InteractiveFly: GeneBrief

rictor: Biological Overview | References

BIOLOGICAL OVERVIEW

Target of rapamycin (TOR) is an evolutionarily conserved serine/threonine protein kinase that functions as a central regulator of cellular growth and metabolism by forming two distinct complexes: TOR complex 1 (TORC1) and TORC2. As well as TORC1, TORC2 plays a key role in regulation of cell growth. But little is known about how TORC2 regulates cell growth. The transcription factor Myc also plays a critical role in cell proliferation and growth. This study reports that TORC2 and Myc regulate cell growth via a common pathway. Expression of Myc fully rescues growth defects associated with lst8 and rictor mutations, both of which encode essential components of TORC2. Furthermore, loss of TORC2 disrupted the nuclear localization of Myc, and inhibited Myc-dependent transcription. Together, these results reveal a Myc-dependent pathway by which TORC2 regulates cell growth (Kuo, 2015).

The Target of rapamycin (TOR) signaling pathway consists of a set of biochemical processes, which in response to environmental cues and growth factors regulates organismal and cellular growth. TOR is an evolutionarily conserved serine/threonine protein kinase and functions as a core catalytic component of two distinct multiprotein complexes, TOR complex 1 (TORC1) and TORC2. TORC1 controls cell autonomous growth in response to nutrient availability and growth factors, whereas mTORC2 mediates cell proliferation and cell survival by activating several kinases within the AGC family, including AKT, serum and glucocorticoid-regulated kinase (SGK), and protein kinase C (PKC). Although the signaling networks regulated by TORC2 are not completely understood, studies in mice, Drosophila, and Caenorhabditis elegans have demonstrated a role for TORC2 in cell growth. Thus, TORC2 is emerging as a pivotal player in many cancers. As a downstream target of TORC2, AKT plays essential roles in several important cellular processes, including growth, proliferation, survival, and metabolism. Studies in multiple systems have demonstrated that TORC2 inactivates FOXO (forkhead transcription factors of the O-class) through AKT signaling, but it is clear that AKT and FOXO do not mediate TORC2-regulated cell growth. It is therefore critical to identify signaling pathways that act downstream of TORC2 to regulate cell growth (Kuo, 2015).

The myc family of proto-oncogenes encode the transcription factors C-myc, N-myc, and L-myc. Myc proteins play pivotal roles in cell growth and proliferation through the transcriptional regulation of a large number of target genes. As such, dysregulation of Myc contributes to the genesis of many human cancers. Drosophila has a single myc gene (dmyc), which plays a key role in controlling cell size and growth rates by regulating the transcription of mRNAs, ribosomal RNA, and small noncoding RNAs. Hypomorphic dmyc mutations reduce the rate of growth and final size of animals, and dMyc overexpression results in cells and animals that are larger than normal (Kuo, 2015).

Myc has been shown to interact with the TOR pathway. In Drosophila, dMyc is an important mediator of TOR-dependent growth and metabolism, and inhibition of TOR leads to the post-transcriptional down-regulation of dMyc. In mammalian cells, a large-scale quantitative phosphoproteomics study has shown that TOR phosphorylates C-Myc at Ser77. Moreover, similar hypogrowth phenotypes are seen in dmyc, lst8, and rictor mutant animals (the latter two encode essential components of TORC2), suggesting that Myc is an essential link between TORC2 and cell growth (Kuo, 2015).

Using the Drosophila model system, this study found that both cellular and organismal growth defects of dmyc mutant animals were not exacerbated by the loss of LST8. Ectopic expression of dMyc completely rescued the growth defects of both lst8 and rictor mutant animals, including reduced body weight and shrunken eyes and wings. Moreover, the nuclear localization of dMyc was disrupted in lst8 or rictor mutant cells. Furthermore, gene expression profiling revealed that a large set of growth related genes was dysregulated in dmyc, lst8, and rictor mutant animals. These findings suggested that Myc functions downstream of TORC2 to regulate cell growth (Kuo, 2015).

TOR kinase is a highly conserved protein kinase and a central regulator of cell growth. TORC1 has been extensively characterized, but the recent identification of a second TOR complex (TORC2) has complicated the TOR-regulated cell growth pathways. TORC2 regulates growth and metabolism in both mammals and invertebrates. Recent findings indicate that ribosomes physically interact with TORC2 and that this interaction is required for mTORC2 activation. This suggests a critical role for TORC2 in cell growth regulation. However, little is known about the regulation of mTORC2 signaling, or the downstream effectors that implement TORC2-mediated cell growth4. Given evidence that TORC2 regulates AKT/FOXO, AKT/FOXO signaling has been considered the major factor acting downstream of TORC2 to control growth. The current findings that AKT- or FOXO does not affect TORC2-mediated cell growth strongly argue against the AKT/FOXO pathway acting downstream of TORC2 in this context (Kuo, 2015).

The present study found that MYC is required for TORC2-regulated cell growth. Mutations in lst8, rictor, or dmyc had similar growth defects, and lst8 dmyc double mutants did not have more severe growth phenotypes than dmyc mutants. Mosaic analysis in multiple cell types showed that lst8 dmyc double mutant cells had similar growth rates and cell sizes as dmyc mutant cells within the same animals. Moreover, this study established that Myc functions downstream of TORC2 in cell growth. Growth phenotypes associated with loss of TORC2 in the retina, wing, fat body, and the entire body were rescued by the overexpression of dMyc (Kuo, 2015).

Myc protein controls metabolism, cell growth, and proliferation by regulating genes transcribed by RNA Polymerase II, and by stimulating transcription by RNA Polymerases I and III. As Myc is a transcription factor, pathways that regulate the subcellular localization of Myc likely affect its ability to regulate growth and metabolism. This study found that the lack of TORC2 activity is associated the cytoplasmic accumulation of dMyc. Consequently, many Myc target genes were dysregulated in lst8 and rictor mutant tissues. TORC2-mediated nuclear localization of Myc may represent a novel mechanism by which Myc activity is regulated (Kuo, 2015).

As master regulators of cell growth and metabolism, TORC1 and Myc exhibit coordinated patterns of activity. TORC1 activity is required for cancer cell survival, and TORC1 inhibition has remarkable therapeutic efficacy in Myc-driven hematological cancers. In flies, inhibition of TORC1 by molecular inhibitors, genetic manipulations, or starvation leads to the post-transcriptional downregulation of dMyc followed by the repression of dMyc target genes. In mammals, it has been reported that TORC1 activity is required for efficient c-MYC translation in TSC2-null Elt3 rat leiomyoma cells, but opposite results have been reported for colorectal cancer cells, in which TORC1 inhibition by rapamycin treatment or knockdown of Raptor results in phosphorylation and accumulation of Myc. Moreover, in Drosophila intestinal stem cells, excessive TORC1-driven growth in TSC mutants blocked dMyc-induced cell division. These results challenge the notion that TORC1 inhibitors can be used as therapeutic drugs in Myc-driven cancers (Kuo, 2015).

