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 In unpublished results, we have made the following discoveries: (1) RASSF5 binding to Mst2 hinders its auto- phosphorylation-dependent activation; (2) we have determined the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