Myc : Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

Gene name - Myc

Synonyms - Myc, dMyc

Cytological map position - 3D5

Function - transcription factor

Keyword(s) - cell cycle, oogenesis, oncogene

Symbol - Myc

FlyBase ID:FBgn0262656

Genetic map position -

Classification - bHLH - leucine zipper

Cellular location - nuclear

NCBI links: Precomputed BLAST | Entrez Gene
Recent literature
Aughey, G. N., Grice, S. J. and Liu, J. L. (2016). The interplay between Myc and CTP synthase in Drosophila. PLoS Genet 12: e1005867. PubMed ID: 26889675
CTP synthase (CTPsyn) is essential for the biosynthesis of pyrimidine nucleotides. Previous studies have shown that CTPsyn is incorporated into a novel cytoplasmic structure which has been termed the cytoophidium. This study reports that Myc regulates cytoophidium formation during Drosophila oogenesis. Myc protein levels correlate with cytoophidium abundance in follicle epithelia. Reducing Myc levels results in cytoophidium loss and small nuclear size in follicle cells, while overexpression of Myc increases the length of cytoophidia and the nuclear size of follicle cells. Ectopic expression of Myc induces cytoophidium formation in late stage follicle cells. Furthermore, knock-down of CTPsyn is sufficient to suppress the overgrowth phenotype induced by Myc overexpression, suggesting CTPsyn acts downstream of Myc and is required for Myc-mediated cell size control. Taken together, these data suggest a functional link between Myc, a renowned oncogene, and the essential nucleotide biosynthetic enzyme CTPsyn.
Strilbytska, O. M., Semaniuk, U. V., Storey, K. B., Edgar, B. A. and Lushchak, O. V. (2016). Activation of the Tor/Myc signaling axis in intestinal stem and progenitor cells affects longevity, stress resistance and metabolism in Drosophila.. Comp Biochem Physiol B Biochem Mol Biol 203: 92-99. PubMed ID: 27693629
The TOR (target of rapamycin) signaling pathway and the transcriptional factor Myc play important roles in growBh control. Myc acts, in part, as a downstream target of TOR to regulate the activity and functioning of stem cells. Tbis study explored the role of TOR-Myc axis in stem and progenitor cells in the regulation of lifespan, stress resistance and metabolism in Drosophila. Goth overexpression of rheb and myc-rheb in midgut stem and progenitor cells decreased the lifespan and starvation resistance of flies. TOR activation caused higher survival under malnutrition conditions. Furthermore, gut-specific activation of JAK/STAT and insulin signaling pathways were demonstrated to control gut integrity. Both genetic manipulations had an impact on carbohydrate metabolism and transcriptional levels of metabolic genes. These findings indicate that activation of the TOR-Myc axis in midgut stem and progenitor cells influences a variety of traits in Drosophila.
Wehr Mathews, K., Cavegn, M. and Zwicky, M. (2017). Sexual dimorphism of body size is controlled by dosage of the X-chromosomal gene Myc and by the sex-determining gene tra in Drosophila. Genetics [Epub ahead of print]. PubMed ID: 28064166
Drosophila females are larger than males. This paper describes how X chromosome dosage drives sexual dimorphism of body size through two means: first, through unbalanced expression of a key X-linked growth regulating gene and second, through female-specific activation of the sex-determination pathway. X-chromosome dosage determines phenotypic sex by regulating the genes of the sex-determining pathway. In the presence of two sets of X-chromosome signal elements (XSEs), Sex-lethal (Sxl) is activated in female (XX) but not male (XY) animals. Sxl activates transformer (tra), a gene that encodes a splicing factor essential for female-specific development. It has previously been shown that null mutations in the tra gene result in only a partial reduction of body size of XX animals, which shows that other factors must contribute to size determination. Whether X dosage directly affects animal size was tested by analyzing males with duplications of X chromosomal segments. Upon tiling across the X chromosome, four duplications were found that increase male size by over 9%. Only one of these, Myc, was found not to be dosage compensated. Together, these results indicate that both Myc dosage and tra expression play crucial roles in determining sex-specific size in Drosophila larvae and adult tissue. Since Myc also acts as an XSE that contributes to tra activation in early, development, a double dose of Myc in females serves at least twice in development to promote sexual size dimorphism.
Ma, X., Huang, J., Tian, Y., Chen, Y., Yang, Y., Zhang, X., Zhang, F. and Xue, L. (2017). Myc suppresses tumor invasion and cell migration by inhibiting JNK signaling. Oncogene [Epub ahead of print]. PubMed ID: 28068320
Tumor metastasis, but not primary overgrowth, is the leading cause of mortality for cancer patients. During the past decade, Drosophila melanogaster has been well-accepted as an excellent model to address the intrinsic mechanism of different aspects of cancer progression, ranging from tumor initiation to metastasis. In a genetic screen aiming to find novel modulators of tumor invasion in Drosophila, this study identified the oncoprotein Myc as a negative regulator. While expression of Myc dramatically blocks tumor invasion and cell migration, loss of Myc promotes cell migration in vivo. The activity of Myc is further enhanced by the co-expression of its transcription partner Max. Mechanistically, Myc/Max directly upregulates the transcription of puc, which encodes an inhibitor of JNK signaling crucial for tumor invasion and cell migration. Furthermore, human cMyc potently suppresses JNK-dependent cell invasion and migration in both Drosophila and lung adenocarcinoma cell lines. These findings provide novel molecular insights into Myc-mediated cancer progression and raise the noteworthy problem in therapeutic strategies as inhibiting Myc might conversely accelerate tumor metastasis.

