Project Summary One of the most fundamental challenges in cell biology is understanding how cells migrate in three- dimensional extracellular matrices (ECM) at specific times and to specific locations. Cell migration helps shape all tissues and organs during development and the disruption of the normal mechanisms that normally control migration dramatically enhance the lethality of cancers. Our recent studies identified members of Rab40 sub- family as important regulators of cell migration. Rab proteins are the largest family of small monomeric GTPases belonging to the Ras oncogene superfamily. Among them, Rab40 sub-family is unique because it contains a suppressor of cytokine signaling (SOCS) box located at the C-terminal end of the protein. Importantly, we and others have shown that Rab40 sub-family of proteins bind to Cullin5 via the SOCS box to form a ubiquitin E3 ligase complex that regulate various aspects of cell migration in vitro and in vivo. Consequently, Rab40 proteins emerged as important coordinators between membrane trafficking, cytoskeleton dynamics and cell signaling, and understanding the molecular machinery governing functions of Rab40 sub- family during cell migration is a major focus of this proposal. In all vertebrates Rab40 family consists of two members, Rab40b and Rab40c. Our recent work has shown that Rab40b and Rab40c are important for targeted secretion of matrix metalloproteinases, as well as regulation of actin dynamics during breast cancer migration, invasion and metastasis. We also have shown that Rab40b/Cullin5 complex mediate ubiquitylation of several regulators of cell migration, including Rap2, while Rab40c/Cullin5 mediates protein phosphatase 6 (PP6) complex ubiquitylation and inactivation. Based on our published and preliminary data, we hypothesize that Rab40b and Rab40c mediate coordination between signaling, membrane trafficking, and actin dynamics during cell migration. The following aims are designed to test this hypothesis by combining of the unique expertise from Dr. Rytis Prekeris (Rab GTPases and actin dynamics), Dr. Kristin Artinger (zebrafish and neural crest cell migration), and Dr. Traci Lyons (breast cancer). First, we will define the roles of Rab40b/Cullin5 and Rap2 complexes in regulating membrane and actin dynamics during cell migration by mapping Rab40b-dependent Rap2 ubiquitylation sites and dissecting how Rap2 regulates actin and focal adhesion site dynamics at the leading edge and/or invadopodia. Second, we will elucidate the roles of Rab40c/Cullin5-PP6 complex in regulating signaling during cell migration. To that end, we will use protein binding assays in combination with protein mutagenesis and various microscopy approaches to determine biochemical properties of PP6 binding to Rab40c and the consequences of this binding on PP6 complex stability and activity. Third, we will determine the functions of Rab40b/Cullin5-Rap2 and Rab40c/Cullin5-PP6 pathways during neural crest cell migration in...