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).

Drosophila salt-inducible kinase (SIK) regulates starvation resistance through cAMP-response element-binding protein (CREB)-regulated transcription coactivator

Salt-inducible kinase (SIK), one of the AMP-activated kinase (AMPK)-related kinases, has been suggested to play important functions in glucose homeostasis by inhibiting the cAMP-response element-binding protein (CREB)-regulated transcription coactivator (CRTC). To examine the role of SIK in vivo, a Drosophila SIK mutant was generated and was found to have higher amounts of lipid and glycogen stores and to be resistant to starvation. Interestingly, SIK transcripts are highly enriched in the brain, and neuron-specific expression of exogenous SIK was found to fully rescued lipid and glycogen storage phenotypes as well as starvation resistance of the mutant. Using genetic and biochemical analyses, it was demonstrated that CRTC Ser-157 phosphorylation by SIK is critical for inhibiting CRTC activity in vivo. Furthermore, double mutants of SIK and CRTC became sensitive to starvation, and the Ser-157 phosphomimetic mutation of CRTC reduced lipid and glycogen levels in the SIK mutant, suggesting that CRTC mediates the effects of SIK signaling. Collectively, these results strongly support the importance of the SIK-CRTC signaling axis that functions in the brain to maintain energy homeostasis in Drosophila (Choi, 2011).

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).

Bipartite functions of the CREB co-activators selectively direct alternative splicing or transcriptional activation

The CREB regulated transcription co-activators (CRTCs) regulate many biological processes by integrating and converting environmental inputs into transcriptional responses. Although the mechanisms by which CRTCs sense cellular signals are characterized, little is known regarding how CRTCs contribute to the regulation of cAMP inducible genes. This study shows that these dynamic regulators, unlike other co-activators, independently direct either pre-mRNA splice-site selection or transcriptional activation depending on the cell type or promoter context. Moreover, in other scenarios, the CRTC co-activators coordinately regulate transcription and splicing. Mutational analyses showed that CRTCs possess distinct functional domains responsible for regulating either pre-mRNA splicing or transcriptional activation. Interestingly, the CRTC1-MAML2 oncoprotein lacks the splicing domain and is incapable of altering splice-site selection despite robustly activating transcription. The differential usage of these distinct domains allows CRTCs to selectively mediate multiple facets of gene regulation, indicating that co-activators are not solely restricted to coordinating alternative splicing with increase in transcriptional activity (Amelio, 2009).

The idea that the CRTC co-activators perform more than one function is in accord with those of other co-regulators that possess multiple activities. For example, the CREB co-activator CBP mediates gene regulation through its intrinsic and associated histone acetyl transferase activity and by promoting the formation of the pre-initiation complex (PIC) through interactions with components of the core transcriptional machinery. Moreover, the transcriptional co-activators PGC, COAA and CAPER function as transcriptional activators, as well as regulators of transcriptionally coupled pre-mRNA processing. However, in contrast to other co-activators involved in splicing, the CRTCs lack any conserved, definable RNA-binding domain (e.g., an RNA recognition motif (RRM), an arginine-serine (RS) domain, or a K homology (KH) domain). Rather, it is hypothesized that CRTCs provide a scaffold for the assembly of a larger complex of proteins that may directly bind the transcript. This idea is supported by CRTC IP MS/MS experiments that identified several proteins involved in pre-mRNA processing (Amelio, 2007). If the CRTCs form the basis for a protein scaffold that mediates RNA processing by recruiting other proteins, then the ability of CRTCs to recruit slightly different complexes (i.e., cell type or promoter specific) may contribute to these independent bipartite functions. Moreover, PKA may contribute to TORC-dependent alternative splicing through phosphorylation of components within the recruited splicing complex (Amelio, 2009).

There is now overwhelming evidence that the subcellular localization, and thus function, of the CRTC2 co-activator is mediated by several cues, including glucagon and insulin. CRTC2 increases fasting blood glucose levels and the induction of key genes directly involved in gluconeogenesis. Collectively, these studies showed that CRTC2 is the rate-limiting step of cAMP signalling and that it alone is sufficient to initiate the early stages of the gluconeogenic program. Interestingly, several of the genes within the gluconeogenic program such as Nurr1 (Nr4A2), as well as those encoding proteins that mediate later stages of glucose production such as PGC-1 (Ppargc1a), are alternatively spliced; this study shows that CRTC2 can promote alternative splicing of Nr4a2. Thus, CRTCs function as integrators of extracellular signals to influence transcript diversity by acting either as a conduit between components of the transcription and splicing machineries or as autonomous regulators of each process. However, although TORCs can selectively activate splicing in some promoter contexts without activating transcription, these promoters possess basal transcriptional activity, therefore, the effects of the TORCs on pre-mRNA splicing could still formally be considered co-transcriptional despite the absence of co-activator-induced transcription (Amelio, 2009).

