有BH3结构域的促凋亡蛋白亚家族成员,其mRNA仅在睾丸中表达,使Bcl-G(Gonad)得以冠名。Bcl-G是Bel-2家族促凋亡蛋白中独特的新成员,其基因编码两个蛋白,长片段BelG_L和短片段BclGs。它依靠非BH3结构域定位于胞内细胞器,基因定位于染色体12p12,此区域在50%的前列腺癌、30%的卵巢癌和30%的儿童急性淋巴细胞白血病中表现为缺失性突变。当BelGs过表达时,显示了很强的细胞毒性。它不但可被共表达的抗凋亡蛋白Bcl-xL所抑制,而且Bcl-xL也能与BelGs发生免疫共沉淀。除了与Bcl-2家族蛋白发生相互作用外,BclGs的调节机制仍需进一步阐明。我们以BclGs为诱饵,利用酵母双杂交的方法,从人睾丸cDNA文库中筛选出长片段BclGL、CSN5/JAB1(c-Jun N端激活结构域结合蛋白)、与DEAF1(变形表皮自调节因子1)相似的蛋白、与LDHA(乳酸脱氢酶)相似的蛋白、与GPx4(谷胱苷肽过氧化物酶4)相似的蛋白和一个新的融合蛋白(未注册)。其中,JAB1又属于COP9复合物的第五组分,负责调节植物中的光信号、线虫中的幼虫发育、整合素信号、细胞周期调控和甾体类激素信号传导。并且,其功能主要是加强AP-1(c-Jun/c-fos)与AP-1结合位点(TRE)即AP-1启动子区的结合力与稳定性,从而增强AP-1反式转录活性。并且,巨嗜细胞移动抑制因子MIF与细胞内蛋白JAB1相互作用,JAB1激活JNK的活性,并增强内源性磷酸化c-Jun的水平,而MIF抑制JAB1所增强的AP-1活性,但不能干扰转录因子NFkappaB的诱导。与之相反,胞外信号通过细胞表面整合素LFA-1在JAB1的介导下,把信号传到核内,激活AP-1。而HPO可进一步加强JAB1对AP-1的激活作用。它增加c-Jun的磷酸化而导致AP-1的激活。并且JAB1是通过非JNK途径加强c-Jun磷酸化的,以非MAPK信号途径,在JAB1的介导下激活AP-1的活性。因此,我们利用酵母双杂交、体外结合实验和细胞共定位进行验证,表明BclGs与CSN/JAB1可以发生相互作用。双报告基因实验表明促凋亡蛋白BclGs与JAB1发生相互作用,通过CSN5/JAB1的介导,降低c-Jun的磷酸化水平,抑制AP-1转录激活活性,从而引发细胞凋亡。在此基础上,进一步研究磷酸化的JNK系列、STAT系列、AKT和LaminA等与细胞增殖和凋亡相关的蛋白,利用Western blot检测其表达,结果表明:BclGs能够降低JNK的磷酸化,但通过非MAPK的途径磷酸化JNK,从而导致下游事件的发生;BclGs还降低STAT3的磷酸化,但不能刺激STAT家族其它三种成员的磷酸化。BclGs使AKT表达降低,而Lamin A水平表达升高。总之,促凋亡蛋白BclGs和辅助激活因子JAB1相互作用所引起的信号转导是对BclGs所致的凋亡机制的补充;其中,在以BclGs-JAB1相互作用作为关键的分子基础的信号转导过程中,JNK和STAT3的磷酸化下降,增加了BclGs所致凋亡的敏感性;AKT的表达降低,而Lamin A的表达增高,仅作为对BclGs反应的结果出现,可能处于细胞凋亡的早期。
BclGs belongs to a member of BH3-only protein, which is structurally distant member of Bcl-2 protein family that trigger apoptosis. BclGs mRNA is uniquely expressed in testis. Apoptosis induction by BlcGs depends on the BH3 domain and is suppressed by coexpression of anti-apoptotic Bcl-xL protein. The molecular targets of BclGs so far remain unclear. Thus, we used the full length BclGs as "bait" in a yeast two-hybrid system to screen a human testis cDNA library. Several types of clones were identified, which interacted specifically with BclGs when tested for nutritional selection and P-galactosidase activity. One clone contained a cDNA insert with almost the entire coding sequence (amino acid 44-335) of human CSN5/JAB1, which is a coactivator of AP-1 transcription and the subunit 5 of CSN. Furthermore, we showed that BclGs specifically interacted with JAB1 by yeast two-hybrid and in vitro binding assay, and co-localized with it in the nucleus of Cos7 cells. By dual-reporter gene assay, BclGs inhibited AP-1 activity through JAB1. Correspondingly, it inhibited the endogenous phospho-c-Jun level through JAB1, and decreased the endogenous phospho-JNK level independent of MAPK pathways. At the same condition, BclGs also inhibited the phosphorylation of STAT3, and not influencing the phospho-STAT 1, 5 and 6. Interestingly, expression level of AKT was decreased, but the level of Lamin A/C was enhanced, and the degradation of Lamin A/C was not observed in COS7 cells. Taken together, it could be concluded that BclGs may inhibit JABl-controlled pathways and that the protein-protein interaction of BclGs-JABl may provide a molecular basis for key activities of BclGs. These results might elucidate a novel mechanism of BclGs signaling by specifically modulating the AP-1 pathway through JAB1 in early apoptosis.
引文
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