Regulation and Roles of Ral GTPase Signaling Components in Oncogenesis

Martin, Timothy Dean

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March 20, 2019

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Martin, Timothy Dean
- Affiliation: School of Medicine, Department of Pharmacology

Abstract

Since their discovery in 1986, Ral (Ras-like) GTPases have emerged as critical regulators of diverse cellular functions. Like Ras, the Ral proteins cycle between an inactive GDP-bound state and an active GTP-bound conformation. Ral guanine nucleotide exchange factors (RalGEFs) facilitate the exchange of GDP for GTP thus activating the Ral proteins. When bound to GTP, Ral can interact with an array of downstream effector proteins and mediate numerous biological processes. Ral GTPase-activating proteins (RalGAPs) catalyze the hydrolysis of the bound GTP returning Ral to an inactive, GDP-bound conformation. RalGEFs function as downstream effectors of the Ras oncoprotein that is mutationally active in approximately one-third of human cancers. The RalGEF-Ral signaling network comprises the third best-characterized effector of Ras-dependent human oncogenesis. The two Ral isoforms, RalA and RalB, have been found to play key roles in both normal and tumor cell biology including regulation of vesicular trafficking, migration and invasion, tumor formation, metastasis, and gene expression. Examination of the contribution of Ral GTPase signaling in colorectal cancer (CRC) cells found opposing roles for RalA and RalB in regulating anchorage-independent growth. Specifically, RalA was necessary for anchorage-independent growth while RalB functioned to suppress anchorage-independent proliferation. We determined that RalA and RalB utilized common and distinct effector proteins to drive their respective growth properties. Lastly, we found that depletion of one Ral isoform resulted in the upregulation of the activity of the remaining isoform indicating that RalA may be a viable therapeutic target to curb the growth of CRC. Previous efforts to understand small GTPase signaling has found that phosphorylation in the membrane-targeting region of a number of small GTPases results in profound changes in their signaling properties. We found that RalB is phosphorylated by PKC! on serine 198 and that this phosphorylation event results in the relocalization of RalB from the plasma membrane to endomembranes concurrent with a change in RalB GTP-loading. Phosphorylation of RalB also results in a change in RalB effector utilization where non-phosphorylated RalB interacting with the exocyst and phosphorylated RalB associating with RalBP1. Interestingly, we found that phosphorylation of RalB controls vesicular trafficking and that the surface expression of !5-integrin is dependent upon RalB phosphorylation/dephosphorylation cycling. Finally we determined that RalGAP signaling functions similar to Tsc1/2 signaling to control the activity of mTORC1, a key regulator of cellular metabolism and homeostasis. Loss of RalGAP in C. elegans resulted in decreased lifespan similar to what has been seen in other organisms upon Tsc1/2 loss or mTORC1 inhibition. We found that RalB but not RalA could directly engage mTORC1 but not mTORC2 and that this association was sensitive to serum but not amino acid stimulation. We show that RalB utilizes the exocyst as the key effector protein responsible for mTORC1 engagement and serum stimulation results in a RalB-dependent translocation of mTORC1 to the plasma membrane. Surprisingly, we found that the tumor suppressor Tsc1/2 complex also regulates Ral GTPase activity and that RalGAP expression can restore mTORC1 signaling in Tsc-deficient cells. In pancreatic cancer (PDAC) cells, where RalB is known to drive invasion and metastasis, loss of RalGAP signaling enhanced RalB activation and led to an increase in cellular invasion. This increase in invasion upon RalGAP loss was blocked by treatment with the mTOR inhibitor rapamycin. Together, these studies have further defined Ral signaling in Ras-driven tumor cells by identifying key signaling events that regulate or are regulated by Ral GTPase signaling. This work provides a more in depth framework for potentially targeting Ral for the treatment of diseases including cancer.

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