Dynamic Adipocyte Phosphoproteome Reveals that Akt Directly Regulates mTORC2

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Prolonged rapamycin treatment inhibits mTORC2 assembly and Akt/PKB

The drug rapamycin has important uses in oncology, cardiology, and transplantation medicine, but its clinically relevant molecular effects are not understood. When bound to FKBP12, rapamycin interacts with and inhibits the kinase activity of a multiprotein complex composed of mTOR, mLST8, and raptor (mTORC1). The distinct complex of mTOR, mLST8, and rictor (mTORC2) does not interact with FKBP12-rapamycin and is not thought to be rapamycin sensitive. mTORC2 phosphorylates and activates Akt/PKB, a key regulator of cell survival. Here we show that rapamycin inhibits the assembly of mTORC2 and that, in many cell types, prolonged rapamycin treatment reduces the levels of mTORC2 below those needed to maintain Akt/PKB signaling. The proapoptotic and antitumor effects of rapamycin are suppressed in cells expressing an Akt/PKB mutant that is rapamycin resistant. Our work describes an unforeseen mechanism of action for rapamycin that suggests it can be used to inhibit Akt/PKB in certain cell types.     

mTORC2/Akt的反馈激活可拮抗隐丹参酮对HepG2细胞的抑制作用

目的:研究隐丹参酮对Akt活性的影响及其在抑制HepG2细胞生长中的作用。方法:Western印迹检测隐丹参酮对Akt磷酸化的影响;CCK-8法检测隐丹参酮与MK2206或PP242联合用药对HepG2的生长抑制作用。结果:Western印迹证明隐丹参酮处理能够增强HepG2细胞Akt的磷酸化,同时发现隐丹参酮对Akt的增强作用依赖于mTORC2的活性;通过MK2206或PP242抑制Akt的反馈激活,能够明显促进隐丹参酮对HepG2细胞的生长抑制作用。结论:通过抑制Akt的反馈激活能够增强隐丹参酮的抗肿瘤作用,为隐丹参酮肿瘤治疗的临床应用联合用药提供了理论基础。     