In some cases, such as hyperactivation of AKT signaling, TORC2 is required for proliferation of tumor cells and subsequent tumor growth. The selective requirement for mTORC2 in tumor development suggests that mTORC2 inhibitors may be of substantial clinical utility. The PI3K/AKT signaling cascade is known to regulate metabolic processes via discrete effectors, such as the TSC (tuberous sclerosis) complex and FOXOs. TORC2 inactivates the FOXO branch without affecting the TSC/TORC1 branch. It has been demonstrated that FOXO inhibits MYC function to decrease mitochondrial function and to reduce ROS production. Moreover, a central role for TORC2 in cancer metabolic reprogramming has been proposed, wherein mTORC2 signaling increases cellular c-Myc levels by acetylating FOXO independent of AKT. This study found that TORC2 controlled cell growth by regulating dMyc via nuclear localization. This regulation of dMyc by TORC2 is likely not through TORC2-mediated inactivation of FOXO, because mutations in lst8 or rictor do not affect dMyc transcription or translation. Moreover, previous reports that TORC2 does not regulate cell growth via AKT/FOXO support the model that TORC2 regulates cell growth via Myc independent of FOXO. This finding suggests that TORC2 inhibitors may represent an effective way of treating Myc-driven cancers (Kuo, 2015).

TORC2 mediates the heat stress response in Drosophila by promoting the formation of stress granules

The kinase TOR is found in two complexes, TORC1, involved in growth control, and TORC2 with less well defined roles. This study asked whether TORC2, disrupted by use of Rictor mutant flies, has a role in sustaining cellular stress. TORC2 inhibition in Drosophila was shown to lead to a reduced tolerance to heat stress. Accordingly, upon heat stress, both in the animal and Drosophila cultured S2 cells, TORC2 is activated and is required for the stability of its known target Akt/PKB. The phosphorylation of the stress activated protein kinases is not modulated by TORC2, nor is the heat-induced upregulation of heat shock proteins. Instead, it was shown, both in vivo and in cultured cells, that TORC2 is required for the assembly of heat-induced cytoprotective ribonucleoprotein particles, the pro-survival stress granules. These granules are formed in response to protein translation inhibition imposed by heat stress that appears less efficient in the absence of TORC2 function. It is proposed that TORC2 mediates heat resistance in Drosophila by promoting the cell autonomous formation of stress granules (Jevtov, 2015).

TOR (Target of rapamycin) is a conserved serine/threonine kinase of the PI3K-related kinase family, and functions in two distinct complexes, TOR complex 1 (TORC1) and TOR complex 2 (TORC2). Each complex comprises the kinase along with specific regulatory subunits that give the kinase its functional specificity and structural distinction. The core adaptor proteins of TORC1 are Raptor and LST8, whereas next to LST8, Rictor and Sin1 are the conserved components of TORC2. Removing either of the proteins from a cell destabilizes the TORC2 complex and inhibits its kinase activity. Since its original discovery in screens for rapamycin suppressors, TOR has been extensively studied in the context of TORC1, and has been shown to stimulate key anabolic cellular processes and inhibit the degradative pathway of autophagy with crucial roles in metabolic diseases, cancer and aging. TORC1 is widely regarded as the central node in cell growth control; its activity is dependent on growth factors and nutrient availability, and it is generally shut down in times of stress (Jevtov, 2015).

Unlike TORC1, TORC2 is less well understood and knowledge on upstream cues regulating its activity is scarce. Its role in growth under normal conditions is minor. In lower eukaryotes, TORC2 is activated upon nitrogen starvation, osmotic, heat and oxidative stress and DNA damage, and the TORC2 response to these environmental stresses is related to its likely ancient role in cellular signalling. TORC2 also has a role in actin cytoskeleton rearrangement. Recently, it has also been implicated in gluconeogenesis and sphingolipid metabolism, as well as apoptosis. Protein kinase B (PKB), also known as Akt, a membrane-associated kinase from the family of AGC kinases, with well described roles in cell growth, metabolism and stress, is one of the best characterized downstream targets of TORC2. In vitro, TORC2 has been shown to directly phosphorylate the hydrophobic loop of Akt (S473 in mammals or S505 in Drosophila), thereby increasing its kinase activity (Jevtov, 2015).

There are three well-studied stress response mechanisms in cells. The first is mediated via the stress-activated protein kinases (SAPKs), p38, JNK and Erk, either to protect the cell or to prime it for apoptosis. The second response is the rapid upregulation of transcription of genes encoding heat shock proteins (HSPs) that act as chaperones for cellular proteins to protect them against misfolding and aggregation in stressful conditions. The third includes branches that regulate translation and mRNA turnover. It is well established that heat exposure, oxidative stress and starvation induce the attenuation of bulk protein translation, polysome disassembly and accumulation of untranslated mRNAs. These are stored in cytoplasmic RNA-protein particles (RNP) known as stress granules along with translation initiation factors and RNA-binding proteins. From stress granules, stalled mRNAs can also be transported to the P-bodies (a different type of RNPs that contain RNA decay machinery) for degradation, or upon stress relief, transferred back to polysomes for translation re-initiation. Besides serving as transient protective storage of translation initiation components, the stress granules have also been suggested to serve as a transient station of the SAPKs and other pro-apoptotic kinases under stress, which is regarded to be a protective cellular mechanism against apoptosis. Whether TORC2 acts on these pathways in stress is not known (Jevtov, 2015).

This study shows that TORC2 is specifically required for heat resistance in vivo as Drosophila melanogaster mutants for TORC2 components are selectively sensitive to heat stress. This sensitivity is accompanied by the reduced phosphorylation of Akt due to the loss of the protein itself. Conversely, Akt phosphorylation is enhanced by heat in wild-type Drosophila larvae and cultured cells, showing that TORC2 is activated. Whereas the stress kinase and the HSP branches of the stress response are not affected, we show that the heat-induced stress granule formation is significantly delayed upon loss of TORC2 function, both in cells and in the animals and an reduction of translation inhibition imposed by heat stress might be a cause for this delay. Taken together, it is proposed that under heat stress conditions, TORC2 promotes survival by enabling stress granule assembly (Jevtov, 2015).

The results show that one key branch of the response to heat stress, the formation of stress granules, is delayed by the loss of TORC2 function both in Drosophila tissues and cultured cells. TORC2 is activated upon heat stress and mediates the formation of stress granules, likely required for heat resistance at the cellular level in Drosophila (Jevtov, 2015).

How TORC2 mediates stress granule formation is not clear. Heat stress is known to stimulate the inhibitory phosphorylation of the initiation factor eIF2 αresulting in protein translation stalling. However, this phosphorylation is not required for stress granule formation in Drosophila upon heat stress, so it is unlikely that TORC2 modulates this event (Jevtov, 2015).

This study shows that stress granule formation is delayed by loss of TORC2 function and it is suggested that this is due to a lift on the overall translation inhibition imposed by heat stress, but also under basal conditions. Depletion of TORC2 components appears to stimulate protein translation. This is in accordance with the observations that depletion of either Rictor or Sin1 from Drosophila S2 cultured cells causes their increased proliferation (115%) and cell diameter, respectively. This activation of translation upon loss of TORC2 function could be due to activation of TORC1, as observed previously in Kc cells, another Drosophila cultured cell line. There, depletion of Rictor elevates levels of the phosphorylated 4E-BP, a known target of TORC (Jevtov, 2015).