Tavares, L., Correia, A., Santos, M. A., Relvas, J. B. and Pereira, P. S. (2017). dMyc is required in retinal progenitors to prevent JNK-mediated retinal glial activation. PLoS Genet 13(3): e1006647. PubMed ID: 28267791
In the nervous system, glial cells provide crucial insulation and trophic support to neurons and are important for neuronal survival. In reaction to a wide variety of insults, glial cells respond with changes in cell morphology and metabolism to allow repair. Additionally, these cells can acquire migratory and proliferative potential. In particular, after axonal damage or pruning the clearance of axonal debris by glial cells is key for a healthy nervous system. Thus, bidirectional neuron-glial interactions are crucial in development, but little is known about the cellular sensors and signalling pathways involved. This study shows that decreased cellular fitness in retinal progenitors caused by reduced Drosophila Myc expression triggers non cell-autonomous activation of retinal glia proliferation and overmigration. Glia migration occurs beyond its normal limit near the boundary between differentiated photoreceptors and precursor cells, extending into the progenitor domain and is stimulated by JNK activation (and the function of its target Mmp1), while proliferative responses are mediated by Dpp/TGF-beta signalling activation.
Chanu, S. I. and Sarkar, S. (2017). Targeted downregulation of dMyc restricts neurofibrillary tangles mediated pathogenesis of human neuronal tauopathies in Drosophila. Biochim Biophys Acta [Epub ahead of print]. PubMed ID: 28529046
Formation of Neurofibrillary Tangles (NFTs) in neuronal tissues has been implicated as the hallmark of disease pathogenesis and tau mediated toxicity in human and mammalian models. However, previous studies had failed to correlate NFT formation with pathogenesis of human neuronal tauopathies in Drosophila disease models. Though, a recent report suggests formation of tau mediated NFTs like structures confined to dopaminergic neurons in Drosophila adult brain, by utilizing various approaches, this study demonstrated distinct and recurrent formation of NFTs in Drosophila neuronal tissues upon expression of wild type or mutant isoforms of human tau, and this appears as the key mediator of the pathogenesis of human neuronal tauopathy in Drosophila. Further, it was shown that tissue specific downregulation of dMyc (Drosophila homolog of human c-myc proto-oncogene) alleviates h-tau mediated cellular and functional deficits by restricting the formation of NFTs in neuronal tissues. Therefore, these findings provide very critical and novel insights about pathogenesis of human neuronal tauopathies in Drosophila disease models.
Paiardi, C., Mirzoyan, Z., Zola, S., Parisi, F., Vingiani, A., Pasini, M. E. and Bellosta, P. (2017). The Stearoyl-CoA Desaturase-1 (Desat1) in Drosophila cooperated with Myc to induce autophagy and growth, a potential new link to tumor survival. Genes (Basel) 8(5). PubMed ID: 28452935
Lipids are an important energy supply in our cells and can be stored or used to produce macromolecules during lipogenesis when cells experience nutrient starvation. Proteomic analysis reveals that the Drosophila homologue of human Stearoyl-CoA desaturase-1 Desat1) is an indirect target of Myc in fat cells. Stearoyl-CoA desaturases are key enzymes in the synthesis of monounsaturated fatty acids critical for the formation of complex lipids such as triglycerides and phospholipids. Their function is fundamental for cellular physiology, however in tumors, overexpression of SCD-1 and SCD-5 has been found frequently associated with a poor prognosis. Another gene that is often upregulated in tumors is the proto-oncogene c-myc, where its overexpression or increased protein stability, favor cellular growth. This study reports a potential link between Myc and Desat1 to control autophagy and growth. Using Drosophila, it was found that expression of Desat1, in metabolic tissues like the fat body, in the gut and in epithelial cells, is necessary for Myc function to induce autophagy a cell eating mechanism important for energy production. In addition, it was observed that reduction of Desat1 affects Myc ability to induce growth in epithelial cells. These data also identify, in prostatic tumor cells, a significant correlation between the expression of Myc and SCD-1 proteins, suggesting the existence of a potential functional relationship between the activities of these proteins in sustaining tumor progression.
Lee, J. E., Rayyan, M., Liao, A., Edery, I. and Pletcher, S. D. (2017). Acute dietary restriction acts via TOR, PP2A, and Myc signaling to boost innate immunity in Drosophila. Cell Rep 20(2): 479-490. PubMed ID: 28700947
Dietary restriction promotes health and longevity across taxa through mechanisms that are largely unknown. This study shows that acute yeast restriction significantly improves the ability of adult female Drosophila melanogaster to resist pathogenic bacterial infections through an immune pathway involving downregulation of target of rapamycin (TOR) signaling, which stabilizes the transcription factor Myc by increasing the steady-state level of its phosphorylated forms through decreased activity of protein phosphatase 2A. Upregulation of Myc through genetic and pharmacological means mimicked the effects of yeast restriction in fully fed flies, identifying Myc as a pro-immune molecule. Short-term dietary or pharmacological interventions that modulate TOR-PP2A-Myc signaling may provide an effective method to enhance immunity in vulnerable human populations.

