InteractiveFly: GeneBrief

TORC: Biological Overview | References

In fasted mammals, glucose homeostasis is maintained through induction of the cAMP response element-binding protein (CREB; see Drosophila CrebB-17A) coactivator transducer of regulated CREB activity 2 (TORC2), which stimulates the gluconeogenic program in concert with the forkhead factor FOXO1 (see Drosophila Foxo). Starvation also triggers TORC activation in Drosophila, where it maintains energy balance through induction of CREB target genes in the brain. TORC mutant flies have reduced glycogen and lipid stores and are sensitive to starvation and oxidative stress. Neuronal TORC expression rescued stress sensitivity as well as CREB target gene expression in TORC mutants. During refeeding, increases in insulin signaling inhibited TORC activity through the salt-inducible kinase 2 (SIK2)-mediated phosphorylation and subsequent degradation of TORC. Depletion of neuronal SIK2 increased TORC activity and enhanced stress resistance. As disruption of insulin signaling also augments TORC activity in adult flies, these results illustrate the importance of an insulin-regulated pathway that functions in the brain to maintain energy balance (Wang, 2008).

Fasting triggers concerted changes in behavior, physical activity, and metabolism that are remarkably well conserved through evolution. In mammals, such responses are often coordinated by transcriptional coactivators that are themselves targets for regulation by environmental cues, but the extent to which these coactivators function in model organisms such as Drosophila is less clear (Wang, 2008).

In the basal state, mammalian TORCs are phosphorylated by salt-inducible kinases (SIKs) and sequestered in the cytoplasm via phosphorylation-dependent association with 14-3-3 proteins (Koo, 2005; Screaton, 2004). During fasting, elevations in circulating pancreatic glucagon promote TORC dephosphorylation via the protein kinase A (PKA)-mediated phosphorylation and inhibition of SIK2 (Wang, 2008).

Increases in intracellular calcium have also been found to stimulate cAMP response element-binding protein (CREB) target gene expression through the activation of calcineurin/PP2B, a calcium/calmodulin-dependent serine/threonine (Ser/Thr) phosphatase that binds directly to and dephosphorylates mammalian TORCs (Koo, 2005; Screaton, 2004). Indeed, cAMP and calcium signals stimulate TORC dephosphorylation cooperatively through their effects on SIKs and PP2B, respectively. Following their liberation from 14-3-3 proteins, dephosphorylated TORCs shuttle to the nucleus, where they mediate cellular gene expression by associating with CREB over relevant promoters (Wang, 2008).

TORC2 is thought to function in parallel with FOXO1 to maintain energy balance during fasting. Knockdown and knockout studies support a critical role for both proteins in regulating catabolic programs in the liver (Dentin, 2007, Koo, 2005; Matsumoto, 2007). In Drosophila, starvation promotes the mobilization of glycogen and lipid stores in response to increases in circulating adipokinetic hormone (AKH), the fly homolog of mammalian glucagon (Kim, 2004; Lee, 2004). In parallel, decreases in insulin/IGF signaling (IIS) also stimulate the dephosphorylation and nuclear translocation of Drosophila FOXO, which in turn stimulates a wide array of nutrient-regulated genes (Wang, 2008).

The accumulation of lipid and glycogen stores in adult flies is highly correlated with resistance to starvation in Drosophila (Djawdan, 1998). Indeed, disruption of the IIS pathway promotes lipid accumulation and correspondingly increases resistance to starvation and oxidative stress. Although FOXO does not appear to be required for starvation resistance in adult flies, overexpression of FOXO has been found to mimic the starvation phenotype in larvae (Wang, 2008).

This study addresses the importance of Drosophila TORC, the single Drosophila homolog of mammalian TORCs, in metabolic regulation. It was found that increases in TORC activity during starvation enhance survival through the activation of CREB target genes in the brain. During feeding, increases in insulin signaling inhibit TORC activity through phosphorylation by a Drosophila homolog of mammalian SIK2. These studies indicate that TORC is part of an insulin-regulated pathway that functions in concert with FOXO to promote energy balance and stress resistance (Wang, 2008).

Drosophila TORC shares considerable sequence homology with mammalian TORCs in its CREB binding domain (CBD), transactivation domain (TAD), calcineurin (Cn) recognition motif, and regulatory site (Ser157), which is phosphorylated by members of the AMPK family of stress- and energy-sensing Ser/Thr kinases in mammals. Drosophila TORC protein is expressed at low levels during larval and pupal stages, with the highest amounts detected in adults. TORC mRNA levels are also increased in adults relative to larvae, although to a lesser extent (Wang, 2008).