However, Rictor and Sin1 mutant flies are smaller in size than control animals (Hietakangas, 2007), suggesting that this translation activation potentially leading to an increase in cell growth and proliferation might be the tissue specific. This might mirror the tissue-specific response in stress granule formation that we report here (Jevtov, 2015).

Such stimulated translation, even upon heat stress might delay or impair stress granule assembly. However, Sin1 depletion has a much stronger effect on translation than Rictor depletion, yet stress granule assembly is inhibited to the same extent in both backgrounds. So whether this lift in translation inhibition is the sole parameter impairing stress granule formation remains to be further investigated. In this regard, Rictor is found at the ribosomes interacting with RACK1 (Zinzalla, 2011), a selective mediator in stress granule function (Arimoto, 2008; Ohn, 2008). Thus, it remains to be determined whether TORC2 senses ribosomal activity and mediates the stress granule assembly on its own, rather than indirectly, by providing balance to TORC1 (Jevtov, 2015).

Interestingly, the ribosomal localization of Rictor activates Akt, the TORC2 downstream kinase and this study shows that Akt is activated upon heat stress both in animals and cell lines, in line with mammalian studies. This heat-mediated activation is in line with the finding that S. pombe mutants for Tor1 (kinase of TORC2), Sin1 and Gad8 (Akt ortholog) are also sensitive to heat stress. This suggests that TORC2 - Akt signalling axis represents an ancient and conserved cellular mechanism to cope with heat stress (Jevtov, 2015).

Surprisingly, however, this study found that TORC2 function not only modulates Akt phosphorylation but also its stability. Strikingly, the absence of TORC2 function both in cells and larvae rapidly and significantly obliterates Akt, probably through increased degradation. It is likely not due to a lower translation during stress since translation is less inhibited in heat stress in the absence of TORC2 components. This correlates well with studies in mammalian cells, where PKCalpha, a second known downstream target of TORC2, and a small fraction of Akt are degraded by the proteasome ubiquitin pathway in cells depleted for TORC2 components. This is due to the lack of phosphorylation by TORC2 that primes PKCalpha and Akt for ubiquitination (Ikenoue, 2008; Oh, 2010). Whether and how Akt plays a role in stress granule formation in Drosophila remains to be investigated (Jevtov, 2015).

The TORC2 based mechanism that this study proposes is different from the one described in mammalian cells (especially cancer cell lines) where TORC1 is a key player. Indeed, depletion of TORC1 components impairs stress granule assembly by reducing the phosphorylation of 4E-BP, subsequently preventing the formation of eIF4E-eIF4GI cap-dependent translational initiation complexes. In S2 cells, however, this study did not observe a direct role for TORC1 in stress granule formation. Neither Raptor depletion nor rapamycin treatment impairs stress granule formation upon heat stress. Whether this differential involvement of TORC1 and TORC2 in stress granule formation is cell- and tissue- dependent and acts via different pathways remains to be tested. Alternatively, the different mechanisms may suggest that mammals have evolved more sophisticated mechanisms to cope with stress. TORC1 has also been shown to be sequestered in stress granules during heat and other stresses where it suppresses its own apoptotic activity, corroborating its role in stress granule function (Jevtov, 2015).

The importance of studying environmental effects on signalling pathways, like the TOR pathway, is illustrated by the central role of these pathways for progression of diseases, such as metabolic and neurological diseases or cancer. Elucidating the modulation of such pathways under different environmental conditions can potentially identify new targets and processes playing roles in the physiological or pathological regulation of cell survival (Jevtov, 2015).

LST8 regulates cell growth via target-of-rapamycin complex 2 (TORC2)

The evolutionarily conserved serine/threonine protein kinase target-of-rapamycin (TOR) controls cell growth as a core component of TOR complexes 1 (TORC1) and 2 (TORC2). Although TORC1 is the more central growth regulator, TORC2 has also been shown to affect cell growth. This study demonstrates that Drosophila LST8, the only conserved TOR-binding protein present in both TORC1 and TORC2, functions exclusively in TORC2 and is not required for TORC1 activity. In mutants lacking LST8, expression of TOR and RAPTOR, together with their upstream activator Rheb, was sufficient to provide TORC1 activity and stimulate cell and organ growth. Furthermore, using an lst8 knockout mutation, this study showed that TORC2 regulates cell growth cell autonomously. Surprisingly, however, TORC2 does not regulate cell growth via its best-characterized target, AKT. These findings support the possible application of TORC2-specific drugs in cancer therapy (Wang, 2012).

LST8 was originally identified genetically as a mutation in yeast that is synthetically lethal with sec13-1, a mutation that causes a sorting defect in the secretory pathway. Later, physical association studies showed that LST8 is a core component of TORC1 and TORC2 in both yeast and mammals. Structural data confirmed that LST8 stably interacts with the kinase domain of TOR without overlapping the Raptor binding site. While it has been clearly demonstrated that LST8 is essential for TORC2 activity, whether or not LST8 functioned in the TORC1 pathway had not been rigorously investigated. In mammalian cells, it was originally reported that the binding of LST8 to mTOR strongly stimulated the binding association between mTOR and Raptor and, as a result, elevated the kinase activity of mTORC1 toward S6K1 and 4E-BP1. In yeast, rapamycin, which is TORC1 specific, mimicked the Gap1p sorting defect of an lst8 mutant, and a temperature-sensitive lst8 mutant was hypersensitive to rapamycin, suggesting that LST8 functions in TORC1. However, the observations that the lst8 knockout did not affect the phosphorylation of S6K1 or 4E-BP1 and that lst8 knockout mice phenocopied rictor knockout mice but not mtor knockout mice suggested that LST8 might not function in TORC1, at least under normal conditions (Wang, 2012).

This report demonstrates that lst8 knockout flies are viable but small, similar to rictor mutants but dissimilar to files with tor or rheb mutations, which are lethal. Neither loss nor overexpression of LST8 affected the kinase activity of TORC1 toward S6K or autophagy, whereas the kinase activity of TORC2 toward AKT was completely lost in the lst8 mutants. Moreover, in the absence of LST8 the overexpression of Rheb still upregulated TORC1 activity and also promoted the growth of cells. Furthermore, the expression of TOR and Raptor was sufficient to drive phosphorylation of S6K and growth of cells when they were supplied with Rheb signaling, even when LST8 was entirely absent. Given these results, it is concluded that LST8 is an essential core component of TORC2 but is dispensable for the regulation and activity of TORC1 (Wang, 2012 and references therein).

Although TOR signaling functions primarily as a central regulator of cell growth, increasing evidence suggests that inappropriate activation of TOR also makes cells sensitive to death signals. For instance, tumor cells in TSC disease, which is caused by mutation of tsc1 or tsc2, exhibit morphological features of apoptosis such as activated caspase-8 and positive terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) results. In a mouse model of TSC disease, conditional knockout of tsc1 induced cell death in the hippocampus and neocortex. In a recent study in flies, it has been shown that activation of TORC1 by either overexpression of Rheb or mutation of Tsc1 led to neurodegeneration. In the latter case the neurodegeneration was specifically attributed to TOR's ability to suppress autophagy (Wang, 2012).

