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

描述（由申请人提供）：Hippo 途径调节多细胞生物体的器官大小并抑制肿瘤发生。该通路的失调会导致人类和小鼠肿瘤的形成。该通路的核心是由 Ste20 家族激酶 Mst1/2、支架蛋白 Salvador/WW45 (Sav)、NDR 家族激酶 Lats1/2 和接头蛋白 Mob1 形成的激酶级联。细胞-细胞接触是开启该通路并激活 Mst1/2-Sav 的上游信号。 Mst1/2 磷酸化并激活 Lats1/2-Mob1，然后磷酸化 YAP。 Phospho-YAP 被 14-3-3 降解或隔离在细胞质中。当 Hippo 途径关闭时，YAP 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，从而促进细胞增殖。我们在这一领域已经取得了关键的初步成果。在发表的结果中，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，我们有了以下发现：（1）RASSF5与Mst2的结合阻碍了其自身磷酸化依赖性激活； (2)我们确定了Mst2-RASSF5复合物的晶体结构； (3)活性Mst2对RASSF5的磷酸化减弱了Mst2-RASSF5相互作用； (4) Mob1是磷酸肽结合模块，与Mst2接头中的自磷酸化T378位点结合； (5)磷酸化的Mob1与Lats1的结合刺激YAP磷酸化而不影响Lats1自身磷酸化。我们的结果支持了这样的中心假设：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 结构域之间的一组复杂的、受调节的相互作用决定了 Mst 的激活状态。活性 Mst2 会自动磷酸化其连接子，为 Mob1 创建对接位点。 Mob1 向 Mst2 的募集促进了 Mob1 的有效磷酸化。磷酸化的 Mob1 与 Lats1 结合并刺激 Lats1 磷酸化 YAP。尽管该领域取得了巨大进展，但中央 Mst-Lats 激酶级联的激活机制尚未完全了解。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。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 Mst1/2 依赖性 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。 Hippo 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激活 Hippo 途径的化合物，从而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 In unpublished results, we have made the following discoveries: (1) RASSF5 binding to Mst2 hinders its auto- phosphorylation-dependent activation; (2) we have determined the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.