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

描述(由申请人提供)：河马途径调节器官大小并抑制多细胞生物体中的肿瘤形成。这一途径的失调会导致人类和小鼠肿瘤的形成。该途径的中心是一个由Ste20家族的Mst1/2、支架蛋白萨尔瓦多/WW45(Sav)、NDR家族的激酶Lats1/2和接头蛋白Mob1组成的激酶级联。细胞-细胞接触是启动这一途径并激活Mst1/2-Sav的上游信号。Mst1/2磷酸化并激活Lats1/2-Mob1，后者再磷酸化YAP。磷酸化的YAP被14-3-3降解或隔离在细胞质中。当河马途径关闭时，YAP移位到细胞核，并与TEAD形成功能性杂交转录因子。YAP-TEAD激活促增殖和促生存基因的转录，使细胞增殖。我们在这方面已经取得了关键的初步成果。在已发表的结果中，我们确定了人TEAD2的YAP结合域的晶体结构，并表征了它与YAP的相互作用。在未发表的结果中，我们发现：(1)RASSF5与Mst2的结合阻碍了其自身磷酸化依赖的激活；(2)我们确定了Mst2-RASSF5复合体的晶体结构；(3)活性Mst2对RASSF5的磷酸化削弱了Mst2-RASSF5的相互作用；(4)Mob1是一个磷酸化的肽结合模块，并结合到Mst2接头上的自动磷酸化T378位点；(5)磷酸化的Mob1与Lats1的结合刺激YAP磷酸化，而不影响Lats1的自动磷酸化。我们的结果支持一个中心假设，即磷酸化调节的蛋白质-蛋白质相互作用是MST-LATS激酶级联激活的基础。MST、RASSF和SAV的Sarah结构域之间一系列复杂的、受调控的相互作用决定了MST的激活状态。活性Mst2自动磷酸化其接头，为Mob1创建一个对接位置。Mob1与Mst2的募集促进了Mob1的高效磷酸化。磷酸化的Mob1与Lats1结合，并通过Lats1刺激YAP的磷酸化。尽管在这一领域取得了巨大的进展，但对中央MST-LATS激酶级联的激活机制还不完全清楚。这项建议旨在填补这一空白，并建立MST-LATS激酶级联的激活机制。在目标1中，我们将研究RASSF和SAV对Mst1/2激活的调节。在目标2中，我们将建立依赖Mst1/2的Mob1磷酸化和激活的机制。在目标3中，我们将剖析Mob1激活Lats1的机制。河马通路的失调与癌症密切相关。我们提出的对MST、LATS及其调节因子的生化和结构研究将建立MST-LATS激酶级联的激活机制。这些知识最终将有助于开发能够激活河马途径的化合物，这可能会成为开发新的抗癌药物的先导。

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 In unpublished results, we have made the following discoveries: (1) RASSF5 binding to Mst2 hinders its auto- phosphorylation-dependent activation; (2) we have determined the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.