Although overactivation of TORC1 can result in cell death, it is not always sufficient for the induction of cell death. For instance, in young flies, overexpression of Rheb alone promoted cell growth but did not drive cell death, whereas neuronal cell death was detected only in aged flies after long-term TOR hyperactivation. This slow neurodegeneration was also dependent on neuronal activity. The present study, however, found that when Rheb and TORC1 are overexpressed together (a condition which very strongly increases TORC1 activity), severe cell death rapidly occurred in all cell types, even in young animals, without any dependency on neuronal activity. Therefore, it is suggested that the regulation of TORC1 activity is crucial for the survival of all cell types and that the incidence of cell death within a tissue may be correlated with TORC1 activity levels. The cell death-promoting function of TORC1 might explain why mutations of tsc1 or tsc2 lead to benign, rather than malignant, tumors (Wang, 2012).

In both mammals and invertebrates, it has been demonstrated that TORC2 regulates growth and metabolism. However, whether TORC2 affects organismal growth cell autonomously or indirectly via systemic effects had not been thoroughly evaluated. In C. elegans, rictor mutants have been shown to be developmentally delayed, small in body size, and short-lived. Targeted expression of Rictor in the intestines of these mutants could rescue these growth defects, and therefore it was suggested that TORC2 might act directly in the intestine to regulate cell mass and growth of the whole animal, non-cell autonomously. This study presents evidence supporting a cell-autonomous role for TORC2 in the growth of several different cell types. In genetic mosaics, cells homozygous for the lst8¹ mutation were smaller than adjacent wild-type control cells in the retina, wing epithelium, and fat body. Moreover, expression of LST8 in posterior wing compartments specially rescued growth of the posterior cells without affecting the anterior (Wang, 2012).

TORC1 consists of TOR, Raptor, LST8, and PRAS40 (proline-rich AKT substrate, 40 kDa), a negative regulator. mTORC1 purified with PRAS40 at substoichiometric levels has been shown to have kinase activity toward S6K1 and 4E-BP1 in vitro. Raptor-free mTORC1 appears unable to support 4E-BP1 phosphorylation but might be capable of phosphorylating full-length S6K1. Interestingly, the phosphorylation of S6K1 by Raptor-free mTORC1 could still be inhibited by FKBP12-rapamycin. Nevertheless, overexpression of TOR and Raptor together has been shown to significantly increase in vitro TORC1 kinase activity compared to overexpressed TOR alone. Overexpressed TOR has been found in some cancer patients, but the significance of this relative to disease progression or TORC1 activity levels has not been assessed. By overexpression of TORC1 components in flies, this study demonstrated that expression of all three positive components of TORC1 (TOR, Raptor, and LST8) cannot drive increased TORC1 activity or cell overgrowth. However, in the presence of the upstream activator, Rheb, overexpression of TOR and Raptor either with or without LST8 increased TORC1 activity toward S6K and dramatically stimulated cell growth. These results further confirm that TOR and Raptor but not LST8 are bone fide components of TORC1. In contrast to results from in vitro studies, expression of TORC1 components did not function in the absence of activating signals, perhaps because cellular translocation of TORC1 is a key event in TORC1 activation. Since expression of TOR alone did not generate functional TORC1, the significance of overexpression of TOR in cancer cells might be reconsidered (Wang, 2012).

The fact that the two TOR complexes display distinct cellular functions and phosphorylate different downstream substrates suggests that they might respond to different upstream signals. It has been well established that TORC1 can be regulated by upstream signals such as amino acids and growth factors, but little is known about whether TORC2 can be activated by such signals. When purified from insulin- or IGF1-stimulated cells and assayed in vitro, TORC2 kinase activity is elevated, suggesting that growth factors can activate TORC2 through PI3K signaling. Data from tissue culture cells suggest that the TSC complex physically associates with TORC2 via Rictor, but surprisingly the regulation of TORC2 by the TSC1/2 complex appeared to be independent of its GTPase-activating protein activity toward Rheb. This study shows that AKT phosphorylation is downregulated when Rheb is overexpressed, suggesting a role for the Rheb GTPase in regulating TORC2. The recent finding that Rictor is directly phosphorylated by the mTORC1-dependent kinase S6K1 might help explain the regulation of TSC2 by Rheb. However, it is also possible that through negative feedback from TORC1, which phosphorylates and deactivates insulin receptor substrate 1, TORC2 is negatively regulated by Rheb signaling (Wang, 2012).

Nutrient availability is one of the major environmental signals influencing growth. Conflicting data have been proffered as to whether amino acids might regulate TORC2 activity. Although amino acids originally were not considered regulation signals for TORC2, it was recently reported that amino acids could increase AKT phosphorylation by TORC2. In addition, leucine addition to starved cells promotes cell migration in a TORC2-dependent manner. The fat body is an insect organ that retains the endocrine and storage functions of the vertebrate liver and adipose tissues and serves as a nutrient sensor that restricts global growth. Thus, fat body cells are very sensitive to nutritional conditions, and since TORC1 is controlled by nutrition, its activity has a profound influence in fat body cells. For instance, downregulation of TORC1 by expression of TSC1/2 strongly decreases fat body cell size relative to other tissues, where it has modest effects. In addition, fat body cells with upregulated TORC1 are capable of massive growth under starvation conditions, essentially bypassing the normal cessation of growth caused by starvation, whereas deregulated TORC1 has mild effects in fat body cells in fed animals. As this study shows, the disruption of TORC2 by mutation of lst8 affected the growth of every tissue to a similar extent. Moreover, starvation did not change the size reduction in lst8¹ cells in the fat body, suggesting that TORC2-regulated cell growth might not be nutrition sensitive (Wang, 2012).

TORC2 is believed to control cell survival, cell growth, and cytoskeletal organization by phosphorylating several AGC kinases including SGK, cPKCα, and AKT. Among these TORC2 substrates, AKT is especially important because of its general role in growth and survival. Therefore, it has been proposed that TORC2 might serve as a drug target in tumors that have lost the expression of PTEN, a tumor suppressor that inhibits AKT activity. However, recent evidence suggests that TORC2-mediated phosphorylation of AKT does not determine its absolute activity but, instead, determines substrate specificity. AKT variants immune to TORC2-mediated phosphorylation retain the ability to control glycogen synthase kinase 3 (GSK3) and TSC2 but show decreased activity toward FOXO1/3a. These observations align with those of this study in supporting the view that TORC2 might only help to inactivate the FOXO branch of the AKT signaling pathway without affecting the TORC1 branch (Wang, 2012).

The results of this study suggest that TORC2-mediated AKT phosphorylation does not regulate the growth of cells and argue against the importance of AKT as an important downstream target of TORC2 in cell growth control. Expression of nonphosphorylatable mutant forms of AKT did not change cellular growth status either with or without TORC2, and a constitutively active phospho-mimetic form of AKT was unable to suppress the growth defect of lst8 mutants. Moreover, mutation of foxo did not compensate for the reduction in cell growth caused by disruption of TORC2. In fact, similar results have been reported in C. elegans, where the reduced size of rictor mutants was proposed not to be a consequence of reduced AKT activity. Meanwhile, it was suggested that TORC2 controls cell growth by affecting SGK activity. However, the lack of an SGK homolog in flies casts some doubt on this mechanism. The unexpected finding of this study that AKT does not mediate TORC2-regulated cell growth emphasizes the importance of finding TORC2's critical substrates and also raises questions about the efficacy of TORC2 inhibitors as drugs for cancer therapy (Wang, 2012).

