CrebB-17A

Targets of Activity

Decapentaplegic (Dpp) is an extracellular signal of the transforming growth factor-beta family with multiple functions during Drosophila development. For example, it plays a key role in the embryo during endoderm induction. During this process, Dpp stimulates transcription of the homeotic genes Ultrabithorax in the visceral mesoderm and labial in the subjacent endoderm. A cAMP response element (CRE) from an Ultrabithorax enhancer mediates Dpp-responsive transcription in the embryonic midgut, and endoderm expression from a labial enhancer depends on multiple CREs. The enhancer, called Ubx B confers Wingless- and Decapentaplegic-dependent expression in the visceral mesoderm. Staining mediated by Ubx B is in two stripes of cells in the visceral mesoderm, a wide prominent one in parasegments 6-9 and a narrow weak one in parasegment 3. The Drosophila CRE-binding protein dCREB-2 binds to the Ultrabithorax CRE. Binding is at a palindromic sequence TGGCGTCA that resembles a typical cAMP response element (CRE) (TGACGTCA). Mutation of this site results in the elimination of response to Dpp, but a maintenance of response to Wg. This residual expression is in parasegment 8 and 9 coinciding with the main source of wg expression in the middle midgut. The Ubx CRE can also mediate response to Dpp signaling in the endoderm. Other transcription factors act through the Ubx B enhancer to confer its tissue-specific response to Dpp in the visceral mesoderm. CRE needs to cooperate with a LEF-1 binding site to respond to the Dpp signal in the visceral mesoderm. Schnurri, a transcription factor implicated in Dpp signaling, fails to interact with Ubx B. Adjacent to the CRE is another palindromic sequence that antagonizes the activating effects of Dpp and Wg signaling on the Ubx B enhancer. Ubiquitous expression of a dominant-negative form of dCREB-2 suppresses CRE-mediated reporter gene expression and reduces labial expression in the endoderm. Therefore, a dCREB-2 protein may act as a nuclear target, or as a partner of a nuclear target, for Dpp signaling in the embryonic midgut (Eresh, 1997).

Long term memory requires de novo gene expression mediated by CREB family genes. Using an inducible transgene that expresses CrebB-17A, the dominant negative member of the CREB family, long term memory has been specifically and completely blocked while short term memory (anasthesia-resistent memory) remains unaffected (Yin 1995b).

Induced expression of a CrebB-17A activator isoform enhances long term memory in Drosophila, so that maximum learning is achieved after only one training session. Memory requires phosphorylation of the activator isoform (Yin 1995b).

Fasciclin2 mutants lead to an increase in number of boutons at neuromuscular synapses without affecting quantal content. Increased cAMP in dunce mutants increases both synaptic structure and quantal content. Thus there must be other elements downstream of cAMP, but not downstream from Fas2, that are involved in increasing quantal content. CREB is a candidate for the cAMP target responsible for increasing quantal content. CREB acts in parallel with Fas2 to cause an increase in synaptic strength. Expression of an endogenous CREB repressor, CrebB-17A-a, in dunce mutants blocks functional but not structural plasticity. Expression of the activator isoform, CrebB-17A-a, increases synaptic strength, by increasing presynaptic transmitter release at single boutons, but only in Fas2 mutants that increase bouton number. Strong overexpression of CrebB-17A-a results in a significant increase in quantal content, independent of genetic background and with little effect on bouton number. Thus CREB-mediated increase in synaptic strength is due to increased presynaptic transmitter release and expression of CrebB-17A-a in a Fas2 mutant background genetically reconstitutes cAMP-dependent plasticity. It is concluded that cAMP initiates parallel changes in CREB and Fas2 to achieve long term synaptic enhancement (Davis, G. W 1996).

Drosophila CREB genes are implicated in regulation of the Drosophila homolog of mammalian JUN. A 43-bp 5' proximal promoter region is necessary for the transcription activity of DJUN (Perkins, 1988a). Deletion of this fragment decreases transcriptional activity 67-fold. This 43-bp sequence alone, containing a Drosophila transcription factor DTF-1 binding site and TATA box, however, is not sufficient for transcription activity. An 80-bp sequence including the start of transcription has considerable basal activity. This intragenic region containing an AP-1 site and a CRE site (presumably binding a Drosophila CREB) modulates or fine tunes activity of the promoter. An extragenic region containing two AP-1 sites similarly affects promoter activity (Wang, 1994).

The responsiveness of DJun to CREB suggests a role for DJun in the preservation of long term memory in the fly. To date, such a role has not been documented.

Protein Interactions

In the cyclic AMP signal transduction pathway, protein kinase A (PKA) activates CREB by phosphorylation (Drain, 1991). The isoform CrebB-17A-a is a PKA dependent activator of transcription (Yin 1994a). The isoform CrebB-17A-b does not function as a PKA dependent activator, but works as a direct antagonist of PKA-dependent activation by CrebB-17A-a (Yin 1995a).