mTORC2 is a tyrosine kinase

The mammalian target of rapamycin (mTOR), a protein kinase, is the centre of huge attention due to its importance in intracellular signaling and in health and disease. In their recent study, Yin et al. show that mTOR can regulate signaling through the insulin-like growth factor 1 receptor and that it possesses a new enzymatic activity — the ability to phosphorylate proteins on tyrosine residues.mTOR is a large, multi-domain protein; its catalytic domain resembles that of lipid kinases such as phosphoinositide 3-kinase (PI 3-kinase), but mTOR actually has protein kinase activity, adding phosphate groups to serine or threonine residues in a growing catalog of substrates, many of which are involved in anabolic pathways.mTOR binds to several protein partners in the cell to form two distinct types of complexes, termed mTOR complexes 1 and 2 (mTORC1/2¹). These differ in their protein components, substrate specificity and regulation. For example, mTORC1 is activated by amino acids, and by hormones and growth factors. mTORC1 contains a protein termed Raptor which allows it to phosphorylate substrates such as the ribosomal protein S6 kinases (S6Ks), and this effect is blocked by rapamycin.mTORC2 contains Rictor in place of Raptor and therefore phosphorylates a distinct set of substrates. These include regulatory (so-called ''hydrophobic'') sites in a family of protein kinases which include Akt, also called protein kinase B (PKB). Rapamycin does not directly inhibit mTORC2 function, but can impair it after longer-term treatment². The regulation of mTORC2 activity remains poorly understood.mTOR complexes play multifaceted roles in insulin signaling. For example, Akt plays key roles in insulin signaling, mediating the regulation of various proteins involved in the effects of this hormone on metabolism, e.g., glucose transport. Akt signaling indirectly activates mTORC1. In turn, mTORC1 regulates key anabolic processes including protein, lipid and ribosome synthesis. However, mTORC1 can, via the S6Ks, inhibit insulin signaling. This involves the phosphorylation of insulin receptor substrates 1 or 2 (IRS1/2), a crucial link between insulin (and related) receptors and downstream signalling protein, e.g., Akt.The receptors for insulin (InsR) and insulin-like growth factor I (IGF-IR) are ligand-activated tyrosine kinases, which undergo autophosphorylation allowing them to phosphorylate additional proteins such as IRS1. In turn, phosphorylated IRS1 binds PI 3-kinase; this leads to enhanced production of phosphatidylinositol 3,4,5-trisphosphate, PIP₃, and to activation of Akt.Yin et al.³ found that rapamycin led to increased phosphorylation of InsR and IGF-IR at key autophosphorylation sites, reflecting increased kinase activity of these receptors.Knockdown of mTOR or Rictor, or treatment of cells with an inhibitor of mTOR kinase activity, Torin 2, decreased the rapamycin-induced phosphorylation of InsR or IGF-IR, while Raptor knockdown had the converse effect. This indicates the effect requires mTORC2; indeed, the authors show that mTORC2 binds to these receptors, apparently via IRS1/2. However, mTORC2 does not appear to directly phosphorylate IRS1/2. One possible way in which mTORC2 increases tyrosine phosphorylation of InsR or IGF-IR is by stimulating the kinase activity of the receptors which then catalyse the phosphorylation of the receptors on tyrosine. The authors ruled this out, by using kinase-dead versions of the receptors or mTOR. Therefore, mTORC2 promotes the tyrosine phosphorylation of InsR/IGF-1R, which is required for downstream signaling from these receptors. While these authors clearly show that rapamycin causes increased phosphorylation of the mTORC2 substrate AKT, earlier studies showed that, at similar time points of treatment in the same cell-type, rapamycin inhibited AKT phosphorylation indicating interference with mTORC2 function². It is not clear how rapamycin promotes mTORC2 function under the conditions used in this study. Another study⁴ found that mTORC2 promotes degradation of IRS1, suggesting, in contrast to the conclusions of Yin et al., that mTORC2 can promote insulin resistance. These and other data suggest that the web of interactions between these signaling components is indeed very complex (Figure 1).Open in a separate windowFigure 1Summary of the signalling connections discussed here, including the new link described by Yin et al.³ between mTORC2 and the insulin/IGF-1 receptors. Phosphorylation sites are shown schematically (not all are indicated) as ''P'' in a yellow background; Y, S and T indicate tyrosine, serine and threonine respectively. Green and red arrows show activating and inhibitory phosphorylation events respectively. The gray arrow and ''?'' indicate potential further tyrosine phosphorylation events catalysed by mTORC2. Solid arrows show direct phosphorylation events; dashed lines are indirect signalling links.mTOR has previously only been reported to act on serine or threonine residues; the present report shows that mTOR can efficiently phosphorylate tyrosines in vitro using either recombinant InsR or peptides as substrate. These data reveal that mTORC2 function is a ''dual-specificity'' protein kinase phosphorylating tyrosine as well as serine/threonine sites. Interestingly, mTORC1 was unable to phosphorylate tyrosines.Does the mTORC2-stimulated phosphorylation of the InsR/IGF-1R play a role in the actions of the ligands for these receptors? To test this, the authors examined the Rictor knockdown on HepG2 cell proliferation. While this had no effect in the absence of insulin or IGF-1, depletion of Rictor did inhibit proliferation in IGF-1- or insulin-stimulated conditions. Rictor overexpression increased proliferation, an effect that requires the activity of the InsR/IGF-1R.What are the main implications of these data? First, rapamycin may actually promote signaling from the InsR/IGF-1R through mTORC2 (as well as via Grb10, a target for mTORC1 itself^5,6) both by the mechanism delineated here and by abrogating the feedback loop from mTORC1 via the S6Ks to IRS1. Second, combining Ins/IGF-1R receptor inhibitors with mTOR inhibitors may be a more effective anti-cancer treatment than inhibiting the individual pathways. Third, mTORC2 may phosphorylate additional, so far unidentified proteins on tyrosine, adding to the growing repertoire of mTOR substrates.     

mTORC2 can associate with ribosomes to promote cotranslational phosphorylation and stability of nascent Akt polypeptide

The mechanisms that couple translation and protein processing are poorly understood in higher eukaryotes. Although mammalian target of rapamycin (mTOR) complex 1 (mTORC1) controls translation initiation, the function of mTORC2 in protein synthesis remains to be defined. In this study, we find that mTORC2 can colocalize with actively translating ribosomes and can stably interact with rpL23a, a large ribosomal subunit protein present at the tunnel exit. Exclusively during translation of Akt, mTORC2 mediates phosphorylation of the nascent polypeptide at the turn motif (TM) site, Thr450, to avoid cotranslational Akt ubiquitination. Constitutive TM phosphorylation occurs because the TM site is accessible, whereas the hydrophobic motif (Ser473) site is concealed in the ribosomal tunnel. Thus, mTORC2 can function cotranslationally by phosphorylating residues in nascent chains that are critical to attain proper conformation. Our findings reveal that mTOR links protein production with quality control.     

