PROJECT SUMMARY Eukaryotic appropriately commence daughter carcinogenesis processes cytokinesis cell division requires dependency relationships in which late events start only after early ones are completed. During cytokinesis, for example, distinct large-scale morphological processes in precise and consistent order to accomplish architectural reorganization that produces two cells. Failure of mechanical division of cells can drastically affect genome stability, contributing to and other important human maladies. mechanisms that coordinate cytokinetic are incompletely understood, and a subject of intense research interest. Notably, machinery of and regulators currently known to control it are highly conserved. Working in budding yeast, we Regulatory have discovered a system that blocks late events of cytokinesis when early ones are delayed or defective, allowing time for corrective mechanisms to function. This “checkpoint” pathway, termed “Enforcement of Cytokinesis Order” (ECO), works by stabilizing a multi-motif protein that blocks a cytokinesis-specific secretion function of the Ndr/Lats protein kinase Cbk1, a key component of a highly conserved “Ndr-hippo” signaling system. Hippo signaling pathways are crucial regulators of morphogenesis, proliferation, and differentiation of eukaryotic cells; Ndr/Lats kinases are their downstream-most signaling components. Our prior research contributed crucial analysis for understanding of these systems. We were was among the first to describe specific functions of a hippo signaling system in vivo, defined the distinctive Ndr kinase phosphorylation motif, reported the first canonical substrate docking by an AGC-family protein kinase, and contributed the first and only crystal structure of an Ndr/Lats kinase bound to a Mob co-activator. This project seeks to determine how the checkpoint mechanism we discovered blocks secretion of late cytokinesis proteins when early stages of the process are defective. It will include exploration of in vivo docking interactions with cytokinesis regulators, assessment of secretion organization during cytokinesis, and evaluation of the importance of Ndr-hippo regulation of key effector proteins of cytokinesis. We will also explore the hypothesis that human Ndr-hippo pathways participate in control of late cytokinesis, using two different approaches. In one, we will determine if human Ndr kinases engage in docking interactions with in vivo phosphorylation targets, using a highly optimized phage display approach to identify possible peptide motifs that interact with Ndr kinase – Mob co-activator complexes. We will use a new ligand footprinting method we developed to map peptide motif associations. In another approach, we will determine if human Ndr-hippo signaling functions in cytokinesis by constructing cell lines expressing only analog sensitive Ndr kinases. This will allow rapid and reversible inhibition of these kinases in cells undergoing both normal and defective cytokinesis.

Project Narrative Cytokinesis is a rapid and precisely orchestrated sequence of events that creates two eukaryotic cells from one; we seek to understand mechanisms that enforce the order of cytokinesis processes that need to happen in an exact sequence. We have discovered a system in budding yeast that keeps late events of cytokinesis from starting when early ones are delayed or disrupted. This project aims to find out how this system works, and to determine if related mechanisms protect cytokinesis in human cells.