AMOT家族蛋白在Hippo信号通路中的功能和分子机制研究

发布时间：2018-07-24 16:49

【摘要】：相对稳定的器官大小是多细胞生物的一个基本特征。近年的研究发现Hippo信号通路是控制器官大小的一条关键通路。Hippo通路通过由mammalianSTE20-like kinases 1/2(MST1/2)-salvador1(SAV1)复合物与 large tumor suppressor kinase 1/2(LATS1/2)-MOB kinase activator 1A/B(MOB1A/B)复合物组成的核心蛋白激酶链磷酸化下游转录辅激活因子 Yes-associated protein(YAP)/transcriptional coactivator with a PDZ-binding domain(TAZ),并促进YAP/TAZ胞质滞留和降解来发挥功能。但是对于Hippo信号通路下游是否有不依赖于YAP/TAZ的功能却知之甚少。我们通过筛选发现angiomotin(AMOT)家族蛋白是Hippo通路LATS1/2激酶的新底物。LATS1/2 响应上游 G protein coupled receptor(GPCR)等信号的调控,磷酸化 AMOT蛋白的剪切体之一 AMOTp130的HXRXXS保守基序。磷酸化抑制AMOTp130与微丝之间的结合,并抑制微丝与黏着斑的形成。我们还发现LATS1/2对AMOTp130的磷酸化在体外抑制血管内皮细胞的迁移,在体内抑制斑马鱼胚胎血管形成。这些结果表明AMOTp130是LATS1/2的直接底物,并且介导Hippo信号通路在内皮细胞迁移和血管生成中的重要功能。另一方面,AMOT家族蛋白作为Hippo通路的成员在调控Hippo通路活性方面发挥重要作用。YAP作为Hippo信号通路中最重要的效应蛋白可以促进细胞增殖、抑制细胞凋亡。但YAP的活性失调会导致肿瘤的形成,所以YAP激活的强度及持续时间都需要受到精确的调控。我们发现AMOT家族蛋白angiomotin-like 2(AMOTL2)和LATS2在YAP激活导致的负反馈调控中具有重要功能。YAP激活后强烈激活上游抑制激酶LATS1/2的活性。这主要是通过YAP直接结合到LATS2和AMOTL2基因的启动子,促进LATS2和AMOTL2的转录,并且通过间接的方式提高neurofibromin2(NF2)蛋白的表达量来实现的。YAP激活导致的负反馈调控在血清刺激、失去细胞间接触抑制等条件下都存在,并且YAP在诱导肿瘤形成过程中也启动负反馈调控。YAP通过激活负反馈机制来精确调节自身的活性,这对机体稳态维持和其失调导致疾病发生非常关键。以上研究表明AMOT家族蛋白在多个层面参与Hippo信号通路。一方面AMOTp130作为Hippo通路LATS1/2激酶的新底物,介导Hippo通路在调节血管生成方面的功能;另一方面AMOTL2作为YAP的靶基因,通过负反馈来抑制YAP的活性,维持Hippo通路在体内的稳态。本课题首次报道了 Hippo通路在生理条件下对血管生成的调控,并发现了维持Hippo通路稳态的机制。
[Abstract]:Relatively stable organ size is a basic feature of multicellular organisms. In recent years, it has been found that Hippo signaling pathway is a key pathway to control organ size. The Hippo pathway is transcripted downstream by phosphorylation of the core protein kinase chain composed of mammalianSTE20-like kinases 1 / 2 (MST1/2) -salvador1 (SAV1) complex and large tumor suppressor kinase 1 / 2 (LATS1/2) -MOB kinase activator 1A/B (MOB1A/B) complex. The active factor Yes-associated protein (YAP) / transcriptional coactivator with a PDZ-binding domain (TAZ), also promotes the retention and degradation of YAP/TAZ cytoplasm to function. But little is known about whether the downstream Hippo signaling pathway is independent of YAP/TAZ. We found that angiomotin (AMOT) family protein is a novel substrate of LATS1/2 kinase in Hippo pathway. LATS 1 / 2 responds to upstream G protein coupled receptor (GPCR) signal regulation and phosphorylated AMOT protein one of the shearing bodies of AMOTp130 conserved the HXRXXS motif. Phosphorylation inhibited the binding between AMOTp130 and microfilament, and the formation of microfilament and adhesion plaque. We also found that LATS1/2 phosphorylation of AMOTp130 inhibited the migration of vascular endothelial cells in vitro and the angiogenesis of zebrafish embryos in vivo. These results suggest that AMOTp130 is a direct substrate of LATS1/2 and mediates the important function of Hippo signaling pathway in endothelial cell migration and angiogenesis. On the other hand, AMOT family proteins, as members of Hippo pathway, play an important role in regulating the activity of Hippo pathway. Yap, as the most important effector protein in Hippo signaling pathway, can promote cell proliferation and inhibit cell apoptosis. However, the imbalance of YAP activity can lead to tumor formation, so the intensity and duration of YAP activation need to be accurately regulated. We found that AMOT family proteins angiomotin-like 2 (AMOTL2) and LATS2 play an important role in the negative feedback regulation induced by YAP activation. This is mainly through the direct binding of YAP to the promoter of LATS2 and AMOTL2 gene, which promotes the transcription of LATS2 and AMOTL2, and indirectly increases the expression of neurofibromin2 (NF2) protein to realize the negative feedback regulation caused by activation of neurofibromin2 (NF2) in serum. In the process of inducing tumor formation, YAP also initiated negative feedback regulation. YAP precisely regulates its activity by activating negative feedback mechanism. This is critical to the homeostasis of the body and its maladjustment leading to disease. These studies suggest that AMOT family proteins are involved in Hippo signaling pathway at multiple levels. On the one hand, AMOTp130 acts as a new substrate of LATS1/2 kinase in Hippo pathway, mediating the function of Hippo pathway in regulating angiogenesis; on the other hand, AMOTL2, as a target gene of YAP, inhibits the activity of YAP through negative feedback and maintains the homeostasis of Hippo pathway in vivo. In this paper, we first reported the regulation of Hippo pathway on angiogenesis under physiological conditions, and found the mechanism of maintaining the steady-state of Hippo pathway.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：Q25

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