Hepatic mTORC2 activates glycolysis and lipogenesis through Akt, glucokinase, and SREBP1c

Mammalian target of rapamycin complex 2 (mTORC2) phosphorylates and activates AGC kinase family members, including Akt, SGK1, and PKC, in response to insulin/IGF1. The liver is a key organ in insulin-mediated regulation of metabolism. To assess the role of hepatic mTORC2, we generated liver-specific rictor knockout (LiRiKO) mice. Fed LiRiKO mice displayed loss of Akt Ser473 phosphorylation and reduced glucokinase and SREBP1c activity in the liver, leading to constitutive gluconeogenesis, and impaired glycolysis and lipogenesis, suggesting that the mTORC2-deficient liver is unable to sense satiety. These liver-specific defects resulted in systemic hyperglycemia, hyperinsulinemia, and hypolipidemia. Expression of constitutively active Akt2 in mTORC2-deficient hepatocytes restored both glucose flux and lipogenesis, whereas glucokinase overexpression rescued glucose flux but not lipogenesis. Thus, mTORC2 regulates hepatic glucose and lipid metabolism via insulin-induced Akt signaling to control whole-body metabolic homeostasis. These findings have implications for emerging drug therapies that target mTORC2.     

mTORC2 protein complex-mediated Akt (Protein Kinase B) Serine 473 Phosphorylation is not required for Akt1 activity in human platelets [corrected

Protein kinase B (PKB, Akt) is a Ser/Thr kinase involved in the regulation of cell survival, proliferation, and metabolism and is activated by dual phosphorylation on Thr(308) in the activation loop and Ser(473) in the hydrophobic motif. It plays a contributory role to platelet function, although little is known about its regulation. In this study, we investigated the role of the mammalian target of rapamycin complex (mTORC)-2 in Akt regulation using the recently identified small molecule ATP competitive mTOR inhibitors PP242 and Torin1. Both PP242 and Torin1 blocked thrombin and insulin-like growth factor 1-mediated Akt Ser(473) phosphorylation with an IC(50) between 1 and 5 nm, whereas the mTORC1 inhibitor rapamycin had no effect. Interestingly, PP242 and Torin1 had no effect on Akt Thr(308) phosphorylation, Akt1 activity, and phosphorylation of the Akt substrate glycogen synthase kinase 3β, indicating that Ser(473) phosphorylation is not necessary for Thr(308) phosphorylation and maximal Akt1 activity. In contrast, Akt2 activity was significantly reduced, concurrent with inhibition of PRAS40 phosphorylation, in the presence of PP242 and Torin1. Other signaling pathways, including phospholipase C/PKC and the MAPK pathway, were unaffected by PP242 and Torin1. Together, these results demonstrate that mTORC2 is the kinase that phosphorylates Akt Ser(473) in human platelets but that this phosphorylation is dispensable for Thr(308) phosphorylation and Akt1 activity.     

Amino acids activate mammalian target of rapamycin complex 2 (mTORC2) via PI3K/Akt signaling

The activity of mammalian target of rapamycin (mTOR) complexes regulates essential cellular processes, such as growth, proliferation, or survival. Nutrients such as amino acids are important regulators of mTOR complex 1 (mTORC1) activation, thus affecting cell growth, protein synthesis, and autophagy. Here, we show that amino acids may also activate mTOR complex 2 (mTORC2). This activation is mediated by the activity of class I PI3K and of Akt. Amino acids induced a rapid phosphorylation of Akt at Thr-308 and Ser-473. Whereas both phosphorylations were dependent on the presence of mTOR, only Akt phosphorylation at Ser-473 was dependent on the presence of rictor, a specific component of mTORC2. Kinase assays confirmed mTORC2 activation by amino acids. This signaling was functional, as demonstrated by the phosphorylation of Akt substrate FOXO3a. Interestingly, using different starvation conditions, amino acids can selectively activate mTORC1 or mTORC2. These findings identify a new signaling pathway used by amino acids underscoring the crucial importance of these nutrients in cell metabolism and offering new mechanistic insights.     