In the basal state, Drosophila TORC is highly phosphorylated at Ser157 and localized to the cytoplasm in Drosophila S2 and KC-167 cells. Demonstrating the importance of Ser157 phosphorylation in sequestering TORC, Ser157Ala mutant TORC shows only low-level binding to 14-3-3 proteins relative to wild-type TORC in HEK293T cells. Exposure to the adenylyl cyclase activator forskolin (FSK) or to staurosporine (STS), an inhibitor of SIKs and other protein kinases, promotes TORC dephosphorylation, liberation from 14-3-3 proteins, and nuclear translocation (Wang, 2008).

Consistent with these changes, overexpression of wild-type Drosophila TORC potentiated CRE-luciferase (CRE-luc) reporter activity following exposure of HEK293T cells to FSK, whereas phosphorylation-defective Ser157Ala TORC stimulated CRE-luc activity under basal as well as FSK-induced conditions. CRE-luc activity is blocked by coexpression of the dominant-negative CREB inhibitor ACREB. Taken together, these results indicate that Drosophila TORC modulates CREB target gene expression following its dephosphorylation at Ser157 and nuclear entry in response to cAMP (Wang, 2008).

Based on the ability of mammalian TORCs to promote fasting metabolism (Koo, 2005), this study examined whether Drosophila TORC performs a similar function in adult flies. Amounts of dephosphorylated, active TORC increased progressively during water-only starvation. Feeding adult flies paraquat, a respiratory chain inhibitor that stimulates the production of reactive oxygen species, also promoted the accumulation of dephosphorylated TORC, suggesting a broader role for this coactivator in stress resistance. Similar to mammalian TORCs (Dentin, 2007), the upregulation of TORC in Drosophila appears to reflect an increase in TORC protein stability, as amounts of TORC mRNA did not change significantly in response to fasting or paraquat treatment (Wang, 2008).

Insulin signaling regulates lipid and glucose metabolism in both C. elegans and Drosophila in part by inhibiting FOXO-dependent transcription. Lipid stores are increased in flies with mutations in the IIS pathway; these animals are resistant to starvation as well as oxidative stress. This study found that TORC enhances survival during starvation in part by stimulating target gene expression in neurons. Although TORC appears to act in parallel with FOXO, the increase in FOXO activity observed in TORC mutant flies indicates that TORC may also impact on this pathway (Wang, 2008).

TORC appears to be required for the expression of genes that promote lipid and glucose metabolism, amino acid transport, and proteolysis. Consistent with this idea, paralogs for a number of TORC-regulated genes (TrxT, CAT, and UCP4c) appear to be required for starvation and oxidative stress resistance (Chen, 2004; Fridell, 2005; Mockett, 2003; Svensson, 2007). Superimposed on these effects, neuronal TORC may also promote systemic resistance to starvation and oxidative stress by modulating the expression of neuropeptide hormones and other circulating factors that regulate peripheral glucose and lipid metabolism (Wang, 2008).

In mammals, refeeding has been found to increase SIK2 kinase activity during refeeding through the AKT-mediated phosphorylation of SIK2 (Dentin, 2007). Phosphorylated TORC2 is subsequently ubiquitinated and degraded through the E3 ligase COP1. Supporting a similar mechanism in Drosophila, RNAi-mediated knockdown of AKT in Drosophila is sufficient to increase TORC activity. Conversely, depletion of neuronal SIK2 enhances TORC activity and increases resistance to both starvation and paraquat feeding. Although a Drosophila homolog for COP1 has not been identified, it is imagined that the ubiquitin-dependent degradation of Drosophila TORC is also critical in modulating its activity in brain as well as other tissues (Wang, 2008).

Based on its ability to potentiate CREB target gene expression in neurons, TORC may function in other biological settings. Indeed, Drosophila CREB appears to have an important role in learning and memory, circadian rhythmicity, rest homeostasis, and addictive behavior. Future studies should reveal the extent to which TORC participates in these contexts as well (Wang, 2008).

