Abstract
Mammalian target of rapamycin complex 1 (mTORC1) is a master regulator of cell growth, proliferation, survival, metabolism, and autophagy. Increases in mTORC1 activity have been linked to cancer, neurodegeneration, and autoimmune diseases. Rapamycin is a known mTORC1 inhibitor that mainly targets the p70S6k phosphorylation pathway. Rapamycin is an FDA-approved drug for the treatment of cancer, lung disease, and for use after kidney transplants. Our laboratory recently discovered a small molecule, CB3A, that inhibits mTORC1 signaling and protein translation by inhibiting the phosphorylation of 4EBP1 and, to a lesser extent, p70S6k. However, the precise mechanism by which CB3A inhibits mTORC1 signaling is unknown. Since mTORC1 inhibitors can increase lifespan and are an effective treatment for cancers and neurodegenerative diseases, elucidation of the precise mechanism of CB3A action may enable us to develop future therapeutic modalities. Tuberous sclerosis complex 2 (TSC2) is the main negative regulator of mTORC1 activity and inhibits mTORC1 by binding to the GTPase Rheb. The amino acid arginine can induce dissociation of TSC2-Rheb, which results in mTORC1 activation. We treated LAM (TSC2 null) and HEK 239T cell lines with CB3A and analyzed 4EBP1 and p70S6k phosphorylation by immunoblotting. We found that CB3A requires TSC2 for mTORC1 inhibition, increases the binding of TSC2 to Rheb, and may interfere with the arginine-induced dissociation of TSC2 and Rheb. Additionally, previous SILAC experiments have shown that ubiquilin 2 is a candidate CB3A binding protein. Therefore, we are currently using the BioID2 method to analyze protein-protein interactions of ubiquilin 2 to determine if CB3A is inducing the degradation of key mTORC1 pathway proteins.