Chang-Chih Wu

mTOR complex 2 regulates proper turnover of insulin receptor substrate-1 via the ubiquitin ligase subunit Fbw8.

The mammalian target of rapamycin (mTOR) integrates signals from nutrients and insulin via two distinct complexes, mTORC1 and mTORC2. Disruption of mTORC2 impairs the insulin-induced activation of Akt, an mTORC2 substrate. Here, we found that mTORC2 can also regulate insulin signaling at the level of insulin receptor substrate-1 (IRS-1). Despite phosphorylation at the mTORC1-mediated serine sites, which supposedly triggers IRS-1 downregulation, inactive IRS-1 accumulated in mTORC2-disrupted cells. Defective IRS-1 degradation was due to attenuated expression and phosphorylation of the ubiquitin ligase substrate-targeting subunit, Fbw8. mTORC2 stabilizes Fbw8 by phosphorylation at Ser86, allowing the insulin-induced translocation of Fbw8 to the cytosol where it mediates IRS-1 degradation. Thus, mTORC2 negatively feeds back to IRS-1 via control of Fbw8 stability and localization. Our findings reveal that in addition to persistent mTORC1 signaling, heightened mTORC2 signals can promote insulin resistance due to mTORC2-mediated degradation of IRS-1.

Mammalian Target of Rapamycin Complex 2 Modulates αβTCR Processing and Surface Expression during Thymocyte Development

An efficient immune response relies on the presence of T cells expressing a functional TCR. Whereas the mechanisms generating TCR diversity for antigenic recognition are well defined, what controls its surface expression is less known. In this study, we found that deletion of the mammalian target of rapamycin complex (mTORC) 2 component rictor at early stages of T cell development led to aberrant maturation and increased proteasomal degradation of nascent TCRs. Although CD127 expression became elevated, the levels of TCRs as well as CD4, CD8, CD69, Notch, and CD147 were significantly attenuated on the surface of rictor-deficient thymocytes. Diminished expression of these receptors led to suboptimal signaling, partial CD4−CD8− double-negative 4 (CD25−CD44−) proliferation, and CD4+CD8+ double-positive activation as well as developmental blocks at the CD4−CD8− double-negative 3 (CD25+CD44−) and CD8–immature CD8+ single-positive stages. Because CD147 glycosylation was also defective in SIN1-deficient fibroblasts, our findings suggest that mTORC2 is involved in the co/posttranslational processing of membrane receptors. Thus, mTORC2 impacts development via regulation of the quantity and quality of receptors important for cell differentiation.

The Target of Rapamycin: Structure and Functions

The target of rapamycin (TOR, also called the mechanistic or mammalian target of rapamycin, mTOR) is an atypical protein kinase that is highly conserved in eukaryotes (Sarbassov et al. 2005; Wullschleger et al. 2006; Jacinto & Lorberg 2008). It modulates cell growth, metabolism, and cell survival in response to diverse extracellular and intracellular signals, such as growth factors, energy levels, and nutrient status (Reiling & Sabatini 2006; Wullschleger et al. 2006; Jacinto 2008). Inhibition of mTOR activity using rapamycin and more recently via mTOR active site inhibitors and disruption of mTOR complexes, has revealed important insights on how mTOR functions under physiological and pathological conditions (Sarbassov et al. 2005; Proud 2011; Zoncu et al. 2011).

Abstract 21: The mTORC2 target Akt is regulated in response to glutamine metabolite levels

Highly proliferating cells are particularly dependent on glucose and glutamine for bioenergetics and to fuel biosynthesis of macromolecules. The signals that respond to the fluctuations of these nutrients and how they control metabolic pathways remain poorly understood. mTOR, as part of mTOR complex 1 (mTORC1), responds to amino acids and plays a central role in metabolism. On the other hand, little is known on how mTORC2, consisting of the core components mTOR, rictor, SIN1 and mLST8 is regulated and its metabolic functions. The phosphorylation of the mTORC2 substrate, Akt, is enhanced in cancer cells, suggesting that mTORC2 becomes deregulated during tumorigenesis. Here we found that the activity of mTORC2 is enhanced by diminishing glutamine-derived metabolites. mTORC2 activity is required by glutamine-requiring biosynthetic pathways such as the hexosamine biosynthetic pathway (HBP). Acute nutrient withdrawal augments Akt phosphorylation but does not affect GFAT1 expression. However, extreme starvation that eventually depletes intracellular glutamine metabolites inactivates mTORC2 and downregulates GFAT1 expression. Thus, while mTORC1 senses glutamine abundance to promote anabolism, mTORC2 responds to declining glutamine catabolites in order to restore metabolic homeostasis. Our findings uncover the role of mTORC2 in metabolic reprogramming and provide insights on more effective therapeutic strategies for glutamine-dependent tumors. Citation Format: Estela Jacinto, Joseph Moloughney, Peter K. Kim, Nicole M. Vega-Cotto, Chang-Chih Wu, Thomas Lynch, Sisi Zhang, Matthew Adlam, Sai Guntaka, Po-Chien Chou, Joshua D. Rabinowitz, Guy Werlen. The mTORC2 target Akt is regulated in response to glutamine metabolite levels. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 21.

mTORC2 modulates the amplitude and duration of GFAT1 Ser243 phosphorylation to maintain flux through the hexosamine pathway during starvation