myc is a mammalian oncogene involved in a wide variety of tumors. Oncogenic activation depends on elevated expression of the short lived Myc protein. In normal cells, myc expression is dependent on mitogenic stimuli. Myc is required for both cell proliferation and to prevent differentiation. Unless growth factors are provided, activation of myc can concomitantly induce programmed cell death (apoptosis).

Before discussing the avid interest Myc holds for developmental biologists, a brief description is given of the interaction of Myc with three other proteins (MAD, MAX and MXI) and their combined effects on both cell activation and quiescence (Amati,1994 and references).

Myc proteins contain two regions characteristic of transcription factors: an N-terminal transactivation domain, and a C-terminal basic helix-loop-helix (bHLH) leucine zipper motif known to mediate dimerization and sequence specific DNA binding. Myc functions as a heterodimer with other bHLH leucine zipper proteins. Max specifically dimerizerizes with Myc, and Myc-Max heterodimers function as transcriptional activators, binding a hexanucleotide motif called the E-box, which is often the target of other bHLH proteins.

Antagonizing the cell cycle promoting activity of Myc-Max in mammals are two other proteins, Mad and Mxi-1. Mad and Mxi can heterodimerize with Max, depriving Myc of a partner. The Max/Mad or Max/Mxi-1 partners either fail to activate or actively repress transcription, leading to a state of growth inhibition or possibly cell differentiation. Max proteins are metabolically stable and constitutively expressed, while Myc, Mad and Mxi-1 are unstable, responding to the level of mitotic stimulation in the cell. Mitogen stimulation induces a rapid rise in Myc levels and thus a shift in the equilibrium from Max/Max homodimers to Myc/Max heterodimers, the combination that promotes entry into cell cycle. Conversely, mitogen withdrawal or differentiation stimuli suppress myc expression and may result in the induction of either or both mad and mxi, leading to a state of growth inhibition. Thus Myc, Max and Mxi function as a protein network leading to alternative states of cell activation or quiescence (Amati, 1994 and references).

