Characterization of Rheb Signaling in Cancer and Disease

Ras homolog enriched in brain (RHEB) is a member of the Ras superfamily of small GTPases that are responsible for the activation of numerous diverse cellular pathways in the cell. Rheb directly activates mTORC1, a crucial signaling pathway that stimulates protein synthesis and cell proliferation. Aberrant Rheb/mTORC1 signaling has been linked to many diseases including cancer. Unfortunately, mTORC1 inhibitors often display only partial antitumor activity, with tumor regrowth upon discontinuation of the drugs. Some reports suggest Rheb may have mTORC1-indpendent functions, but studies to identify additional Rheb effector proteins are lacking. The aim of this dissertation is to identify new effector proteins of Rheb, and characterize the signaling pathways affected by Rheb interaction with these proteins. To accomplish this, I employ various biochemical techniques. The main approach in identifying new effector proteins is to look for proteins that do not interact with Rheb effector domain mutants, show similar co-localization with Rheb, and whose function is altered upon Rheb interaction.In this dissertation I present results identifying four different effector proteins of Rheb, and explore the biological relevance of these interactions. First I confirm Rheb interaction with mTOR and TSC. Second, I show a novel interaction between Rheb and CAD, the protein responsible for carrying out the first three enzymatic steps in de novo pyrimidine biosynthesis. In this section I present results demonstrating Rheb stimulates pyrimidine biosynthesis through CAD. Third, I show Rheb interaction with AMPK, a kinase complex that shuts down cell growth and proliferation. Fourth, I show RHEB interaction with BRAF that results in the inhibition of the MAPK pathway. In this section I identify a RHEB mutation from cancer genome database analysis, and demonstrate its ability to transform normal cells into cancer cells through stimulation of the RAF/MEK/ERK signaling pathway. In conclusion, this dissertation identifies new effector proteins of Rheb and significantly expands our current knowledge of Rheb signaling