筛选和鉴定Hippo信号转导途径中抑制Hippo蛋白激酶活性的新成员

Hippo pathway is emerging as a highly conserved tumour suppressor pathway. Studies over the past decade have uncovered that Hippo pathway controls organ size during development by balancing cell proliferation and apoptosis. It also has been reported that, Hippo pathway coordinates with other known pathways that involve cell fate decision to regulate tumorigenesis and stem cell proliferation and differentiation. Although the signaling of the core kinase cascade is relatively well understood, less is known about the upstream inputs and regulation mechanism of the Hippo kinase activity. By large scale genetic screening and analyses using molecular and cellular techniques, we identify and uncover the Par-1 polarity kinase as a negative regulator of Hippo. Par-1 inhibits Hippo kinase activity by direct mono-phosphorylation, and also regulates the stability of core kinase complex of Hippo signaling to suppress Hippo signaling, leading to organ growth, which makes it a potential oncogene. By using in vitro experiments and animal models, we are going to expound the regulation mechanism of Hippo kinase activity by Par-1 and hence uncover the regulatory relationship between cell polarity and Hippo signaling. Considering the close relationship between malfunction of Hippo signaling and tumorigenesis, our research may not only help to uncover the basic principle of growth control during development, but also enrich our understanding of the molecular mechanism of tumorigenesis, and hence provide new ideas for diagnosis and treatment of human cancers.

Hippo(Hpo)信号通路是近年来发现的在物种间非常保守的肿瘤抑制信号通路，能通过抑制细胞增殖，促进细胞凋亡来调控器官发育的大小。已有研究证明，Hpo通路直接调控了肿瘤的发生及干细胞的增殖发育。Hpo激酶作为Hpo通路中激酶复合物的核心，其活性调节机制一直知之甚少。通过大规模遗传学筛选，并结合分子、生化、遗传等手段进行分离、鉴定，首次发现了参与细胞极性维持的Par-1激酶，能通过直接磷酸化Hpo抑制其激酶活性，并同时调控了Hpo激酶复合物的稳定性，进而抑制了Hpo信号，促进了组织器官的生长，是一个潜在的原癌基因。我们将利用体外实验和动物模型，阐明Par-1调控Hpo活性的机制，同时揭示细胞极性与Hpo信号的调控关系。由于Hpo信号通路的活性异常与癌症的发生密切相关，本研究不仅能回答发育生长调控的基本问题，还将有助于我们认识癌症发生的分子机理，为人类癌症的诊断和治疗提供理论基础和新的思路。

器官大小的调控机制一直是长久以来大家比较关注的问题。近年来发现的Hippo信号通路可能是解开这个问题的一个重要切入点。自从在果蝇内发现Hippo信号通路后，越来越多证据表明，Hippo信号通路在物种间高度保守。Hippo信号通路的失活能导致器官增大，相反激活Hippo信号通路能够抑制组织增生。其他研究也表明Hippo信号通路在肿瘤发生，干细胞维持过程中起重要作用。虽然Hippo信号通路的核心成员已经比较清楚，但作为一条近年来才被定义的新的信号通路，参与其中的蛋白组分及其介导的生理功能还有待进一步发掘。基于此，在本论文中，我们深入探讨了Hippo信号通路的其它调节组分及其未知的功能。.我们利用果蝇为模式生物，通过大规模的遗传筛选，发现Par-1蛋白能够调节Hippo信号通路。研究表明，在果蝇S2细胞内，Par-1过表达能够抑制Hippo蛋白的活化，从而激活Hippo信号通路转录转录辅因子Yorkie (Yki)，最终活化Yki/ Scalloped (Sd) 复合体的转录活性。我们进一步的实验表明，在果蝇成体内，Par-1过表达通过抑制Hippo信号通路促进组织增生。与之相反，RNAi干扰或者敲除par-1能激活Hippo信号通路，从而抑制组织增生。通过遗传学实验，我们发现Par-1蛋白在Fat/Expanded (ex) 蛋白下游及Hippo/Salvador (Sav) 蛋白复合体上游发挥功能。通过体外生化实验，我们最终发现Par-1能够导致Hippo蛋白第30位点丝氨酸磷酸化，从而导致Hippo蛋白失活。除此以外，我们还发现 Par-1能够导致Hippo/Salvador (Sav) 蛋白复合体解离。在哺乳动物系统中的研究表明， Par-1蛋白的同源物微管亲和调节激酶 (Microtubule Affinity Regulating Kinase，MARK) 能在哺乳动物细胞内通过影响激酶MST调节Hippo信号通路活性。因此，通过以上实验我们认为par-1基因是一个新的Hippo信号通路调节基因。