Of particular interest to developmental biologists is the question of whether Myc, Mad and Max play a role in normal development. This question became more complex and interesting with the discovery in vertebrates of multiple Myc genes. There is a neurally expressed myc (N-myc), for example. In mammals N-myc and myc show contrasting expression patterns during gastrulation. myc is most abundant in extraembryonic cells; in contrast, N-myc is found at highest levels in the expanding primitive streak and other portions of the embryonic mesoderm. Differentiation of mesoderm to epithelioid cells is accompanied by diminished expression of N-myc. Expression of myc is not an inevitable correlate of cellular proliferation. Instead, the gene appears to be regulated in concert with changes that affect diverse cellular properties, including proliferation, invasiveness and differentiation. For example, myc is selectively down-regulated in the primitive ectoderm, the most highly proliferative tissue of the embryo. A decline in myc expression correlates with terminal differentiation of brown adipose tissue and liver. In contrast, N-myc continues to be expressed in post-mitotic cells of neuronal origin (Downs, 1989 and Hirning, 1991).

Isolation of Drosophila Myc, properly termed Diminutive but referred to as dMyc, was accomplished by means of its conserved affinity for Max. Human Max was used to screen a two-hybid library prepared from Drosophila cDNAs. A second yeast two-hybrid screen was performed using the bHLH leucine zipper region of dMyc as bait. This experiment resulted in the identification of dMax (Gallant, 1996).

What is the phenotypic effect of loss of dmyc function? The results are inconclusive, but it is believed that a Drosophila mutation (known as diminutive), resulting in an abnormally small body size and female sterility, is due to a hypomorphic mutation in dmyc. Defects are observed in both germ cell nuclei and follicle cells; they fail to migrate and aberrantly undergo the transition to columnar epithelium. This phenotype suggests dysfunction in either follicle or nurse cells, or in communication between these two cell types. Interestingly the degeneration of the egg chamber in dm mutant females occurs at stage 8, at a time when cell division does not occur.

It is thought that a stage-specific downregulation of dmyc expression in diminished mutants results in a loss of the capacity of the follicle cells to grow and migrate. A possibly related effect has been observed in mice: hypomorphic N-myc mutation results in a loss of induction of tissue specific differentiation (Moens, 1992). In both cases diminished Myc expression in progenitor cells may result in their inability to respond to inductive signals. In addition, the smaller size of the diminished mutants may also result from partial loss of dMyc function in other tissues (Gallant, 1996). Recent studies in mice demonstrate that alterations in the cell cycle can significantly influence overall size. Mice lacking p27Kip1, an inhibitor of cyclin dependent kinase, display increased body size without an increase in growth hormone levels (Fero, 1996).

Is the small body size of diminutive mutants due to a hormonal effect or is it due to cell autonomous dmyc deficiency? Mosaic experiments should yield clues as to dMyc's influence on specific organs and developmental timing.

Cell mixing induced by myc is required for competitive tissue invasion and destruction

Cell-cell intercalation is used in several developmental processes to shape the normal body plan. There is no clear evidence that intercalation is involved in pathologies. This study used the proto-oncogene myc to study a process analogous to early phase of tumour expansion: myc-induced cell competition. Cell competition is a conserved mechanism driving the elimination of slow-proliferating cells (so-called 'losers') by faster-proliferating neighbours (so-called 'winners') through apoptosis and is important in preventing developmental malformations and maintain tissue fitness. Using long-term live imaging of myc-driven competition in the Drosophila pupal notum and in the wing imaginal disc, this study showed that the probability of elimination of loser cells correlates with the surface of contact shared with winners. As such, modifying loser-winner interface morphology can modulate the strength of competition. Elimination of loser clones requires winner-loser cell mixing through cell-cell intercalation. Cell mixing is driven by differential growth and the high tension at winner-winner interfaces relative to winner-loser and loser-loser interfaces, which leads to a preferential stabilization of winner-loser contacts and reduction of clone compactness over time. Differences in tension are generated by a relative difference in F-actin levels between loser and winner junctions, induced by differential levels of the membrane lipid phosphatidylinositol (3,4,5)-trisphosphate. These results establish the first link between cell-cell intercalation induced by a proto-oncogene and how it promotes invasiveness and destruction of healthy tissues (Levayer, 2015).

