SEPT1受Aurora B磷酸化调节的生物学意义及SEPT12的生物学功能探讨

【作者】 丁向明；

【导师】 余龙；

【作者基本信息】 复旦大学 ， 遗传学， 2008， 博士

【摘要】 Septin是一类进化保守的GTP结合蛋白家簇,最早是作为引起胞质分裂缺陷的细胞周期突变体在芽酵母中被发现的。后来相继在真菌、线虫、果蝇及哺乳动物中都相继有septin被发现,但植物中未发现此类蛋白质编码基因的存在。目前已知的septin除了与胞质分裂相关外,还参与协调核分裂、膜运输、细胞骨架组建、凋亡等生物学过程。在我们实验室先前研究工作中已发现SEPT1能和Aurora B相互作用,还发现Aurora B在体外能磷酸化SEPT1。在本项研究中我们发现SEPT1与Aurora B在细胞分裂末期及胞质分裂内源性共定位于中间体处,同时质谱分析证明AuroraB磷酸化SEPTI位点是64位苏氨酸和248位丝氨酸,并进一步证明这两位点在体内也是被磷酸化修饰的。由于我们的另一项研究已证明SEPT1特异性在淋巴造血系统表达,于是采用RNAi实验发现SEPT1参与了Jurkat细胞的胞质分裂过程,并用点突变的方法证实Aurora B对SEPT1的磷酸化也影响了这一过程。此外,我们还进行了SEPT12的生化和细胞学研究。SEPT12是最近在人基因组序列分析中新发现的一个septin,它与SEPT3和SEPT9同源度最高。在本研究中我们发现,SEPT12在体外能够结合GTP,而56位甘氨酸到天冬酰氨的突变能破坏SEPT12结合GTP的能力。免疫细胞化学分析显示野生型SEPT12能形成细胞骨架纤维,而SEPT12^G56A突变体却丧失这种能力,在胞浆中呈现为大的聚集物。SEPT12在分裂间期和分裂期有着不同的亚细胞定位,在分裂间期形成纤维而在分裂后期及胞质分裂期则分别移位于中央纺锤体和中间体处。SEPT12在体内和体外都可以和SEPT6及SEPT11相互作用。综上所述,本文为研究淋巴细胞胞质分裂机制提供了一个新的线索,SEPT1作为淋巴细胞特异表达的蛋白,参于调控淋巴细胞胞质分裂,而SEPT1这种调控作用又受Aurora B激酶的调节。此外,还初步阐述了SEPT12蛋白的生化和细胞学行为。更多还原

【Abstract】 The septins are an evolutionarily conserved family of polymerizing GTP binding proteins originally discovered in budding yeast as a group of cell cycle mutants which cause defects in cytokinesis.It is now clear that septins are present in the fungi and animals,although apparently not in plants.In addition to their original role in cytokinesis,septins have been shown to have roles in coordinating nuclear division, membrane trafficking,organizing the cytoskeleton,apoptosis.In our previous study,we found that SEPT1 interact with Aurora B and is phosphorylated by Aurora B in vitro.In this study we found that SEPT1 colocalizated endogenously with Aurora B in the midbody during the cytokinesis of Jurkat T cell.Mass spectrum analysis revealed that SEPT1 was phosphorylated by Aurora B at T64 and S248 in vitro and further study showed that the two residues were also phosphorylate in vivo.In addition,our another study showed that SEPT1 was expressed specificly in haematopoietic system.,thus SEPT1 RNAi experiment in Jurkat cells indicated that SEPT1 was involved in the cytokinesis of Jurkat cells.We also found that the Aurora B phosphorylation of SEPT1 at Thr 64 and Ser 248 was associated with the cytokinesis of Jurkat cells.In addition,we reported the biochemical and immunocytochemical characterization of a recently identified mammalian septin,SEPT12,sharing closest homology to SEPT3 and SEPT9.SEPT12 can bind GTP in vitro and the mutation(Gly56 to Asn) in the GTP-bindng motif abrogated this binding.Immunocytochemical analysis revealed that the wild-type SEPT12 formed the filament structure and the SEPT12G56A appearared as large aggregates when transiently expressed in Hela cells. It was revealed that subcelluar localization of SEPT12 varied at interphase and mitotic phase.While SEPT12 formed filamentous structures at interphase,it was localized to the central spindle and to midbody during anaphase and cytokinesis,respectively.In addition,we found that SEPT12 can interact with SEPT6 and SEPT11 in vitro and in vivo.更多还原