Tuberous sclerosis complex regulates Drosophila neuromuscular junction growth via the TORC2/Akt pathway

Mutations in the tuberous sclerosis complex (TSC) are associated with various forms of neurodevelopmental disorders, including autism and epilepsy. The heterodimeric TSC complex, consisting of Tsc1 and Tsc2 proteins, regulates the activity of the TOR (target of rapamycin) complex via Rheb, a small GTPase. TOR, an atypical serine/threonine kinase, forms two distinct complexes TORC1 and TORC2. Raptor and Rictor serve as specific functional components of TORC1 and TORC2, respectively. Previous studies have identified Tsc1 as a regulator of hippocampal neuronal morphology and function via the TOR pathway, but it is unclear whether this is mediated via TORC1 or TORC2. In a genetic screen for aberrant synaptic growth at the neuromuscular junctions (NMJs) in Drosophila, Tsc2 mutants showed increased synaptic growth. Increased synaptic growth was also observed in rictor mutants, while raptor knockdown did not phenocopy the TSC mutant phenotype, suggesting that a novel role exists for TORC2 in regulating synapse growth. Furthermore, Tsc2 mutants showed a dramatic decrease in the levels of phosphorylated Akt, and interestingly, Akt mutants phenocopied Tsc2 mutants, leading to the hypothesis that Tsc2 and Akt might work via the same genetic pathway to regulate synapse growth. Indeed, transheterozygous analysis of Tsc2 and Akt mutants confirmed this hypothesis. Finally, the data also suggest that while overexpression of rheb results in aberrant synaptic overgrowth, the overgrowth might be independent of TORC2. Thus, it is proposed that at the Drosophila NMJ, TSC regulates synaptic growth via the TORC2-Akt pathway (Natarajan, 2013).

The target of rapamycin complex 2 controls dendritic tiling of Drosophila sensory neurons through the Tricornered kinase signalling pathway

To cover the receptive field completely and non-redundantly, neurons of certain functional groups arrange tiling of their dendrites. In Drosophila class IV dendrite arborization (da) neurons, the NDR family kinase Tricornered (Trc) is required for homotypic repulsion of dendrites that facilitates dendritic tiling. This study reports that Sin1, Rictor, and target of rapamycin (TOR), components of the TOR complex 2 (TORC2), are required for dendritic tiling of class IV da neurons. Similar to trc mutants, dendrites of sin1 and rictor mutants show inappropriate overlap of the dendritic fields. TORC2 components physically and genetically interact with Trc, consistent with a shared role in regulating dendritic tiling. Moreover, TORC2 is essential for Trc phosphorylation on a residue that is critical for Trc activity in vivo and in vitro. Remarkably, neuronal expression of a dominant active form of Trc rescues the tiling defects in sin1 and rictor mutants. These findings suggest that TORC2 likely acts together with the Trc signalling pathway to regulate the dendritic tiling of class IV da neurons, and thus uncover the first neuronal function of TORC2 in vivo (Koike-Kumagai, 2009).

mTORC2 controls actin polymerization required for consolidation of long-term memory

A major goal of biomedical research is the identification of molecular and cellular mechanisms that underlie memory storage. This study reports a previously unknown signaling pathway that is necessary for the conversion from short- to long-term memory. The mammalian target of rapamycin (mTOR) complex 2 (mTORC2), which contains the regulatory protein Rictor (rapamycin-insensitive companion of mTOR), was discovered only recently and little is known about its function. Conditional deletion of Rictor in the postnatal murine forebrain were found to greatly reduced mTORC2 activity and selectively impaired both long-term memory (LTM) and the late phase of hippocampal long-term potentiation (L-LTP). A comparable impairment of LTM in was observed dTORC2-deficient flies, highlighting the evolutionary conservation of this pathway. Actin polymerization was reduced in the hippocampus of mTORC2-deficient mice and its restoration rescued both L-LTP and LTM. Moreover, a compound that promoted mTORC2 activity converted early LTP into late LTP and enhanced LTM. Thus, mTORC2 could be a therapeutic target for the treatment of cognitive dysfunction (Huang, 2013).

Re-evaluating AKT regulation: role of TOR complex 2 in tissue growth

Phosphatidylinositol-3-kinase (PI3K)/AKT signaling is essential for growth and metabolism and is elevated in many cancers. Enzymatic activity of AKT has been shown to depend on phosphorylation of two conserved sites by PDK1 and TOR (target of rapamycin) complex 2 (TORC2) in a PI3K-dependent manner. This study analyzed the role of TORC2-mediated AKT phosphorylation in Drosophila. Mutants removing critical TORC2 components, rictor and sin1, strongly reduced AKT hydrophobic motif (HM) phosphorylation and AKT activity, but showed only minor growth impairment. A mutant form of AKT lacking the HM phosphorylation site displayed comparable activity. In contrast to the mild effects of removing HM site phosphorylation at normal levels of PI3K activity, loss of TORC2 activity strongly inhibited hyperplasia caused by elevated pathway activity, as in mutants of the tumor suppressor PTEN. Thus, TORC2 acts as a rheostat to broaden the range of AKT signaling at the high end of its range (Hietakangas, 2007).

The PI3K/AKT signaling pathway is conserved between Drosophila and mammalian species. The lack of genetic redundancy among pathway components makes Drosophila a useful system in which to dissect the roles of the individual pathway members in vivo. Earlier analyses of other pathway members have shown that the Insulin receptor, PI3K, PDK1, and AKT are each essential for viability, and that mutant tissue displays severe undergrowth. Mutants of Drosophila insulin receptor substrate homolog, chico, are semiviable but severely growth impaired. Although individual AKT mutants are viable in mouse, the essential nature of AKT is likely to be masked by genetic redundancy among the three AKT genes. Previous studies in cultured cells have suggested that TORC2 is an important regulator of AKT phosphorylation and activity, and that this phosphorylation event is required for AKT kinase activity. It was recently shown that loss of TORC2 activity in rictor mutant mice leads to loss of AKT HM phosphorylation and to embryonic lethality, suggesting that HM phosphorylation is essential for AKT activity in the mouse. In contrast, the current findings show that TORC2-mediated phosphorylation on the HM site is not essential for AKT activity in vivo. Indeed, although AKT activity was reduced, considerable residual activity was found in flies lacking TORC2 activity. Flies expressing a mutant form of AKT lacking the HM phosphorylation site also showed considerable AKT activity in vivo. These findings indicate that the maximal level of AKT activity is limited in the absence of HM phosphorylation. Under normal physiological conditions in Drosophila, this reduced level of AKT activity is almost sufficient to support normal growth. But without HM phosphorylation, AKT cannot transduce the higher-than-normal levels of PI3K pathway activity that result from mutation of the tumor suppressor PTEN or increased insulin stimulation. When considered in this context, the lethality of rictor mutant mice could reflect a higher threshold in the requirement for AKT activity in some biological process in mouse than in fly, but the possibility of essential TORC2 targets other than AKT cannot be excluded (Hietakangas, 2007).

