Administrative Supplement for Control and Function of Ndr/LATS Signaling Systems (GM084223)

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DESCRIPTION (provided by applicant): Signaling pathways in which GCK group "Mst/hippo" kinases regulate AGC group "Ndr/LATS" kinases are ancient controllers of growth, proliferation, and architecture of eukaryotic cells. Our broad goal is to define the diverse intracellular processes these pathways regulate and determine the mechanisms underlying this control. Forms of Mst/hippo signaling ("hippo-warts" pathways) that suppress metazoan cell proliferation in metazoans by inhibiting YAP/yorkie-related transcriptional co-activators have been the subject of intensive recent interest. However, the distinct and highly conserved "hippo-trc" form of this pathway in which large "furry" related proteins mediate Mst/hippo kinase activation of Ndr/tricornered kinases have dramatically different functions; comparatively little is known about them, and they are the focus of this project. The hippo-trc pathways are important for polarized growth and organization of cellular extensions, neuron morphogenesis, mitotic spindle organization, and positive regulation of cell proliferation. We have successfully studied the system in budding yeast, which use a conserved hippo-trc pathway known as the "RAM network" to control cell division and polarized growth. Under close regulation by mitotic exit machinery, this pathway directly drives asymmetric localization and activity of a transcription factor that turns on expression of genes involved in the final step of cytokinesis. In addition to this primordial cell fate decision, budding yeast hippo-trc signaling promotes maintenance of cell polarity and regulates translation of proteins required for physical expansion of the cell during rapid growth. This project aims to define the regulatory mechanisms and downstream targets of Cbk1. Through combined computational and experimental work we have discovered that a novel "docking motif" peptide recruits this Ndr/LATS kinase to in vivo substrates through interaction with the kinase catalytic domain. We have crystallized the Mob2-Cbk1 complex and solved its structure, the first for any Ndr/LATS kinase, and will use this information to guide analysis of the kinase's activation mechanisms. We will define how the docking motif binds to Cbk1's kinase domain, analyze effects caused by disruption of this interaction in vivo, and determine if the novel substrate docking behavior we have discovered in budding yeast also occurs with metazoan orthologs. When combined with existing interaction and phosphoproteomic data, our analysis of substrate docking and consensus motif conservation at least triples the number of high confidence Cbk1 targets. In addition to extending our analysis of the pathway's regulation of mRNA translation, we will explore this expanded regulatory network to gain a more comprehensive mechanistic understanding of this hippo-trc pathway's control of cell division and morphogenesis.