Activation of cAMP response element-mediated gene expression by regulated nuclear transport of TORC proteins

The CREB family of proteins are critical mediators of gene expression in response to extracellular signals and are essential regulators of adaptive behavior and long-term memory formation. The TORC proteins were recently described as potent CREB coactivators, but their role in regulation of CREB activity remained unknown. TORC proteins were found to be exported from the nucleus in a CRM1-dependent fashion. A high-throughput microscopy-based screen was developed to identify genes and pathways capable of inducing nuclear TORC accumulation. Expression of the catalytic subunit of PKA and the calcium channel TRPV6 relocalized TORC1 to the nucleus. Nuclear accumulation of the three human TORC proteins was induced by increasing intracellular cAMP or calcium levels. TORC1 and TORC2 translocation in response to calcium, but not cAMP, is mediated by calcineurin, and TORC1 is directly dephosphorylated by calcineurin. TORC function was shown to be essential for CRE-mediated gene expression induced by cAMP, calcium, or GPCR activation, and nuclear transport of TORC1 is sufficient to activate CRE-dependent transcription. Drosophila TORC is translocated in response to calcineurin activation in vivo. Thus, TORC nuclear translocation is an essential, conserved step in activation of cAMP-responsive genes (Bittinger, 2004: Full text of article).

The subcellular localization of the single ancestral Drosophila TORC (dTORC) was examined to determine whether regulated nuclear translocation is a general property of TORC proteins. An inducible eGFP-dTORC transgene was constructed and stably integrated into the Drosophila genome. Examination of intact salivary tissue from transgenic larvae demonstrated that dTORC is predominantly present in the cytoplasm in untreated cells and rapidly translocates to the nucleus upon ionomycin exposure, with the majority of the dTORC shuttling to the nucleus 10 min after ionomycin exposure. Cotreatment with calcineurin inhibitor CsA completely blocked dTORC translocation in response to ionomycin, indicating that participation of calcineurin in the calcium-mediated cAMP response element binding protein (CREB) response is conserved between vertebrates and insects (Bittinger, 2004).

Search PubMed for articles about Drosophila Torc

Bittinger, M. A., et al. (2004). Activation of cAMP response element-mediated gene expression by regulated nuclear transport of TORC proteins. Curr. Biol. 14(23): 2156-61. PubMed Citation: 15589160

Chen, L., Rio, D. C., Haddad, G. G. and Ma, E. (2004). Regulatory role of dADAR in ROS metabolism in Drosophila CNS. Brain Res. Mol. Brain Res. 131: 93-100. PubMed Citation: 15530657

Dentin, R., et al. (2007). Insulin modulates gluconeogenesis by inhibition of the coactivator TORC2. Nature 449: 366-369. PubMed Citation: 1780530

Djawdan, M., et al. (1998). Metabolic reserves and evolved stress resistance in Drosophila melanogaster. Physiol. Zool. 71: 584-594. PubMed Citation: 9754535

Fridell, Y. W., et al. (2005). Targeted expression of the human uncoupling protein 2 (hUCP2) to adult neurons extends life span in the fly. Cell Metab. 1: 145-152. PubMed Citation: 16054055

Kim, S. K. and Rulifson, E. J. (2004). Conserved mechanisms of glucose sensing and regulation by Drosophila corpora cardiaca cells. Nature 431: 316-320. PubMed Citation: 15372035

Koo, S. H., et al. (2005). The CREB coactivator TORC2 is a key regulator of fasting glucose metabolism. Nature 437: 1109-1111. PubMed Citation: 16148943

Lee, G. and Park, J. H. (2004). Hemolymph sugar homeostasis and starvation-induced hyperactivity affected by genetic manipulations of the adipokinetic hormone-encoding gene in Drosophila melanogaster. Genetics 167: 311-323. PubMed Citation: 15166157

Matsumoto, M., et al. (2007). Impaired regulation of hepatic glucose production in mice lacking the forkhead transcription factor foxo1 in liver. Cell Metab. 6: 208-216. PubMed Citation: 17767907

Mockett, R. J., et al (2003). Ectopic expression of catalase in Drosophila mitochondria increases stress resistance but not longevity. Free Radic. Biol. Med. 34: 207-217. PubMed Citation: 12521602

Screaton, R. A., et al. (2004). The CREB coactivator TORC2 functions as a calcium- and cAMP-sensitive coincidence detector. Cell 119: 61-74. PubMed Citation: 15454081

Svensson, M. J. and Larsson, J. (2007). Thioredoxin-2 affects lifespan and oxidative stress in Drosophila. Hereditas 144: 25-32. PubMed Citation: 17567437

Wang, B., et al. (2008).The insulin-regulated CREB coactivator TORC promotes stress resistance in Drosophila. Cell Metab. 7(5): 434-44. PubMed Citation: 18460334

date revised: 28 March 2008

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