导　　师： 杨晓明

学科专业： 071010

授予学位： 博士

作　　者： ;

机构地区： 军事医学科学院

摘　　要： AKT1即蛋白激酶B是PI3K//AKT信号转导通路的核心分子,具有调控物质代谢、细胞生长、分化、凋亡和迁移等多种重要的生物学功能,我们实验室前期利用串联亲和纯化联合质谱技术/( TAP-MS /),以AKT1为诱饵从HepG2细胞中纯化鉴定出一批AKT1可能的候选相互作用蛋白质。 HEB是鉴定到的AKT1候选相互作用蛋白质之一,属于bHLH转录因子家族,参与调控细胞生长和分化过程。本论文研究了AKT1与HEB的相互作用和AKT1对HEB活性的调控作用。 通过免疫共沉淀、GST-pull down、共定位实验验证了HEB与AKT1之间的相互作用;采用体内体外实验证明AKT1可磷酸化HEB,证明HEB是AKT1新的底物分子;活化PI3K途径可增强HEB磷酸化,PI3K特异抑制剂可抑制胰岛素、肝细胞生长因子诱导的HEB磷酸化,表明HEB的磷酸化依赖PI3K//AKT信号通路。HEB蛋白质序列的Ser~/(559/)位点在不同物种同源分子间高度保守,并符合AKT1磷酸化底物的保守氨基酸序列特征/(RGRTSS~/(559/)/),我们推测该位点是AKT1的磷酸化位点,并得到突变体实验的验证。突变Ser~/(559/)位点不改变HEB的细胞核定位,但减低了HEB上调p21表达、诱发细胞周期G0-G1期阻滞、进而导致细胞生长抑制的活性、表明该位点是HEB发挥功能的重要位点。HEB通常与转录因子E47形成异源二聚体发挥转录调控作用, Ser~/(559/)位点突变导致HEB丧失了与E47发生相互作用的能力,并降低了两者协同调控SRG3启动子的活性。综上,我们的实验结果证明HEB是AKT1新的作用底物,AKT1可通过Ser~/(559/)位点磷酸化HEB调控其生物学功能。 AKT1, also known as PKB, is a key molecular in PI3K//AKT signaling pathway. It regulates multiple cellular functions, including intermediary metabolism, cell proliferation, differentiation, apoptosis and migration. In our previous study, using a TAP-MS-based approach, we isolated AKT1 complex and identified several novel binding proteins of AKT1 in human hepatocellular carcinoma line HepG2. HEB is one of the identified interactions, and it belongs to bHLH transcription factors family which are involved in cell proliferation and differentiation. In this study, the interaction between AKT1 and HEB and the regulation of HEB activity by AKT1 were investigated. The protein-protein interaction between AKT1 and HEB was confirmed by co-IP and GST-pull down assays. AKT1 can phosphoralate HEB in vitro and in vivo, suggesting that HEB is a novel phosphorylated substrate of AKT1. The activation of PI3K signaling pathway increased the phosphorylation of HEB, and the specific inhibitor of PI3K suppressed the phosphorylation of HEB by insulin and HGF, indicating that the phosphorylation of HEB depends on PI3K//AKT pathway activation. Bioinformation analysis of HEB sequence revealed that the Ser~/(559/) site is highly conserved in most of the animal species and the flanking sequence surrounding Ser~/(559/) containing a putative AKT1 phosphorylationmotif /(RGRTSS/). So we speculate this site can be phosphorylated by AKT1. We constructed a mutant of HEB whose phosphorylation site is mutated, and confirmed Ser~/(559/) is the phosphorylation site of HEB by AKT1. Mutation of Ser~/(559/) doesn’t change the nuclear localization of HEB by immunofluorescence assay, but led to abolishement of the activation of p21 expression induced by wild type HEB /(HEBwt/). Also, the arrest of G0-G1 phase was attenuated and the suppression of cell growth was released by HEBmt. HEB and E47 usually form heterodimers to regulate the transcription of targets, and we found that HEBmt failed to form hetero-oligomers with E47 to transactivate the promoter activity of SRG3. In conclusion, HEB is a novel phosphorylated substrate of AKT1, and the phosphoralation of the Ser~/(559/) site by AKT1 is essential for the biological function and activity of HEB.

关 键 词： 磷酸化 细胞增殖

分 类 号： [Q75]

领　　域： [生物学]