The central nervous system is incredibly complex, consisting of billions of neurons, some of which form thousands of synaptic connections with other neurons. The critical feature of the nervous system is its ability to propagate information, and this requires that synaptic connections be formed at the correct location and with the correct density. Construction of functional neural circuits results from the coordination and integration of a series of developmental events. Neurons extend an axon outward and navigate to a target cell, form synapses, and terminate outgrowth in a spatially and temporally precise manner. These developmental events are often analyzed independently of one another, although several studies suggest that they are coordinated. At present, the intracellular signaling mechanisms that regulate coordination of these different events in development remain poorly understood. The Pam/Highwire/RPM-1 (PHR) proteins are conserved intracellular regulators of axon guidance, synapse formation, and axon termination, and have emerged as candidates that mediate the coordination of these developmental events. Despite significant progress in understanding how the PHR proteins function, more knowledge is needed to fully understand how these proteins integrate information and regulate multiple signaling pathways. In the following dissertation, I outline several studies that focus on the identification of novel downstream molecules and pathways that mediate the function of the C. elegans PHR protein RPM-1. These studies have led to the discovery of additional components of PHR signaling that have strengthened their candidacy as intracellular signaling proteins that mediate the coordination between different developmental events. In the first study, we identify the PP2C phosphatase PPM-2 as an RPM-1 binding protein, and show that PPM-2 functions independent of the RPM-1/FSN-1 ubiquitin ligase complex to negatively regulate DLK-1 during neuronal development. This study demonstrates that RPM-1 functions through both phosphatase and ubiquitin ligase mechanisms to inhibit DLK-1, thus making PHR proteins more accurate and sensitive regulators of DLK-1 than originally thought. In the second study, we identify an MLK-1 MAP kinase pathway that regulates neuronal development, and show that RPM-1 negatively regulates this pathway. Overall, these findings expand our understanding of how neurons employ both the ubiquitin ligase activity of RPM-1 and PP2C phosphatase activity to inhibit the DLK-1 and MLK-1 pathways. In a third study study, we detail structure-function analysis of RPM-1 and the F-box protein FSN-1. Our biochemical and genetic analysis led to the identification of RIP, an in vivo inhibitor of the RPM-1/FSN-1 ubiquitin ligase complex that to our knowledge represents the first inhibitor of a PHR ubiquitin ligase complex.
University of Minnesota Ph.D. dissertation. December 2015. Major: Pharmacology. Advisor: Brock Grill. 1 computer file (PDF); viii, 148 pages.
The C. elegans PHR protein RPM-1: Deciphering the molecular mechanisms of neuronal development by examining a central regulatory molecule.
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