The 3.2-Å resolution structure of human mTORC2.

The protein kinase mammalian target of rapamycin (mTOR) is the central regulator of cell growth. Aberrant mTOR signaling is linked to cancer, diabetes, and neurological disorders. mTOR exerts its functions in two distinct multiprotein complexes, mTORC1 and mTORC2. Here, we report a 3.2-Å resolution cryo-EM reconstruction of mTORC2. It reveals entangled folds of the defining Rictor and the substrate-binding SIN1 subunits, identifies the carboxyl-terminal domain of Rictor as the source of the rapamycin insensitivity of mTORC2, and resolves mechanisms for mTORC2 regulation by complex destabilization. Two previously uncharacterized small-molecule binding sites are visualized, an inositol hexakisphosphate (InsP6) pocket in mTOR and an mTORC2-specific nucleotide binding site in Rictor, which also forms a zinc finger. Structural and biochemical analyses suggest that InsP6 and nucleotide binding do not control mTORC2 activity directly but rather have roles in folding or ternary interactions. These insights provide a firm basis for studying mTORC2 signaling and for developing mTORC2-specific inhibitors.

Repression of Human Papillomavirus Oncogene Expression under Hypoxia Is Mediated by PI3K/mTORC2/AKT Signaling.

Hypoxia is linked to therapeutic resistance and poor clinical prognosis for many tumor entities, including human papillomavirus (HPV)-positive cancers. Notably, HPV-positive cancer cells can induce a dormant state under hypoxia, characterized by a reversible growth arrest and strong repression of viral E6/E7 oncogene expression, which could contribute to therapy resistance, immune evasion and tumor recurrence. The present work aimed to gain mechanistic insights into the pathway(s) underlying HPV oncogene repression under hypoxia. We show that E6/E7 downregulation is mediated by hypoxia-induced stimulation of AKT signaling. Ablating AKT function in hypoxic HPV-positive cancer cells by using chemical inhibitors efficiently counteracts E6/E7 repression. Isoform-specific activation or downregulation of AKT1 and AKT2 reveals that both AKT isoforms contribute to hypoxic E6/E7 repression and act in a functionally redundant manner. Hypoxic AKT activation and consecutive E6/E7 repression is dependent on the activities of the canonical upstream AKT regulators phosphoinositide 3-kinase (PI3K) and mechanistic target of rapamycin (mTOR) complex 2 (mTORC2). Hypoxic downregulation of E6/E7 occurs, at least in part, at the transcriptional level. Modulation of E6/E7 expression by the PI3K/mTORC2/AKT cascade is hypoxia specific and not observed in normoxic HPV-positive cancer cells. Quantitative proteome analyses identify additional factors as candidates to be involved in hypoxia-induced activation of the PI3K/mTORC2/AKT signaling cascade and in the AKT-dependent repression of the E6/E7 oncogenes under hypoxia. Collectively, these data uncover a functional key role of the PI3K/mTORC2/AKT signaling cascade for viral oncogene repression in hypoxic HPV-positive cancer cells and provide new insights into the poorly understood cross talk between oncogenic HPVs and their host cells under hypoxia.IMPORTANCE Oncogenic HPV types are major human carcinogens. Under hypoxia, HPV-positive cancer cells can repress the viral E6/E7 oncogenes and induce a reversible growth arrest. This response could contribute to therapy resistance, immune evasion, and tumor recurrence upon reoxygenation. Here, we uncover evidence that HPV oncogene repression is mediated by hypoxia-induced activation of canonical PI3K/mTORC2/AKT signaling. AKT-dependent downregulation of E6/E7 is only observed under hypoxia and occurs, at least in part, at the transcriptional level. Quantitative proteome analyses identify additional factors as candidates to be involved in AKT-dependent E6/E7 repression and/or hypoxic PI3K/mTORC2/AKT activation. These results connect PI3K/mTORC2/AKT signaling with HPV oncogene regulation, providing new mechanistic insights into the cross talk between oncogenic HPVs and their host cells.

Insulin resistance causes inflammation in adipose tissue.

Obesity is a major risk factor for insulin resistance and type 2 diabetes. In adipose tissue, obesity-mediated insulin resistance correlates with the accumulation of proinflammatory macrophages and inflammation. However, the causal relationship of these events is unclear. Here, we report that obesity-induced insulin resistance in mice precedes macrophage accumulation and inflammation in adipose tissue. Using a mouse model that combines genetically induced, adipose-specific insulin resistance (mTORC2-knockout) and diet-induced obesity, we found that insulin resistance causes local accumulation of proinflammatory macrophages. Mechanistically, insulin resistance in adipocytes results in production of the chemokine monocyte chemoattractant protein 1 (MCP1), which recruits monocytes and activates proinflammatory macrophages. Finally, insulin resistance (high homeostatic model assessment of insulin resistance [HOMA-IR]) correlated with reduced insulin/mTORC2 signaling and elevated MCP1 production in visceral adipose tissue from obese human subjects. Our findings suggest that insulin resistance in adipose tissue leads to inflammation rather than vice versa.

mTORC2 sustains thermogenesis via Akt-induced glucose uptake and glycolysis in brown adipose tissue.

Activation of non-shivering thermogenesis (NST) in brown adipose tissue (BAT) has been proposed as an anti-obesity treatment. Moreover, cold-induced glucose uptake could normalize blood glucose levels in insulin-resistant patients. It is therefore important to identify novel regulators of NST and cold-induced glucose uptake. Mammalian target of rapamycin complex 2 (mTORC2) mediates insulin-stimulated glucose uptake in metabolic tissues, but its role in NST is unknown. We show that mTORC2 is activated in brown adipocytes upon ß-adrenergic stimulation. Furthermore, mice lacking mTORC2 specifically in adipose tissue (AdRiKO mice) are hypothermic, display increased sensitivity to cold, and show impaired cold-induced glucose uptake and glycolysis. Restoration of glucose uptake in BAT by overexpression of hexokinase II or activated Akt2 was sufficient to increase body temperature and improve cold tolerance in AdRiKO mice. Thus, mTORC2 in BAT mediates temperature homeostasis via regulation of cold-induced glucose uptake. Our findings demonstrate the importance of glucose metabolism in temperature regulation.

Making new contacts: the mTOR network in metabolism and signalling crosstalk.

More than 20 years after its discovery, our understanding of target of rapamycin (TOR) signalling continues to grow. Recent global 'omics' studies have revealed physiological roles of mammalian TOR (mTOR) in protein, nucleotide and lipid synthesis. Furthermore, emerging evidence provides new insight into the control of mTOR by other pathways such as Hippo, WNT and Notch signalling. Together, this progress has expanded the list of downstream effectors and upstream regulators of mTOR signalling.