The overall goal of this proposal is to gain a better understanding of the functions of the Ras related Ral- GTPase family. RalA and RalB have been implicated in diverse cell functions including the control of vesicle sorting, gene expression and cell proliferation. As GTPases, Ral proteins function as molecular switches to transmit extracellular signals to specific intracellular signaling cascades. Ral proteins reach the active GTP bound state in cells by interacting with one of a family of Ral-specific guanine nucleotide exchange factors (Ral-GEFs). One class of Ral-GEFs binds to and is activated by GTP-bound Ras, and a growing body of evidence supports the idea that elevated Ral-GEF/Ral signaling has the potential to contribute to human oncogenesis. This proposal will attempt to reveal the mechanisms underlying two newly identified processes by which the Ral-GEF, Ral-GDS, is regulated. One process positively regulates Ral-GEF activity through interaction with the PDK1 protein kinase, and the other negatively regulates Ral-GEF activity through protein kinase D-mediated phosphorylation. Active GTP-bound Ral proteins bind to and alter the activity of a set of downstream """"""""effector"""""""" proteins to influence cellular processes. Studies in this proposal will expand upon our recent finding that RalA but not RalB functions in the maintenance of cellular polarity by enhancing the rate of delivery of membrane proteins to the basolateral surface of epithelial cells through its newly identified effector, the exocyst, and possibly through an exocyst-independent mechanism. Thus, one set of goals is to define the biochemical basis for the difference in activities of these two closely related Ral family members. Another goal is to identify the additional RalA """"""""effector"""""""" that participates in basolateral membrane delivery. We also plan to define how RalA binding to the exocyst or other Ral effectors promotes membrane delivery in MDCK epithelial cells. Understanding how Ral functions in this process is important because faulty delivery and polarization of membrane proteins can lead to serious diseases including cystic fibrosis, I cell disease, familial, polycystic kidney disease and possibly cancer. Finally, there is a growing appreciation that Ral-GEFs contribute to downstream signaling from GTPases by mechanisms that are independent from their ability to activate GTPases. Therefore, another aim of this proposal is to evaluate the contribution of Ral-GEF binding proteins for their ability to contribute functions that complement those of active Ral in cell processes mediated by the Ral-GEF/Ral signaling cascade.
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