Hypercholesterolemia is associated with hyperactive cardiac mTORC1 and mTORC2 signaling

Nutritional excess and hyperlipidemia increase the heart’s susceptibility to ischemic injury. Mammalian target of rapamycin (mTOR) controls the cellular response to nutritional status and may play a role in ischemic injury. To explore the effect of hypercholesterolemia on cardiac mTOR signaling, we assessed mTOR signaling in hypercholesterolemic swine (HC) that are also susceptible to increased cardiac ischemia-reperfusion injury. Yucatan pigs were fed a high-fat/high-cholesterol diet for 4 weeks to induce hypercholesterolemia, and mTOR signaling was measured by immunoblotting and immunofluorescence in the non-ischemic left ventricular area. Total myocardial mTOR and raptor levels were markedly increased in the HC group compared to the normocholesterolemic group, and directly correlated with serum cholesterol levels. mTOR exhibited intense perinuclear staining in myocytes only in the HC group. Hypercholesterolemia was associated with hyperactive signaling upstream and downstream of both mTOR complexes, including myocardial Akt, S6K1, 4EBP1, S6, and PKC-alpha, increased levels of cardiac hypertrophy markers, and a trend toward lower levels of myocardial autophagy. Hypercholesterolemia can now be added to the growing list of conditions associated with aberrant mTOR signaling. Hypercholesterolemia produces a unique profile of alterations in cardiac mTOR signaling, which is a potential target in cardiac diseases associated with hypercholesterolemia and nutritional excess.     

DNMT1, a Novel Regulator Mediating mTORC1/mTORC2 Pathway-Induced NGF Expression in Schwann Cells

Schwann cells play an important role in maintaining the normal function of peripheral nerves via the secretion of nerve growth factor (NGF). The mTOR signaling pathway is known as a kind of Ser/Thr protein kinase that regulates various cell functions. DNA methyltransferase 1 (DNMT1) is an epigenetic regulator and downstream target of the mTOR pathway. In the present study, we explored the relationship between NGF expression and the mTOR pathway/DNMT1 in RSC96 cells. The results showed that both rapamycin and Torin 1 downregulated NGF expression via the inhibition of phospho-mTOR (Ser 2448) and phospho-S6K1 (Thr 389). Similarly, the silencing of RAPTOR and RICTOR decreased NGF expression by 56.7% and 52.4%, respectively, in RSC96 cells compared with the control siRNA treatment, which was accompanied by reduced phospho-S6K1 (Thr 389). The mTOR/S6K1 activator MHY1485 increased NGF expression by 28.7% and 17.1% 1 day and 2 day after stimulation, respectively, compared to the corresponding control group in RSC96 cells. Furthermore, DNMT1 was enhanced by 94.5% and 42.5% with mTOR pathway inhibitor (rapamycin and Torin 1, respectively) treatment for 3 day compared with the control group. Additionally, the inhibition of DNMT1 with a chemical inhibitor or a specific shRNA plasmid upregulated NGF in RSC96 cells. In summary, our findings suggest that DNMT1 is the downstream target of the mTOR pathway and mediates the mTOR pathway inhibition-induced reduction in NGF expression in Schwann cells. Activation of the mTOR signaling pathway and/or inhibition of DNMT1 increased NGF expression, which may benefit patients suffering from NGF deficiencies, such as diabetic peripheral neuropathy.     

Multiple site acetylation of Rictor stimulates mammalian target of rapamycin complex 2 (mTORC2)-dependent phosphorylation of Akt protein

The serine/threonine protein kinase Akt is a critical regulator of cell growth and survival in response to growth factors. A key step in Akt activation is phosphorylation at Ser-473 by the mammalian target of rapamycin (mTOR) complex 2 (mTORC2). Although Rictor is required for the stability and activity of mTORC2, little is known about functional regions or post-translational modifications within Rictor that are responsible for regulating mTORC2. Here, we demonstrate that Rictor contains two distinct central regions critical for mTORC2 function. One we refer to as the stability region because it is critical for interaction with Sin1.1 and LST8, and a second adjacent region is required for multisite acetylation. p300-mediated acetylation of Rictor increases mTORC2 activity toward Akt, whereas site-directed mutants within the acetylation region of Rictor exhibit reduced insulin-like growth factor 1 (IGF-1)-stimulated mTORC2 kinase activity. Inhibition of deacetylases, including the NAD+-dependent sirtuins, promotes Rictor acetylation and IGF-1-mediated Akt phosphorylation. These results suggest that multiple-site acetylation of Rictor signals for increased activation of mTORC2, providing a critical link between nutrient-sensitive deacetylases and mTORC2 signaling to Akt.     