Interactions of Creb-binding Protein

Drosophila CBP (Nejire) is a co-activator of cubitus interruptus in hedgehog signaling. Drosophila CBP predicts a protein of relative molecular mass 332,000; the gene maps to position 8F/9A on the X chromosome. Mutants for dCBP gene, nejire, die at stages 9 or 10 during embryogenesis, although some embryos survive to hatching. The most severe phenotype of the nej hemizygotes is the twisting of the embryo at germband elongation. The expression of wingless is strikingly reduced at the posterior margin of each parasegment in mutants. In addition, engrailed expression, which is maintained by WG protein, is significantly lower than in wild type. These observations suggest the Drosophila CBP might contribute to the functioning of some transcription factors involved in the wingless-engrailed signaling pathway. Cubitus interruptus protein physically interacts with Drosophila CBP (dCBP). A series of deletion mutants of ci indicate that a region of CI between amino acids 1020 and 1160 is required for phosphorylation independent interaction with dCBP. This region is part of the CI transactivation domain, C-terminal to five putative PKA sites. dCBP expression augments transactivation by CI up to a maximum of 62 fold. The dominant gain-of-function ciD mutant phenotype in which the longitudinal vein 4 of the adult wing is shortened, some posterior row hairs are missing, and the posterior wing margin is flattened, can be explained by the inappropriate expression of ci in the posterior compartment of the wing imaginal disc, where it is usually repressed by Engrailed. A subset of the ciD wing defects is suppressed by haploinsufficiency of dCBP. Thus dCBP is required for the activation of Cubitus interruptus target genes such as patched, and CBP is required for the activator function of CI but not for the repressor function. dCBP binds to dCREB2, the Drosophila homolog of CREB, in a phosphorylation-dependent manner, whereas the dCBP-CI interaction is phosphorylation-independent. These findings raise the possiblilty that a limited amount of dCBP might be recruited to PKA-phosphorylated dCREB2, resulting in a decrease in CI activity, explaining the antagonistic actions of PKA and Hedgehog (Akimaru, 1997a).

Attempts to demonstrate trans-activation activity by the Drosophila Myb gene product have been unsuccessful so far. Co-transfection of Schneider cells with a plasmid expressing the Drosophila homolog of transcriptional co-activator CBP (dCBP) results in transactivation by Myb. Using this assay system, the functional domains of Myb have been analyzed. Two domains located in the N-proximal region, one of which is required for DNA binding and the other for dCBP binding, are both necessary and sufficient for trans-activation. In this respect, D-Myb is similar to c-Myb and A-Myb, but different from mammalian B-Myb. These results shed light on how the myb gene diverged during the course of evolution (Hou, 1997).

Although CREB-binding protein (CBP) functions as a co-activator of many transcription factors, relatively little is known about the physiological role of CBP. Mutations in the human CBP gene are associated with Rubinstein-Taybi syndrome, a haplo-insufficiency disorder characterized by abnormal pattern formation. Drosophila CBP is maternally expressed, suggesting that it plays a role in early embryogenesis. Mesoderm formation is one of the most important events during early embryogenesis. To initiate the differentiation of the mesoderm in Drosophila, multiple zygotic genes such as twist (twi) and snail (sna), which encode a basic-helix-loop-helix and a zinc finger transcription factor, respectively, are required. The transcription of these genes is induced by maternal Dorsal protein, a transcription factor that is homologous to the NF-kappa B family of proteins. Drosophila CBP mutants fail to express twi and generate twisted embryos. This is explained by results showing that dCBP is necessary for Dorsal-mediated activation of the twi promoter (Akimaru, 1997b).

T-cell factor (TCF), a high-mobility-group domain protein, is the transcription factor activated by Wnt/Wingless signaling. When signaling occurs, TCF binds to its coactivator, beta-catenin/Armadillo, and stimulates the transcription of the target genes of Wnt/Wingless by binding to TCF-responsive enhancers. Inappropriate activation of TCF in the colon epithelium and other cells leads to cancer. It is therefore desirable for unstimulated cells to have a negative control mechanism to keep TCF inactive. Drosophila CREB-binding protein (dCBP) binds to Drosophila TCF (Pangolin). dCBP mutants show mild Wingless overactivation phenotypes in various tissues. Consistent with this, dCBP loss-of-function suppresses the effects of armadillo mutation. Moreover, dCBP is shown to acetylate a conserved lysine in the Armadillo-binding domain of dTCF, and this acetylation lowers the affinity of Armadillo binding to dTCF. Although CBP is a coactivator of other transcription factors, these data show that CBP represses TCF (Waltzer, 1998).

The insulin-regulated CREB coactivator TORC promotes stress resistance in Drosophila

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. 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, 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. 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. 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).

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