Perhaps the most intriguing implication of this study lies in the area of cancer biology. Elevated AKT activity is a hallmark of human cancer, with a substantial proportion of human tumors depending on AKT pathway activation, for example, due to PTEN mutations. The current findings suggest that inhibiting TORC2 activity, rather than AKT itself, may prove to be a promising strategy for cancer therapy (Hietakangas, 2007).

Discrete functions of rictor and raptor in cell growth regulation in Drosophila

The TOR signaling pathway regulates cell growth and metabolism in response to various nutrient signals by forming complexes with either rictor or raptor. To distinguish the physiological roles of the complexes formed by the two different TOR partners in vivo functions of rictor and raptor were compared in Drosophila. In rictor-null mutants, Akt-induced tissue hyperplasia was reduced and Akt-Ser-505 phosphorylation was decreased. Furthermore, FOXO-dependent apoptosis, which is inhibited by Akt, was augmented in the rictor-null background, indicating that rictor is essential for the Akt-FOXO signaling module. Neither S6K-dependent cell growth nor S6K-Thr-398 phosphorylation was affected in rictor-null mutants. However, the knockdown of another TOR binding partner, raptor, decreased S6K-Thr-398 phosphorylation and inhibited S6K-induced cell overgrowth. Collectively, these findings strongly support that the association of TOR with rictor or raptor plays pivotal roles in TOR-mediated cell apoptosis and growth control by differentially regulating Akt- and S6K-dependent signaling pathways, respectively (Lee, 2007).

Phosphorylation and regulation of Akt/PKB by the rictor-mTOR complex

Deregulation of Akt/protein kinase B (PKB) is implicated in the pathogenesis of cancer and diabetes. Akt/PKB activation requires the phosphorylation of Thr308 in the activation loop by the phosphoinositide-dependent kinase 1 (PDK1) and Ser473 within the carboxyl-terminal hydrophobic motif by an unknown kinase. This study shows in Drosophila and human cells the target of rapamycin (TOR) kinase and its associated protein rictor are necessary for Ser473 phosphorylation and that a reduction in rictor or mammalian TOR (mTOR) expression inhibits an Akt/PKB effector. The rictor-mTOR complex directly phosphorylated Akt/PKB on Ser473 in vitro and facilitated Thr308 phosphorylation by PDK1. Rictor-mTOR may serve as a drug target in tumors that have lost the expression of PTEN, a tumor suppressor that opposes Akt/PKB activation (Sarbassov, 2005a).

The Akt/PKB kinase is a well-characterized effector of phosphoinositide 3-kinase (PI3K), and its deregulation plays important roles in the pathogenesis of human cancers. PI3K is necessary for the activation of Akt/PKB, and current models suggest that phosphatidylinositol-3,4,5-triphosphates produced upon growth factor stimulation recruit Akt/PKB to the plasma membrane by binding to its N-terminal pleckstrin homology (PH) domain. At the membrane, Akt/PKB is phosphorylated on two key residues: Thr³⁰⁸ (T308) of the activation loop by PDK1 and Ser⁴⁷³ (S473) in the hydrophobic motif of the C-terminal tail by a kinase whose identity has been elusive. The role of S473 phosphorylation is controversial, but there is an emerging view that it precedes the phosphorylation of T308 and is important for the recognition and activation of Akt/PKB by PDK1 (Sarbassov, 2005a and references therein).

The molecular identity of the S473 kinase (S473K), at times referred to as 'PDK2' or the 'hydrophobic motif (HM) kinase,' has been hotly debated for many years. Several candidate S473Ks have been proposed, including PDK1, integrin-linked kinase (ILK), Akt/PKB itself, and, most recently, DNA-PK_cs. Many lines of evidence argue that neither PDK1, ILK, nor Akt/PKB is the physiological S473K, and for several reasons, DNA-PK_cs is also unlikely to have this function. There is no Drosophila ortholog of DNA-PK_cs, and, thus, if DNA-PK_cs is a physiological S473K in mammals, a distinct kinase must play that role in flies even though all other core components of the pathway (e.g., PI3K, Akt/PKB, PDK1, and PTEN) are well conserved. Moreover, it has not been shown that DNA-PK_cs phosphorylates full-length Akt/PKB, and DNA-PK_cs null mice do not suffer the growth retardation or insulin signaling defects associated with Akt1/PKB1 or Akt2/PKB2 (Sarbassov, 2005a).

Mammalian TOR (mTOR) is a large protein kinase that exists in two distinct complexes within cells: one that contains mTOR, GβL, and raptor and another containing mTOR, GβL, and rictor. The raptor-containing complex is sensitive to the drug rapamycin and regulates cell growth, in part by phosphorylating the hydrophobic motif of S6K1, a member of the same family of kinases to which Akt/PKB belongs. The rictor-containing complex does not appear to be rapamycin-sensitive, and its cellular function is just beginning to be understood. Despite its structural similarity to S6K1, Akt/PKB phosphorylation is not sensitive to acute rapamycin treatment, and thus mTOR has not previously been considered as the S473K (Sarbassov, 2005a).

This study used RNA interference (RNAi) in cultured Drosophila cells to determine the role of TOR pathway components in the phosphorylation of the hydrophobic motif sites of Drosophila Akt/PKB (dAKT/dPKB) and S6K (dS6K). In mammals and Drosophila, S6K suppresses signaling through the PI3K/Akt pathway so that inhibition of S6K boosts Akt/PKB phosphorylation. Knockdown of dS6K or Drosophila Raptor expression with double-stranded RNAs (dsRNAs) inhibited the phosphorylation and activity of dS6K and increased the phosphorylation of dAkt/dPKB. Despite reducing dS6K phosphorylation to the same extent as did dRaptor dsRNA, the dTOR dsRNA failed to increase dAkt/dPKB phosphorylation and, surprisingly, decreased it by a small amount. The contrasting effects on dAkt/dPKB phosphorylation by the dTOR and dRaptor dsRNAs suggest that dTOR has an unexpected positive role in dAkt/dPKB signaling that is not shared with dRaptor and that dTOR is required for the increase in dAkt/dPKB phosphorylation caused by dS6K inhibition. Consistent with the dRaptor-independent role for dTOR in dAkt/dPKB phosphorylation, a knockdown of dRictor reduced dAkt/dPKB phosphorylation (Sarbassov, 2005a).

Because basal dAkt/dPKB phosphorylation is low in Drosophila Kc₁₆₇ cells, the roles of dRictor and dTOR were verified in cells in which dAkt/dPKB phosphorylation was enhanced by decreasing the expression of dPTEN, the negative regulator of the PI3K/Akt pathway. Knockdown of dS6K or dRaptor expression in dPTEN-depleted cells further boosted dAkt/dPKB phosphorylation. In contrast, knockdown of dRictor expression almost completely prevented the dramatic increase in dAkt/dPKB phosphorylation caused by a dPTEN knockdown, whereas the knockdown of dTOR expression caused a slightly smaller suppression. Also, dRictor and dTOR were required for the increase in phosphorylation of dAkt/dPKB caused by a knockdown in the expression of dRaptor (Sarbassov, 2005a).