mSin1 is necessary for Akt/PKB phosphorylation, and its isoforms define three distinct mTORC2s

The mammalian target of rapamycin (mTOR) is a serine/threonine kinase that participates in at least two distinct multiprotein complexes, mTORC1 and mTORC2 . These complexes play important roles in the regulation of cell growth, proliferation, survival, and metabolism. mTORC2 is a hydrophobic motif kinase for the cell-survival protein Akt/PKB and, here, we identify mSin1 as a component of mTORC2 but not mTORC1. mSin1 is necessary for the assembly of mTORC2 and for its capacity to phosphorylate Akt/PKB. Alternative splicing generates at least five isoforms of the mSin1 protein , three of which assemble into mTORC2 to generate three distinct mTORC2s. Even though all mTORC2s can phosphorylate Akt/PKB in vitro, insulin regulates the activity of only two of them. Thus, we propose that cells contain several mTORC2 flavors that may phosphorylate Akt/PKB in response to different signals.     

mTORC2 regulates neutrophil chemotaxis in a cAMP- and RhoA-dependent fashion

We studied the role of the target of rapamycin complex 2 (mTORC2) during neutrophil chemotaxis, a process that is mediated through the polarization of actin and myosin filament networks. We show that inhibition of mTORC2 activity, achieved via knock down (KD) of Rictor, severely inhibits neutrophil polarization and directed migration induced by chemoattractants, independently of Akt. Rictor KD also abolishes the ability of chemoattractants to induce cAMP production, a process mediated through the activation of the adenylyl cyclase 9 (AC9). Cells with either reduced or higher AC9 levels also exhibit specific and severe tail retraction defects that are mediated through RhoA. We further show that cAMP is excluded from extending pseudopods and remains restricted to the cell body of migrating neutrophils. We propose that the mTORC2-dependent regulation of MyoII occurs through a cAMP/RhoA-signaling axis, independently of actin reorganization during neutrophil chemotaxis.     

ERK and Akt signaling pathways function through parallel mechanisms to promote mTORC1 signaling

The mammalian target of rapamycin (mTOR) is a protein kinase that, when present in a complex referred to as mTOR complex 1 (mTORC1), acts as an important regulator of growth and metabolism. The activity of the complex is regulated through multiple upstream signaling pathways, including those involving Akt and the extracellular-regulated kinase (ERK). Previous studies have shown that, in part, Akt and ERK promote mTORC1 signaling through phosphorylation of a GTPase activator protein (GAP), referred to as tuberous sclerosis complex 2 (TSC2), that acts as an upstream inhibitor of mTORC1. In the present study we extend the earlier studies to show that activation of the Akt and ERK pathways acts in a synergistic manner to promote mTORC1 signaling. Moreover, we provide evidence that the Akt and ERK signaling pathways converge on TSC2, and that Akt phosphorylates residues on TSC2 distinct from those phosphorylated by ERK. The results also suggest that leucine-induced stimulation of mTORC1 signaling occurs through a mechanism distinct from TSC2 and the Akt and ERK signaling pathways. Overall, the results are consistent with a model in which Akt and ERK phosphorylate distinct sites on TSC2, leading to greater repression of its GAP activity, and consequently a magnified stimulation of mTORC1 signaling, when compared with either input alone. The results further suggest that leucine acts through a mechanism distinct from TSC2 to stimulate mTORC1 signaling.     

