Our long-term goal is to understand the molecular mechanisms by which cell growth is controlled in eukaryotic cells, in particular by the rapamycin-sensitive TOR kinase. Recent studies in several laboratories, including my own, have demonstrated that TOR functions as part of two distinct protein complexes, TORC1 and TORC2, where TORC1 is uniquely inhibited by rapamycin. Rapamycin is proving to have many beneficial therapeutic applications, including as a potential anti-cancer treatment, and so there is great interest in understanding more about the cellular role of TOR. Because it is not a direct target for rapamycin, however, the cellular role of TORC2 has remained much less well characterized than TORC1. Accordingly, alternative approaches are needed to study TORC2. To this end, we have discovered recently that a mutation within a TORC2-specific component affects dramatically the earliest steps of the sphingolipid pathway, in particular the de novo formation of ceramides, in budding yeast. More recently, we have extended this observation to mammalian cells, where we have shown that inhibiting mTORC2 function in HEK393T cells also leads to a reduction in the activity of the enzyme ceramide synthase. Ceramides, as well as their immediate precursors, the fatty acid long chain bases (LCBs), represent classes of lipids that are increasingly recognized as playing crucial roles in malignant cell growth, tumorigenesis, as well as aging. Thus, both TOR and ceramide biosynthesis represent important areas important for biomedical research, and our findings indicate for the first time that they are intimately linked. In the experiments proposed here, we will identify the mechanism by which ceramide synthesis is regulated by TORC2, including a detailed analysis of the ceramide synthase, and will begin to delineate the signaling pathway involved in this regulation. For this purpose, we will use both yeast and mammalian cells as complementary experimental systems. We will also address the role that mTORC2 signaling plays in the response of certain cancer cells to chemotherapeutic drugs, which are known to increase the de novo synthesis of ceramides and contribute to the cell killing action of these drugs by inducing ceramide-mediated apoptosis.
The proposed experiments address the role of a highly conserved signaling pathway, defined by the TOR kinase, in the regulation of synthesis of precursors to complex sphingolipids, in particular ceramides, using both yeast and mammalian cell culture. Both TOR signaling and ceramides are known to play important roles in cell proliferation, stress, cancer, as well as aging. Thus, our work brings together these two areas of investigation that are very important for understanding human health and disease and we expect to gain novel insights into these processes through our studies.
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