The mechanistic target of rapamycin (mTOR) controls metabolic pathways in response to nutrients. Recently, we have shown that mTOR complex 2 (mTORC2) modulates the hexosamine biosynthetic pathway (HBP) by promoting the expression of the key enzyme of the HBP, glutamine:fructose-6-phosphate aminotransferase 1 (GFAT1). Here, we found that GFAT1 Ser-243 phosphorylation is also modulated in an mTORC2-dependent manner. In response to glutamine limitation, active mTORC2 prolongs the duration of Ser-243 phosphorylation, albeit at lower amplitude. Blocking glycolysis using 2-deoxyglucose robustly enhances Ser-243 phosphorylation, correlating with heightened mTORC2 activation, increased AMPK activity, and O-GlcNAcylation. However, when 2-deoxyglucose is combined with glutamine deprivation, GFAT1 Ser-243 phosphorylation and mTORC2 activation remain elevated, whereas AMPK activation and O-GlcNAcylation diminish. Phosphorylation at Ser-243 promotes GFAT1 expression and production of GFAT1-generated metabolites including ample production of the HBP end-product, UDP-GlcNAc, despite nutrient starvation. Hence, we propose that the mTORC2-mediated increase in GFAT1 Ser-243 phosphorylation promotes flux through the HBP to maintain production of UDP-GlcNAc when nutrients are limiting. Our findings provide insights on how the HBP is reprogrammed via mTORC2 in nutrient-addicted cancer cells.

Abstract 1150: mTORC2 enhances flux through the hexosamine biosynthetic pathway by regulation of GFAT1 expression

Metabolic and biosynthetic pathways drive cell growth and proliferation in response to nutrients and growth factors. Highly proliferating cells utilize glucose and glutamine to fuel biosynthetic processes. These two nutrients serve as substrates for the glutamine:fructose-6-phosphate amidotransferase (GFAT1), the rate-limiting enzyme in the hexosamine biosynthetic pathway (HBP), which ultimately produces UDP-GlcNAc that is necessary for protein glycosylation. Despite a role for the HBP in insulin resistance and lifespan extension, the mechanisms underlying GFAT1 regulation in vivo has remained elusive. We found mTOR complex 2 (mTORC2) controls flux through the HBP via regulation of GFAT1 expression levels in response to glucose. In the absence of mTORC2, GFAT1 expression is reduced and highly sensitive to glucose starvation. Furthermore, UDP-GlcNAc is highly diminished and glycosylation of specific transmembrane proteins such as CD147 is defective upon mTORC2 disruption. However, mTORC2 is also required for glycolysis and other biosynthetic pathways whose metabolites feed into the HBP. Thus, although exogenous UDP-GlcNAc can partially rescue glycosylation defects, it does not rescue the metabolic deficiencies in the absence of mTORC2. Like GFAT1, key enzymes of biosynthetic pathways have decreased expression in mTORC2-disrupted cells. Thus, by regulating levels of metabolic enzymes, mTORC2 coordinates flux through biosynthetic pathways in response to glucose availability. Our findings have implications for therapeutic targeting of mTORC2 in insulin resistance and cancer metabolism. Citation Format: Estela Jacinto, Joseph Moloughney, Thomas Lynch, Chang-Chih Wu, Olufunmilola Ibironke, Aixa Navia, Po-Chien Chou, Sisi Zhang, Joshua Rabinowitz, Guy Werlen. mTORC2 enhances flux through the hexosamine biosynthetic pathway by regulation of GFAT1 expression. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1150. doi:10.1158/1538-7445.AM2015-1150

Abstract 2441: mTOR complex 2 modulates glycosylation of CD147 via the hexosamine biosynthetic pathway

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Cell surface proteins transduce extracellular signals into the cell to control metabolism and growth. In turn, their expression is linked to nutrient availability and other growth signals by mechanisms that are poorly understood. The mammalian target of rapamycin (mTOR) regulates cell growth and metabolism and is part of two distinct protein complexes, mTOR complex 1 (mTORC1) and mTORC2. We found that mTORC2 is involved in the processing and maturation of cell surface receptors such as CD147. CD147 is a highly glycosylated receptor that has been linked to tumor progression via its role in activating matrix metalloproteinases and maturation of lactate transporters. In breast cancer cells, CD147 glycosylation is highly sensitive to glucose starvation and mTOR inhibition. In mTORC2-disrupted cells CD147 is misprocessed and occurs predominantly in a low glycosylated form. CD147 misprocessing can be partly rescued by addition of exogenous UDP-GlcNAc, the end product of the hexosamine biosynthetic pathway (HBP). However, UDP-GlcNAc cannot restore the abnormal growth and metabolism in mTORC2-disrupted cells due to defects in expression of other key metabolic enzymes in these cells. Our findings define a role for mTORC2 in regulating receptor glycosylation via the HBP and reveal a broader role for mTORC2 in controlling other biosynthetic pathways that become deregulated in cancer. Citation Format: Chang-Chih Wu, Thomas Lynch, Joseph Moloughney, Aixa Navia, Olufunmilola Ibironke, Po-Chien Chou, Nicole M. Vega-Cotto, Sisi Zhang, Joshua Rabinowitz, Guy Werlen, Estela Jacinto. mTOR complex 2 modulates glycosylation of CD147 via the hexosamine biosynthetic pathway. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2441. doi:10.1158/1538-7445.AM2014-2441