Livers with Constitutive mTORC1 Activity Resist Steatosis Independent of Feedback Suppression of Akt

Insulin resistance is an important contributing factor in non-alcoholic fatty liver disease. AKT and mTORC1 are key components of the insulin pathway, and play a role in promoting de novo lipogenesis. However, mTORC1 hyperactivity per se does not induce steatosis in mouse livers, but instead, protects against high-fat diet induced steatosis. Here, we investigate the in vivo mechanism of steatosis-resistance secondary to mTORC1 activation, with emphasis on the role of S6K1-mediated feedback inhibition of AKT. Mice with single or double deletion of Tsc1 and/or S6k1 in a liver-specific or whole-body manner were generated to study glucose and hepatic lipid metabolism between the ages of 6–14 weeks. Following 8 weeks of high-fat diet, the Tsc1-/-;S6k1-/- mice had lower body weights but higher liver TG levels compared to that of the Tsc1-/- mice. However, the loss of S6k1 did not relieve feedback inhibition of Akt activity in the Tsc1-/- livers. To overcome Akt suppression, Pten was deleted in Tsc1-/- livers, and the resultant mice showed improved glucose tolerance compared with the Tsc1-/- mice. However, liver TG levels were significantly reduced in the Tsc1-/-;Pten-/- mice compared to the Pten-/- mice, which was restored with rapamycin. We found no correlation between liver TG and serum NEFA levels. Expression of lipogenic genes (Srebp1c, Fasn) were elevated in the Tsc1-/-;Pten-/- livers, but this was counter-balanced by an up-regulation of Cpt1a involved in fatty acid oxidation and the anti-oxidant protein, Nrf2. In summary, our in vivo models showed that mTORC1-induced resistance to steatosis was dependent on S6K1 activity, but not secondary to AKT suppression. These findings confirm that AKT and mTORC1 have opposing effects on hepatic lipid metabolism in vivo.     

IBP-mediated suppression of autophagy promotes growth and metastasis of breast cancer cells via activating mTORC2/Akt/FOXO3a signaling pathway

Interferon regulatory factor-4 binding protein (IBP) is a novel upstream activator of Rho GTPases. Our previous studies have shown that ectopic expression of IBP was correlated with malignant behaviors of human breast cancer cells, and invasive human breast cancer had high expression of IBP that promoted the proliferation of these cells. However, it remains unknown whether autophagy inhibition contributes to IBP-mediated tumorigenesis. In this study, we for the first time, reported that upregulation of IBP expression significantly suppressed the autophagy of breast cancer cells, and downregulation of IBP expression markedly induced autophagy of these cells. Further investigation revealed that IBP effectively counteracted autophagy by directly activating mammalian target of rapamycin complex 2 (mTORC2) and upregulating phosphorylation of Akt on ser473 and FOXO3a on Thr32. Moreover, IBP-mediated suppression of autophagy was dependent on mTORC2/Akt/FOXO3a signaling pathway. Finally, our results demonstrated that IBP-mediated breast cancer cell growth in vitro and in vivo was strongly correlated with suppression of mTORC2-dependent autophagy. These findings suggest that the anti-autophagic property of IBP has an important role in IBP-mediated tumorigenesis, and IBP may serve as an attractive target for treatment of breast cancer.     

Differential dependence of hypoxia-inducible factors 1 alpha and 2 alpha on mTORC1 and mTORC2

Constitutive expression of hypoxia-inducible factor (HIF) has been implicated in several proliferative disorders. Constitutive expression of HIF1 alpha and HIF2 alpha has been linked to a number of human cancers, especially renal cell carcinoma (RCC), in which HIF2 alpha expression is the more important contributor. Expression of HIF1 alpha is dependent on the mammalian target of rapamycin (mTOR) and is sensitive to rapamycin. In contrast, there have been no reports linking HIF2 alpha expression with mTOR. mTOR exists in two complexes, mTORC1 and mTORC2, which are differentially sensitive to rapamycin. We report here that although there are clear differences in the sensitivity of HIF1 alpha and HIF2 alpha to rapamycin, both HIF1 alpha and HIF2 alpha expression is dependent on mTOR. HIF1 alpha expression was dependent on both Raptor (a constituent of mTORC1) and Rictor (a constitutive of mTORC2). In contrast, HIF2 alpha was dependent only on the mTORC2 constituent Rictor. These data indicate that although HIF1 alpha is dependent on both mTORC1 and mTORC2, HIF2 alpha is dependent only on mTORC2. We also examined the dependence of HIF alpha expression on the mTORC2 substrate Akt, which exists as three different isoforms, Akt1, Akt2, and Akt3. Interestingly, the expression of HIF2 alpha was dependent on Akt2, whereas that of HIF1 alpha was dependent on Akt3. Because HIF2 alpha is apparently more critical in RCC, this study underscores the importance of targeting mTORC2 and perhaps Akt2 signaling in RCC and other proliferative disorders in which HIF2 alpha has been implicated.