To analyse quantitatively loser cell elimination, long-term live imaging was performed of clones showing a relative decrease of the proto-oncogene myc in the Drosophila pupal notum, a condition known to induce cell competition in the wing disc. Every loser cell delamination was counted over 10 h, and the probability of cell elimination was calculated for a given surface of contact shared with winner cells. A significant increase was observed of the proportion of delamination with winner-loser shared contact, whereas this proportion remained constant for control clones. The same correlation was observed in ex vivo culture of larval wing disc. Cell delamination in the notum was apoptosis dependent and expression of flowerlose (fwelose), a competition-specific marker for loser fate, was necessary and sufficient to drive contact-dependent delamination. Moreover it was confirmed that contact-dependent death is based on the computation of relative differences of fwelose between loser cells and their neighbours. Thus, cell delamination in the notum recapitulates features of cell competition (Levayer, 2015).

This suggests that winner-loser interface morphology could modulate the probability of eliminating loser clones. Using the wing imaginal disc, winner-loser contact was reduced by inducing adhesion- or tension-dependent cell sorting and observed a significant reduction of loser clone elimination. This rescue was not driven by a cell-autonomous effect of E-cadherin (E-cad) or active myosin II regulatory light chain (MRLC) on growth, death or cell fitness but rather by a general diminution of winner-loser contact. Competition is ineffective across the antero-posterior compartment boundary, a frontier that prevents cell mixing through high line tension. Accordingly, there was no increase in death at the antero-posterior boundary in wing discs overexpressing fweloseA in the anterior compartment. However, reducing tension by reducing levels of myosin II heavy chains was sufficient to increase the shared surface of contact between cells of the anterior and posterior compartments, and induced fwelose death at the boundary. Altogether, it is concluded that the reduction in surface contact between winners and losers is sufficient to block competition, which explains how compartment boundaries prevent competition (Levayer, 2015).

Loser clones have been reported to fragment more often than controls, whereas winner clones show convoluted morphology, suggesting that winner-loser mixing is increased during competition. This could affect the outcome of cell competition by increasing the surface shared between losers and winners. Clone splitting was used as a readout for loser–winner mixing. Two non-exclusive mechanisms can drive clone splitting: cell death followed by junction rearrangement, or junction remodelling and cell–cell intercalation independent of death. To assess the contribution of each phenomenon, the proportion of clones fragmented 48 h after clone induction (ACI) was systematically counted. A twofold increase was observed in the frequency of split clones in losers (wild type (WT) in tub-dmyc) versus WT in WT controls. Overexpressing E-cad or active myosin II was sufficient to prevent loser clone splitting, whereas blocking apoptosis or blocking loser fate by silencing fwelose did not reduce splitting. Finally, the proportion of split clones was also increased for winner clones either during myc-driven competition or during Minute-dependent competition. Altogether, this suggested that winner–loser mixing is increased independently of loser cell death or clone size by a factor upstream of fwe, and could be driven by cell–cell intercalation. Accordingly, junction remodelling events leading to disappearance of a loser–loser junction were three times more frequent at loser clone boundaries than control clone boundaries in the pupal notum. The rate of junction remodelling was higher in loser–loser junctions and in winner–winner junctions than in winner–loser junctions. The preferential stabilization of winner–loser interfaces should increase the surface of contact between winner and loser cells over time. Accordingly, loser clone compactness in the notum decreased over time whereas it remains constant on average for WT clones in WT background. Similarly, the compactness of clones in the notum also decreased over time for conditions showing high frequency of clone splitting in the wing disc, whereas clone compactness remained constant for conditions rescuing clone splitting. Altogether, it is concluded that both Minute- and myc-dependent competition increase loser–winner mixing through cell–cell intercalation (Levayer, 2015).

