PROJECT SUMMARY The long-term goal of our research is to understand the molecular mechanisms that control eukaryotic signaling pathways. The objective of this application is to elucidate the principles that regulate the core kinases in the Hippo pathway. The Hippo pathway controls organ size during development, tumorigenesis, and maintains the stem-cell niche through the coordinated activity of a kinase cassette that includes two kinases, Mst1/2 and Lats1/2, and two accessory proteins, Mob1 and hSalvador. In Aim 1, we will establish a molecular basis for understanding complex formation between SARAH domain proteins (hSalvador, Mst1/2, and RassF5) to inform on how these proteins regulate pathway activity. In Aim 2, our investigations will further understanding of Mst1/2 activation and provide a mechanistic link between upstream pathway components and kinase activation. In Aim 3 we will elucidate the molecular mechanisms governing the activity of Lats1 and Mst2 activity. Outcomes from the proposed work will fundamentally alter our picture of Hippo signaling in normal and disease states and stimulate new directions of cellular and therapeutic investigation.