There are two prevailing models of promoter-dependent coordination of transcription and pre-mRNA processing that could apply to CRTC-mediated alternative splicing. These include splicing factor recruitment to the carboxyl terminal domain (CTD) of RNA polymerase II (RNAP II) (recruitment model) and regulation of RNAP II elongation rates (kinetic model). These models embody a 'forward coupling' mechanism that links RNAP II transcription to pre-mRNA splicing. However, ‘reverse coupling' mechanisms have been described, whereby pre-mRNA splicing exerts an influence back on transcription. Indeed, promoter-proximal 5' splice sites seem to promote reverse coupling by increasing transcription initiation through recruitment of the PIC (Damgaard, 2008). CRTCs interact with TAFII130, a component of the PIC (Conkright, 2003). Therefore, the finding that CRTCs can induce splicing independent of their effects on transcription may reflect the failure of CRTCs to recruit components of the PIC to TATA-less promoters. Accordingly, although parts of both models help to explain mechanistically how the CRCT co-activators mediate alterative splicing, neither model can fully account for the current observations, suggesting that further refinement to the current working models may be necessary. Apart from CRTCs mechanistic role in pre-mRNA processing, CRTC-mediated splicing probably has physiological significance. This is supported by the vast number of genes that are alternatively spliced, the large cast of promoters that contain CRE elements (roughly 10%), and by the fact that the CRTC recruitment to promoters (e.g. through interactions with CREB) is sufficient to augment alternative splicing. The inability of the CRTC1-MAML2 oncoprotein to mediate CREB-dependent splice-site selection, despite retaining robust transcriptional activation properties, suggests that this fusion product may also promote cellular transformation through aberrant pre-mRNA splicing of CRE-containing genes. However, transcriptional activators have been shown to promote pre-mRNA splicing, therefore, it is possible that the MAML2 transactivation domain in the CRTC1-MAML2 fusion can mediate splicing in other promoter contexts. Furthermore, although the current data clearly show that the CRTC co-activators have the potential to regulate alternative splicing, this does not mandate that transcripts from CRE-containing genes will be alternatively spliced in a CRTC-dependent manner. Indeed, several studies have shown that the significance of the CREB co-activators, CBP/p300, is dictated by gene context and varies among tissues. Similar, cell-type specificity is also observed in CRTC-mediated alternative splicing of the endogenous Nr4a2 transcript. It is speculated that the generation of select transcript isoforms may differ between cell types, in part, due to the many diverse signalling events that converge on the CRTC co-activators (Amelio, 2009).

Traditional methods, such as expression arrays or TaqMan PCR focus on gene expression to examine gene regulation in response to signalling cascades. In these technologies, the probes and primer sets intentionally do not account for alternatively spliced products. As a result the canonical analysis of cAMP responsive genes has been restricted to the analysis of transcription induction effectively missing any affects of cAMP activation on pre-mRNA processing. The current findings establish that CRTCs direct alternative splicing independently or in coordination with transcriptional activation, thereby, linking the cAMP signalling cascade with the regulation of pre-mRNA processing. Future experiments will monitor alternative splicing in addition to transcriptional activation when identifying tissue-specific CRTC target promoters (Amelio, 2009).

Search PubMed for articles about Drosophila Torc

Amelio, A. L., Caputi, M. and Conkright, M. D. (2009). Bipartite functions of the CREB co-activators selectively direct alternative splicing or transcriptional activation. EMBO J. 28(18): 2733-47. PubMed Citation: 19644446

Amelio, A. L., et al. (2007) A coactivator trap identifies NONO (p54nrb) as a component of the cAMP-signaling pathway. Proc. Natl. Acad. Sci. 104: 20314-20319. PubMed Citation: 18077367

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

Choi, S., Kim, W. and Chung, J. (2011). Drosophila salt-inducible kinase (SIK) regulates starvation resistance through cAMP-response element-binding protein (CREB)-regulated transcription coactivator (CRTC). J. Biol. Chem. 286(4): 2658-64. PubMed Citation: 21127058

Conkright, M. D., et al. (2003) TORCs: transducers of regulated CREB activity. Mol. Cell 12: 413-423. PubMed Citation: 14536081

Damgaard, C. K., et al. (2008). A 5' splice site enhances the recruitment of basal transcription initiation factors in vivo. Mol. Cell 29: 271-278. PubMed Citation: 18243121

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: 15 December 2011

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