The results in Drosophila cells suggest that dTOR and dRictor have a shared positive role in the phosphorylation of the hydrophobic motif site of dAkt/dPKB. This finding was unexpected, because previously no decrease was observed in the phosphorylation of the hydrophobic motif site of Akt/PKB after reducing mTOR expression in human cells with small interfering RNAs (siRNAs). In retrospect, however, these experiments were undertaken when RNAi-mediated knockdowns of expression in mammalian cells were relatively inefficient. In this study, with the use of a lentiviral short hairpin RNA (shRNA) expression system that robustly suppresses gene expression, results in human cell lines were obtained analogous to those in Drosophila cells. In human HT-29 colon and A549 lung cancer cells, knockdown of rictor or mTOR expression using two different sets of shRNAs decreased phosphorylation of both S473 and T308 of Akt/PKB. Mammalian cells may try to compensate for the effects of the rictor and mTOR knockdowns by boosting Akt/PKB expression. The decrease in T308 phosphorylation is consistent with the importance of S473 phosphorylation for T308 phosphorylation and with the fact that the Ser⁴⁷³ --> Asp⁴⁷³ mutant of Akt/PKB is a better substrate than the wild-type protein for T308 phosphorylation by PDK1. Knockdown of raptor expression increased the phosphorylation of both S473 and T308 despite reducing Akt/PKB expression. Knockdown of rictor or mTOR expression also decreased S473 phosphorylation in HeLa and HEK-293T cells, two human cell lines that, like A549 and HT-29 cells, contain wild-type PTEN. In addition, the knockdowns also decreased S473 phosphorylation in the PTEN-null PC-3 prostate cancer cell line, a result reminiscent of that in Drosophila cells with reduced dPTEN expression. Furthermore, the knockdowns decreased S473 phosphorylation in M059J glioblastoma cells that are null for DNA-PK_cs, a proposed S473K candidate. Thus, in six distinct human cell lines, rictor and mTOR but not raptor are necessary for the phosphorylation of the hydrophobic motif of Akt/PKB (Sarbassov, 2005a).

Because the rictor and mTOR knockdowns inhibit phosphorylation events critical for Akt/PKB activity, they should affect Akt/PKB-regulated effectors. In HeLa cells, a reduction in the expression of rictor or mTOR but not raptor decreased phosphorylation of AFX (Foxo4a), a forkhead family transcription factor that is a direct substrate of Akt/PKB. Because the raptor-mTOR complex directly phosphorylates the hydrophobic motif site of S6K1, whether rictor-mTOR has an analogous function for Akt/PKB was determined. Rictor-mTOR complexes isolated from HEK-293T or HeLa phosphorylated S473 but not T308 of full-length, wild-type Akt/PKB in vitro. Immunoprecipitates of raptor, the ataxia telagiectasia mutated (ATM) protein, or protein kinase C α (PKCα) did not phosphorylate either site, and Akt/PKB did not autophosphorylate S473. Importantly, the raptor immunoprecipitates also contain mTOR but did not phosphorylate Akt/PKB, suggesting that for mTOR to phosphorylate Akt/PKB, it must be bound to rictor and that raptor cannot substitute. This lack of phosphorylation holds even in the raptor immunoprecipitates isolated from HEK-293T cells that contain as much mTOR as the rictor immunoprecipitates. Consistent with a key role for rictor, mTOR immunoprecipitates prepared from the rictor knockdown cells did not phosphorylate Akt/PKB despite containing a similar amount of mTOR as the controls. To verify that mTOR is the S473K in the rictor immunoprecipitates, immunoprecipitates were prepared from control cells and from two different lines of mTOR knockdown cells. Although rictor levels were equivalent in all the immunoprecipitates, only those prepared from cells expressing mTOR phosphorylated Akt/PKB in vitro. Both the LY294002 and wortmannin mTOR kinase inhibitors blocked the in vitro phosphorylation of Akt/PKB by rictor-mTOR, and LY294002 acted at concentrations that inhibit S473 phosphorylation in cells. Staurosporine, an inhibitor of Akt/PKB kinase activity, did not decrease the phosphorylation of Akt/PKB by rictor-mTOR. Thus, in vitro the rictor-mTOR complex phosphorylates S473 of Akt/PKB in a rictor- and mTOR-dependent fashion and with a drug sensitivity profile consistent with mTOR being the phosphorylating kinase (Sarbassov, 2005a).

To determine whether the phosphorylation of Akt/PKB on S473 by rictor-mTOR activates Akt/PKB activity, rictor-mTOR was used to phosphorylate Akt/PKB on S473, and then PDK1 was added to the assay to phosphorylate T308. Prior phosphorylation of Akt/PKB on S473 boosted subsequent phosphorylation by PDK1 of T308, consistent with the importance of S473 phosphorylation for T308 phosphorylation and with the inhibitory effects of the rictor and mTOR knockdowns on T308 phosphorylation. After phosphorylation with rictor-mTOR and PDK1, Akt1/PKB1 had about four- to fivefold more activity than after phosphorylation with PDK1 alone, confirming the important role of S473 in fully activating Akt/PKB. Because growth factors control the phosphorylation of Akt/PKB on S473, it was determined whether the concentration of serum in the cell media regulated the in vitro kinase activity of rictor-mTOR toward Akt/PKB. Rictor-mTOR had decreased activity in HeLa cells deprived of serum and was reactivated by serum stimulation for 30 min, indicating that modulation of the intrinsic kinase activity of rictor-mTOR may be a mechanism for regulating S473 phosphorylation (Sarbassov, 2005a).

These results indicate that the rictor-mTOR complex is a hydrophobic motif kinase for Akt/PKB. Rictor-TOR has essential roles in Akt/PKB hydrophobic motif site phosphorylation in Drosophila and human cells and in vitro phosphorylates full-length, wild-type Akt/PKB in a serum-sensitive fashion. No other proposed hydrophobic motif kinase has been shown to fulfill all these criteria. With hindsight, clues are seen in the literature to the important role of mTOR in Akt/PKB activation. Prolonged but not acute treatment of certain human cells with rapamycin partially inhibits Akt/PKB phosphorylation, and the current findings provide a possible rationale to explain these results. Although rapamycin does not bind to a preformed rictor-mTOR complex, during long-term rapamycin treatment the drug should eventually sequester many of the newly synthesized mTOR molecules within cells. Thus, as the rictor-mTOR complex turns over, rapamycin may interfere with its reassembly or over time become part of the new complexes. It is reasonable to expect then that prolonged rapamycin treatment may partially inhibit rictor-mTOR activity, which would explain why rapamycin is particularly effective at suppressing the proliferation of tumor cells with hyperactive Akt/PKB. The PI3K/Akt pathway is frequently deregulated in human cancers that have lost the expression of the PTEN tumor suppressor gene, and the current findings suggest that direct inhibitors of mTOR-rictor should strongly suppress Akt/PKB activity. Thus, the rictor-mTOR complex, like its raptor-mTOR sibling, may be a valuable drug target (Sarbassov, 2005a).

