The Hippo pathway is crucial for tumor suppression and is genetically altered in 10% of all human cancers. Hippo signaling regulates tissue proliferation, development, and apoptosis, and is a leading target for anticancer therapeutic development. Angiomotin (AMOT) functions as the central signaling platform that integrates Hippo signaling inputs and transduces them into biological outputs that either consolidate tight junctions and cell homeostasis (HIPPO “on”) or promote actin assembly and proliferative gene transcription (HIPPO “off”). In the Sundquist lab we propose to define the biochemistry and structural biology of central AMOT assemblies, both free and in complex with actin, inhibitory kinases, tumor suppressors, transcriptional co-activators, and ubiquitation enzymes. The completion of these aims will: 1) reveal the architectures of AMOT assemblies, 2) provide insights into how these platforms promote actin polymerization and thereby activate proliferation, and 3) position us well to obtain external funding for more comprehensive structural and functional studies of different motin family members, including other AMOT family isoforms and their complexes with the Merlin tumor suppressor, the HECT ubiquitin E3 ligase NEDD4L, inhibitory LATS1/2 kinases, and YAP/TAZ transcriptional coactivators.