Activation mechanism of the tumor-suppressing MST-LATS kinase cascade

描述（由申请人提供）：Hippo通路调节多细胞生物体的器官大小并抑制肿瘤发生。该途径的失调驱动人类和小鼠中的肿瘤形成。该途径的中心是由Ste 20家族激酶Mst 1/2、支架蛋白萨尔瓦多/WW 45（Sav）、NDR家族激酶Lats 1/2和衔接蛋白Mob 1形成的激酶级联。细胞-细胞接触是开启该通路并激活Mst 1/2-Sav的上游信号。Mst 1/2磷酸化并激活Lats 1/2-Mob 1，然后Lats 1/2-Mob 1磷酸化雅普。磷酸-YAP被14-3-3降解或隔离在细胞质中。当Hippo通路关闭时，雅普易位到细胞核并与TEAD形成功能性杂合转录因子。YAP-TEAD激活促增殖和促存活基因的转录，使细胞增殖。我们在这一领域取得了重要的初步成果。在已发表的结果中，我们确定了人TEAD 2的YAP结合结构域的晶体结构，并表征了其与雅普的相互作用。在未发表的结果中，我们有以下发现：（1）RASSF 5与Mst 2的结合阻碍了其自身磷酸化依赖性激活;（2）我们已经确定了Mst 2-RASSF 5复合物的晶体结构;（3）RASSF 5被活性Mst 2磷酸化减弱了Mst 2-RASSF 5相互作用;（4）Mob 1是磷酸化肽结合模块，并与Mst 2接头中的自磷酸化T378位点结合;和（5）磷酸化Mob 1与Lats 1的结合刺激雅普磷酸化而不影响Lats 1自磷酸化。我们的研究结果支持了中心假设，磷酸化调节的蛋白质-蛋白质相互作用的基础激活的Mst-Lats激酶级联。Mst、RASSF和Sav的SARAH结构域之间的一组复杂的、受调节的相互作用决定了Mst激活的状态。活性Mst 2自磷酸化其接头，为Mob 1创建对接位点。将Mob 1募集到Mst 2促进有效的Mob 1磷酸化。磷酸化的Mob 1与Lats 1结合，并通过Lats 1刺激雅普磷酸化。尽管在这一领域取得了巨大的进展，中央Mst-Lats激酶级联的激活机制尚未完全理解。该提案旨在填补这一空白，并建立Mst-Lats激酶级联的激活机制。目的1：研究RASSF和Sav对Mst 1/2激活的调控。目的2：建立Mst 1/2依赖的Mob 1磷酸化和激活机制。在目标3中，我们将剖析Mob 1激活Lats 1的机制。Hippo通路的失调与癌症密切相关。我们提出的Mst，Lats，和他们的监管机构的生化和结构的研究将建立Mst-Lats激酶级联的激活机制。这些知识最终将有助于开发激活Hippo通路的化合物，这可以作为开发新抗癌药物的先导。

DESCRIPTION (provided by applicant): The Hippo pathway regulates organ size and suppresses tumorigenesis in multicellular organisms. Dysregulation of this pathway drives tumor formation in humans and mice. Central to this pathway is a kinase cascade formed by a Ste20 family kinase Mst1/2, a scaffolding protein Salvador/WW45 (Sav), an NDR family kinase Lats1/2, and an adaptor protein Mob1. Cell-cell contact is an upstream signal that turns on this pathway and activates Mst1/2-Sav. Mst1/2 phosphorylates and activates Lats1/2-Mob1, which then phosphorylates YAP. Phospho-YAP is either degraded or sequestered in the cytoplasm by 14-3-3. When the Hippo pathway is off, YAP translocates to the nucleus and forms a functional hybrid transcriptional factor with TEAD. YAP-TEAD activates the transcription of pro-proliferative and pro-survival genes, enabling cell proliferation. We have obtained key preliminary results in this area. In published results, we have determined the crystal structure of the YAP-binding domain of human TEAD2 and characterized its interaction with YAP. In unpublished results, we have made the following discoveries: (1) RASSF5 binding to Mst2 hinders its auto- phosphorylation-dependent activation; (2) we have determined the crystal structure of the Mst2-RASSF5 complex; (3) phosphorylation of RASSF5 by active Mst2 weakens the Mst2-RASSF5 interaction; (4) Mob1 is a phospho-peptide-binding module and binds to the auto-phosphorylation T378 site in the Mst2 linker; and (5) binding of phosphorylated Mob1 to Lats1 stimulates YAP phosphorylation without affecting Lats1 auto- phosphorylation. Our results support the central hypothesis that phosphorylation-regulated protein-protein interactions underlie the activation of the Mst-Lats kinase cascade. A set of intricate, regulated interactions among the SARAH domains of Mst, RASSF, and Sav determine the status of Mst activation. Active Mst2 autophosphorylates its linker, creating a docking site for Mob1. Recruitment of Mob1 to Mst2 promotes efficient Mob1 phosphorylation. Phosphorylated Mob1 binds to Lats1 and stimulates YAP phosphorylation by Lats1. Despite the tremendous progress in this area, the activation mechanisms of the central Mst-Lats kinase cascade are not fully understood. This proposal aims to fill this void and establish the activation mechanisms of the Mst-Lats kinase cascade. In Aim 1, we will study the regulation of Mst1/2 activation by RASSF and Sav. In Aim 2, we will establish the mechanism of Mst1/2-dependent Mob1 phosphorylation and activation. In Aim 3, we will dissect the mechanism of Lats1 activation by Mob1. Dysregulation of the Hippo pathway is intimately linked to cancer. Our proposed biochemical and structural studies on Mst, Lats, and their regulators will establish the activation mechanisms of the Mst-Lats kinase cascade. This knowledge will ultimately aid the development of chemical compounds that activate the Hippo pathway, which can serve as leads for the development of new anti-cancer drugs.