It was then asked what could modulate the rate of junction remodelling during competition. The rate of junction remodelling can be cell-autonomously increased by myc. Interestingly, downregulation of the tumour suppressor PTEN is also sufficient to increase the rate of junction remodelling through the upregulation of phosphatidylinositol (3,4,5)-trisphosphate (PIP3). It was reasoned that differences in PIP3 levels could also modulate junction remodelling during competition. Using a live reporter of PIP3 that could detect modulations of PIP3 in the notum, a significant increase of PIP3 was observed in the apico-lateral membrane of tub-dmyc–tub-dmyc interfaces compared with WT–WT and WT–tub-dmyc interfaces (Fig. 3a, b). Moreover, increasing/reducing Myc levels in a full compartment of the wing disc was sufficient to increase/decrease the levels of phospho-Akt (a downstream target of PIP3, whereas fweloseA overexpression had no effect. Similarly, levels of phospho-Akt were relatively higher in WT clones than in the surrounding M-/+ cells. Thus differences in PIP3 levels might be responsible for winner–loser mixing. Accordingly, reducing PIP3 levels by overexpressing a PI3 kinase dominant negative (PI3K-DN) or increasing PIP3 levels by knocking down PTEN (UAS-pten RNAi) were both sufficient to induce a high proportion of fragmented clones and to reduce clone compactness over time in the notum , whereas increasing PIP3 in loser clones was sufficient to prevent cell mixing. Moreover, abolishing winner–loser PIP3 differences through larval starvation prevented loser clone fragmentation, the reduction of clone compactness over time in the notum and could rescue WT clone elimination in tub-dmyc background. It is therefore concluded that differences in PIP3 levels are necessary and sufficient for loser–winner mixing and required for loser cell elimination (Levayer, 2015).

It was then asked which downstream effectors of PIP3 could affect junction stability. A relative growth decrease can generate mechanical stress that can be released by cell-cell intercalation. Accordingly, growth reduction through Akt downregulation is sufficient to increase clone splitting and could contribute to loser clone splitting. However, Akt is not sufficient to explain winner-loser mixing because, unlike PIP3, increasing Akt had no effect on clone splitting. PIP3 could also modulate junction remodelling through its effect on cytoskeleton and the modulation of intercellular adhesion or tension. No obvious modifications of E-cad, MRLC or Dachs (another regulator of tension) was detected in loser cells. However, a significant reduction of F-actin levels and a reduction of actin turnover/polymerization rate were observed in loser-loser and loser-winner junctions in the notum. Similarly, modifying Myc levels in a full wing disc compartment was sufficient to modify actin levels, and F-actin levels were higher in WT clones than M-/+ cells. This prompted a test of the role of actin organization in winner-loser mixing. Downregulating the formin Diaphanous (Dia, a filamentous actin polymerization factor) by RNA interference (RNAi) or by using a hypomorphic mutant was sufficient to obtain a high proportion of fragmented clones and to reduce clone compactness over time, whereas overexpressing Dia in loser clones prevented clone splitting (UAS-dia::GFP) and compactness reduction. This effect was specific to Dia as modulating Arp2/3 complex (a regulator of dendritic actin network) had no effect on clone splitting. Thus, impaired filamentous actin organization was necessary and sufficient to drive loser-winner mixing. These actin defects were driven by the differences in PIP3 levels between losers and winners. Thus Dia could be an important regulator of competition through its effect on cell mixing. Overexpression of Dia was indeed sufficient to reduce loser clone elimination significantly (Levayer, 2015).

Filamentous actin has been associated with tension regulation. It was therefore asked whether junction tension was modified in winner and loser junctions. The maximum speed of relaxation of junction after laser nanoablation (which is proportional to tension) was significantly reduced in loser-loser and winner-loser junctions compared with winner-winner junctions. This distribution of tension has been proposed to promote cell mixing. Accordingly, decreasing PIP3 in clones reduced tension both in low-PIP3-low-PIP3 and low-PIP3-normal-PIP3 junctions, whereas overexpressing Dia in loser clones or starvation were both sufficient to abolish differences in tension, in agreement with their effect on winner-loser mixing and the distribution of F-actin. Thus the lower tension at winner-loser and loser-loser junctions is responsible for winner-loser mixing. Altogether, it is concluded that the relative PIP3 decrease in losers increases winner-loser mixing through Akt-dependent differential growth and the modulation of tension through F-actin downregulation in winner-loser and loser-loser junctions (Levayer, 2015).

