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的器官大小并抑制肿瘤发生。在人类和小鼠中，这一通路的失调驱动肿瘤的形成。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组成的激酶级联。细胞-细胞接触是一个上游信号，它开启了这条通路并激活了Mst1/2-Sav。Mst1/2磷酸化并激活Lats1/2-Mob1，然后使YAP磷酸化。Phospho-YAP被14-3-3降解或隔离在细胞质中。当Hippo通路关闭时，YAP易位到细胞核并与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。我们已经在这方面取得了关键的初步成果。在已发表的结果中，我们确定了人类TEAD2的YAP结合域的晶体结构，并表征了其与YAP的相互作用。在未发表的结果中，我们有以下发现：(1)RASSF5与Mst2的结合阻碍了其自磷酸化依赖性激活；(2)确定了Mst2-RASSF5配合物的晶体结构；(3)活性Mst2对RASSF5的磷酸化会减弱Mst2-RASSF5的相互作用；(4) Mob1是一个磷酸化肽结合模块，与Mst2连接体中的自磷酸化T378位点结合；(5)磷酸化的Mob1与Lats1结合刺激YAP磷酸化而不影响Lats1的自磷酸化。我们的研究结果支持了磷酸化调节的蛋白-蛋白相互作用是Mst-Lats激酶级联激活的基础这一中心假设。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激活的状态。活性Mst2自磷酸化其连接体，为Mob1创建一个对接位点。Mob1向Mst2的募集促进了Mob1的高效磷酸化。磷酸化的Mob1结合Lats1并刺激Lats1对YAP的磷酸化。尽管在这一领域取得了巨大的进展，但中央Mst-Lats激酶级联的激活机制尚不完全清楚。本研究旨在填补这一空白，建立Mst-Lats激酶级联的激活机制。在Aim 1中，我们将研究RASSF和Sav对Mst1/2活化的调控。在Aim 2中，我们将建立mst1 /2依赖性Mob1磷酸化和激活的机制。在Aim 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Mst， Lats及其调控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通路的化合物，这可以作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 In unpublished results, we have made the following discoveries: (1) RASSF5 binding to Mst2 hinders its auto- phosphorylation-dependent activation; (2) we have determined the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.