Fellowship/Rudolph: The Role of Rit GTPase Signaling in Neurons

Rit is a member of a unique subfamily of Ras-like GTPase proteins. It is highly expressed in multiple tissues including the brain and expression of an activated version of Rit in pheochromocytoma 6 (PC6) neuronal precursor cells induces potent differentiation evidenced by the formation of numerous, highly-branched neurites. Expression of Rit in PC6 cells also protects the cells from trophic factor withdrawal-induced apoptosis. The goals of this proposal are 1) to generate a series of effector loop point mutations in a constitutively activated version of Rit (Rit79L) and to determine the ability of these mutants to induce neurite outgrowth and survival in PC6 cells 2) to determine the ability of each point mutant to activate the ERK pathway, the p38 MAPK pathway, and Rho family proteins and 3) to determine the ability of each point mutant to bind the known Rit effectors B-Raf and Par6. To address these aims, a series of single amino acid effector domain mutants will be generated. The effector domain is the region of the GTPase responsible for physical interaction with downstream targets, and single amino acid mutations have previously been used to distinguish the relative importance of a particular pathway to the biological activity of the GTPase. Numerous pathways have been implicated in neuronal differentiation and survival studies, but the precise roles they play are disputed. To attempt to tease apart the role of signaling pathways known to be downstream of Rit, the neurite outgrowth inducing ability and survival promoting ability of the mutants will be tested in PC6 cells. These mutants will then be screened for their ability to stimulate the ERK and p38 MAPK pathways which are thought to be critical for survival and differentiation from a variety of stimuli. Because cytoskeletal rearrangements are necessary for the formation of neurites, it is likely that Rit activates members of the Rho family, but their activation by Rit has not been tested. Finally, the mutants will be examined for their ability to bind to known Rit interacting partners B-Raf kinase, a regulator of ERK signaling, and Par6, a Rho family interacting protein critical for establishing cellular polarization. Integration of this data should determine which binding partners and pathways are essential and dispensable for Rit-mediated differentiation and survival signaling.