Several modes of tissue invasion by cancer cells have been described, most of them relying on the departure of the tumour cells from the epithelial layer. This study suggests that some oncogenes may also drive tissue destruction and invasion by inducing ectopic cell intercalation between cancerous and healthy cells, and subsequent healthy cell elimination. myc-dependent invasion could be enhanced by other mutations further promoting intercalation (such as PTEN). Stiffness is increased in many tumours, suggesting that healthy cell-cancer cell mixing by intercalation might be a general process (Levayer, 2015).

Drosophila insulin and target of rapamycin (TOR) pathways regulate GSK3 beta activity to control Myc stability and determine Myc expression in vivo

Genetic studies in Drosophila reveal an important role for Myc in controlling growth. Similar studies have also shown how components of the insulin and target of rapamycin (TOR) pathways are key regulators of growth. Despite a few suggestions that Myc transcriptional activity lies downstream of these pathways, a molecular mechanism linking these signaling pathways to Myc has not been clearly described. Using biochemical and genetic approaches this study tried to identify novel mechanisms that control Myc activity upon activation of insulin and TOR signaling pathways. Biochemical studies show that insulin induces Myc protein accumulation in Drosophila S2 cells, which correlates with a decrease in the activity of glycogen synthase kinase 3-β (GSK3β) a kinase that is responsible for Myc protein degradation. Induction of Myc by insulin is inhibited by the presence of the TOR inhibitor rapamycin, suggesting that insulin-induced Myc protein accumulation depends on the activation of TOR complex 1. Treatment with amino acids that directly activate the TOR pathway results in Myc protein accumulation, which also depends on the ability of S6K kinase to inhibit GSK3β activity. Myc upregulation by insulin and TOR pathways is a mechanism conserved in cells from the wing imaginal disc, where expression of Dp110 and Rheb also induces Myc protein accumulation, while inhibition of insulin and TOR pathways result in the opposite effect. Functional analysis, aimed at quantifying the relative contribution of Myc to ommatidial growth downstream of insulin and TOR pathways, revealed that Myc activity is necessary to sustain the proliferation of cells from the ommatidia upon Dp110 expression, while its contribution downstream of TOR is significant to control the size of the ommatidia. This study presents novel evidence that Myc activity acts downstream of insulin and TOR pathways to control growth in Drosophila. At the biochemical level it was found that both these pathways converge at GSK3β to control Myc protein stability, while genetic analysis shows that insulin and TOR pathways have different requirements for Myc activity during development of the eye, suggesting that Myc might be differentially induced by these pathways during growth or proliferation of cells that make up the ommatidia (Parisi, 2011).

Previous studies in vertebrates have indicated a critical function for Myc downstream of growth factor signaling including insulin-like growth factor, insulin and TOR pathways. In Drosophila, despite a few notes that Myc transcriptional activity acts downstream of insulin and TOR pathways, no clear molecular mechanisms linking these pathways to Myc have been elucidated yet (Parisi, 2011).

It has been demonstrated that inhibition of GSK3β prevents Myc degradation by the proteasome pathway. This study further unravels the pathways that control Myc protein stability and shows that signaling by insulin and TOR induce Myc protein accumulation by regulating GSK3β activity in S2 cells. GSK3β is a constitutively active kinase that is regulated by multiple signals and controls numerous cellular processes. With the biochemical data it is proposed that GSK3β acts as a common point where insulin and TOR signaling converge to regulate Myc protein stability (see Model showing the proposed relationship between Myc and the insulin and TOR signaling pathways). In particular, activation of insulin signaling was shown to induce activation of Akt, an event that is accompanied by GSK3β phosphorylation on Ser 9 that causes its inactivation and Myc protein to stabilize. Interestingly, insulin-induced Myc protein accumulation, when GSK3β activity was blocked by the presence of LiCl or by expression of GSK3β-KD, was similar to that obtained with insulin alone. Since it was shown that activation of insulin signaling leads to GSK3β inhibition and to an increase in Myc protein, if insulin and GSK3β signaling were acting independently, it would be expected that activation of insulin signaling concomitantly with the inhibition of GSK3β activity would result in a higher level of Myc than that obtained with insulin or LiCl alone. The results instead showed a similar level of Myc protein accumulation with insulin in the presence of GSK3β inhibitors as compared to insulin alone, supporting the hypothesis that GSK3β and insulin signaling, at least in the current experimental condition, depend on each other in the mechanism that regulates Myc protein stability (Parisi, 2011).

