br Results br Discussion By using

Results
Discussion By using an unbiased proteomic screen for xCT binding partners, followed by functional validation, we have made the surprising discovery that mTORC2 regulates amino EGFR Inhibitor sale metabolism in tumor cells by phosphorylating serine 26 of the cystine-glutamate antiporter xCT on its cytosolic N terminus to suppress glutamate secretion. Aberrant growth factor receptor signaling and/or c-MYC activation increase glutamine uptake, converting it to glutamate to provide tumor cells with a carbon source for tricarboxylic acid cycle (TCA) anaplerosis as well as a nitrogen source for protein and nucleotide synthesis (Altman et al., 2016, DeBerardinis et al., 2008, Masui et al., 2015a). Thus, when microenvironmental nutrient levels are sufficient to support tumor cell proliferation, it would be disadvantageous for cancer cells to secrete glutamate. The mechanism identified here ensures that glutamine-derived glutamate can be used primarily for tumor growth when extracellular nutrient levels can support it. However, when nutrients become scarce, it would be advantageous for tumor cells to increase xCT-dependent cystine uptake at the expense of glutamate efflux, enabling tumor cells to buffer cellular redox stress by synthesizing glutathione from xCT-derived cystine. Therefore, the mechanism described here enables tumor cells to adapt to changing nutrient levels, linking proliferative signals to environmental conditions. It is interesting to note that mTORC2 has recently been shown to require either glucose or acetate in order to phosphorylate its downstream substrates (Masui et al., 2015b), raising the possibility that under nutrient poor conditions, lower mTORC2 signaling could tilt the balance from proliferation to survival, at least in part by favoring glutamate efflux, cystine uptake, and glutathione synthesis to protect tumor cells from cellular stress. xCT is a 12-pass transmembrane protein that has two serine residues preceded by an arginine at the −3 position (RXXS/T), S26, S51 on its N terminus that may serve as consensus phosphorylation sites for mTORC2. Unlike S51, which lies in the transmembrane domain, S26 is predicted to reside on the cytoplasmic face of the membrane, where it could be engaged by mTORC2 (Gasol et al., 2004, UniProt Consortium, 2015). Interestingly, in a SILAC-based mass spectrometric screen of TSC-null MEFs to identify mTOR-regulated proteins, Yu and colleagues identified serine 26 of xCT as a site whose phosphorylation is inhibited by the mTOR kinase inhibitor Ku-0062794, but not by rapamycin (Yu et al., 2011), consistent with our finding that xCT serine 26 is an mTORC2 substrate. mTORC2 is thought to promote its biological activity by phosphorylating AGC kinases such as AKT, PKC, and SGK1, which, in turn, phosphorylate their downstream substrates (Laplante and Sabatini, 2009, Laplante and Sabatini, 2012). It is interesting to note that we found no evidence of xCT binding to these AGC kinases either by SILAC mass spectrometry, or in the coIP studies, suggesting that mTORC2 may directly regulate xCT serine 26 phosphorylation. High xCT levels are associated with poor outcome in a number of cancer types, including GBM (Robert et al., 2015) and triple-negative breast cancer (Timmerman et al., 2013). The mTORC2-dependent mechanism reported here, in addition to a recently described paracrine mechanism of xCT reported by Briggs et al. (2016), suggests that regulation of xCT function by post-translational modification may be critical for its tumor-promoting effects. In triple-negative breast cancer cells, high extracellular glutamate levels were demonstrated to suppress xCT function, depleting tumor cells of intracellular cysteine. Intracellular cysteine depletion was shown to cause oxidation of specific cysteine residues of the prolyl hydroxylase EglN1, thereby suppressing EglN1-dependent HIF1α degradation, thus elevating intra-tumoral HIF1α levels to drive tumor growth (Briggs et al., 2016). In addition, a recent study suggests that xCT plays an important role in regulating nutrient flexibility (Shin et al., 2017). Our results identified an important molecular mechanism linking growth factor signaling with anapleurotic flux through phosphorylation of xCT on serine 26. Future studies will be needed to determine whether there is any cooperation between these complementary post-translational regulatory mechanisms.