Rictor, a novel binding partner of mTOR, defines a rapamycin-insensitive and raptor-independent pathway that regulates the cytoskeleton

The mammalian TOR (mTOR) pathway integrates nutrient- and growth factor-derived signals to regulate growth, the process whereby cells accumulate mass and increase in size. mTOR is a large protein kinase and the target of rapamycin, an immunosuppressant that also blocks vessel restenosis and has potential anticancer applications. mTOR interacts with the raptor and GΔL proteins to form a complex that is the target of rapamycin. mTOR is also part of a distinct complex defined by the novel protein rictor (rapamycin-insensitive companion of mTOR). Rictor shares homology with the previously described pianissimo from D. discoidieum, STE20p from S. pombe, and AVO3p from S. cerevisiae. Interestingly, AVO3p is part of a rapamycin-insensitive TOR complex that does not contain the yeast homolog of raptor and signals to the actin cytoskeleton through PKC1. Consistent with this finding, the rictor-containing mTOR complex contains GΔL but not raptor, and it neither regulates the mTOR effector S6K1 nor is it bound by FKBP12-rapamycin. The rictor-mTOR complex modulates the phosphorylation of Protein Kinase C alpha (PKCa) and the actin cytoskeleton, suggesting that this aspect of TOR signaling is conserved between yeast and mammals (Sarbassov, 2004).

Phosphorylation and regulation of Akt/PKB by the rictor-mTOR complex

Deregulation of Akt/protein kinase B (PKB) is implicated in the pathogenesis of cancer and diabetes. Akt/PKB activation requires the phosphorylation of Thr308 in the activation loop by the phosphoinositide-dependent kinase 1 (PDK1) and Ser473 within the carboxyl-terminal hydrophobic motif by an unknown kinase. This study shows that in Drosophila and human cells the target of rapamycin (TOR) kinase and its associated protein rictor are necessary for Ser473 phosphorylation and that a reduction in rictor or mammalian TOR (mTOR) expression inhibited an Akt/PKB effector. The rictor-mTOR complex directly phosphorylated Akt/PKB on Ser473 in vitro and facilitated Thr308 phosphorylation by PDK1. Rictor-mTOR may serve as a drug target in tumors that have lost the expression of PTEN, a tumor suppressor that opposes Akt/PKB activation (Sarbassov, 2005a).

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. This study shows 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. This work describes an unforeseen mechanism of action for rapamycin that suggests it can be used to inhibit Akt/PKB in certain cell types (Sarbassov, 2006).

Search PubMed for articles about Drosophila Rictor

Arimoto, K., Fukuda, H., Imajoh-Ohmi, S., Saito, H. and Takekawa, M. (2008). Formation of stress granules inhibits apoptosis by suppressing stress-responsive MAPK pathways. Nat Cell Biol 10: 1324-1332. PubMed ID: 18836437

Hietakangas, V. and Cohen, S. M. (2007). Re-evaluating AKT regulation: role of TOR complex 2 in tissue growth. Genes Dev 21: 632-637. PubMed ID: 17369395

Huang, W., Zhu, P. J., Zhang, S., Zhou, H., Stoica, L., Galiano, M., Krnjevic, K., Roman, G. and Costa-Mattioli, M. (2013). mTORC2 controls actin polymerization required for consolidation of long-term memory. Nat Neurosci 16: 441-448. PubMed ID: 23455608

Ikenoue, T., Inoki, K., Yang, Q., Zhou, X. and Guan, K. L. (2008). Essential function of TORC2 in PKC and Akt turn motif phosphorylation, maturation and signalling. EMBO J 27: 1919-1931. PubMed ID: 18566587

Jevtov, I., et al. (2015). TORC2 mediates the heat stress response in Drosophila by promoting the formation of stress granules. J Cell Sci 128(14): 2497-508. PubMed ID: 26054799

Koike-Kumagai, M., Yasunaga, K., Morikawa, R., Kanamori, T. and Emoto, K. (2009). The target of rapamycin complex 2 controls dendritic tiling of Drosophila sensory neurons through the Tricornered kinase signalling pathway. EMBO J 28: 3879-3892. PubMed ID: 19875983

Kuo, Y., Huang, H., Cai, T. and Wang, T. (2015). Target of Rapamycin Complex 2 regulates cell growth via Myc in Drosophila. Sci Rep 5: 10339. PubMed ID: 25999153

Lee, G. and Chung, J. (2007). Discrete functions of rictor and raptor in cell growth regulation in Drosophila. Biochem Biophys Res Commun 357: 1154-1159. PubMed ID: 17462592

Natarajan, R., Trivedi-Vyas, D. and Wairkar, Y. P. (2013). Tuberous sclerosis complex regulates Drosophila neuromuscular junction growth via the TORC2/Akt pathway. Hum Mol Genet 22: 2010-2023. PubMed ID: 23393158

Oh, W. J., Wu, C. C., Kim, S. J., Facchinetti, V., Julien, L. A., Finlan, M., Roux, P. P., Su, B. and Jacinto, E. (2010). mTORC2 can associate with ribosomes to promote cotranslational phosphorylation and stability of nascent Akt polypeptide. EMBO J 29: 3939-3951. PubMed ID: 21045808

Ohn, T., Kedersha, N., Hickman, T., Tisdale, S. and Anderson, P. (2008). A functional RNAi screen links O-GlcNAc modification of ribosomal proteins to stress granule and processing body assembly. Nat Cell Biol 10: 1224-1231. PubMed ID: 18794846

Sarbassov, D. D., et al. (2004), Rictor, a novel binding partner of mTOR, defines a rapamycin-insensitive and raptor-independent pathway that regulates the cytoskeleton. Curr. Biol. 14: 1296-1302. 15268862

Sarbassov, D., Guertin, D. A., Ali, S. M. and Sabatini, D. M. (2005a). Phosphorylation and regulation of Akt/PKB by the rictor-mTOR complex. Science 307: 1098-1101. Medline abstract: 15718470

Sarbassov, D. D. and Sabatini, D. M. (2005b). Redox regulation of the nutrient-sensitive raptor-mTOR pathway and complex. J. Biol. Chem. 280(47): 39505-9. Medline abstract: 16183647

Sarbassov, D. D., Ali, S. M., Sengupta, S., Sheen, J. H., Hsu, P. P., Bagley, A. F., Markhard, A. L. and Sabatini, D. M. (2006). Prolonged rapamycin treatment inhibits mTORC2 assembly and Akt/PKB. Mol Cell 22: 159-168. PubMed ID: 16603397

Wang, T., Blumhagen, R., Lao, U., Kuo, Y. and Edgar, B. A. (2012). LST8 regulates cell growth via target-of-rapamycin complex 2 (TORC2). Mol Cell Biol 32: 2203-2213. PubMed ID: 22493059

Zinzalla, V., Stracka, D., Oppliger, W. and Hall, M. N. (2011). Activation of mTORC2 by association with the ribosome. Cell 144: 757-768. PubMed ID: 21376236

date revised: 12 July 2015

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