In a similar biochemical approach, the effect of AAs was analyzed on Myc protein stability and how TOR signaling is linked to mechanisms that inactivate GSK3β to stabilize Myc protein in S2 cells. In these experiments it was possible to demonstrate that treatment with amino acids (AAs) increased Myc protein stability, and it was also shown that treatment with rapamycin, an inhibitor of TORC1, reduced insulin-induced Myc upregulation. The reduction of Myc protein accumulation by rapamycin was blocked by inhibition of the proteasome pathway, linking TOR signaling to the pathway that controls Myc protein stability. TORC1 is a central node for the regulation of anabolic and catabolic processes and contains the central enzyme Rheb-GTPase, which responds to amino acids by activating TOR kinase to induce phosphorylation of p70-S6K and 4E-BP1. Analysis of the molecular mechanisms that act downstream of TOR to regulate Myc stability shows that AA treatment induces p70-S6K to phosphorylate GSK3β on Ser 9, an event that results in its inactivation and accumulation of Myc protein (Parisi, 2011).

Reducing GSK3β activity with LiCl, in medium lacking AAs, resulted in a slight increase in Myc protein levels. Adding back AAs lead to a substantial increase in Myc protein levels, which did not further increase when AAs where added to cells in the presence of the GSK3β inhibitor LiCl. These events were accompanied by phosphorylation of S6K on Thr 398, which correlated with phosphorylation of GSK3β on Ser 9. From these experiments it is concluded that TOR signaling also converges to inhibit GSK3β activity to regulate Myc protein stability. However, it has to be pointed out that since AAs alone increased Myc protein levels to a higher extent than that observed with LiCl alone, the experiments also suggest that Myc protein stability by TOR signaling is not solely directed through the inhibition of GSK3β activity, but other events and/or pathways contribute to Myc regulation. In conclusion, the biochemical experiments demonstrate that GSK3β acts downstream of insulin and TOR pathways to control Myc stability, however it cannot be excluded that other pathways may control Myc protein stability upon insulin and amino acids stimulation in S2 cells (Parisi, 2011).

Reduction of insulin and TOR signaling in vivo reduces cell size and proliferation, and clones mutant for chico, the Drosophila orthologue of IRS1-4, or for components of TOR signaling, are smaller due a reduction in size and the number of cells. The experiments showed that reducing insulin signaling by expression of PTEN or using TORTED, a dominant negative form of TOR, decreased Myc protein levels in clones of epithelial cells of the wing imaginal discs, while the opposite was true when these signals were activated using Dp110 or RhebAV4 . Those experiments suggested that the mechanism of regulation of Myc protein by insulin and TOR pathways was conserved also in vivo in epithelial cells of the larval imaginal discs (Parisi, 2011).

During these experiments it was also noted that Myc protein was induced in the cells surrounding and bordering the clones (non-autonomously), particularly when clones where positioned along the dorsal-ventral axis of the wing disc. This upregulation of Myc protein was not restricted to components of the insulin signaling pathway since it was also observed in cells surrounding the clones mutant for components of the Hippo pathway or for the tumor suppressor lethal giant larvae (lgl), which upregulates Myc protein cell-autonomously. It is suspected that this non-autonomous regulation of Myc may be induced by a novel mechanism that controls proliferation of cells when 'growth' is unbalanced. It can be speculated that clones with different growth rates, caused by different Myc levels, might secrete factors to induce Myc expression in neighboring cells. As a consequence, these Myc-expressing cells will speed up their growth rate in an attempt to maintain proliferation and tissue homeostasis. Further analysis is required to identify the mechanisms responsible for this effect (Parisi, 2011).

In order to distinguish if Myc activity was required downstream of insulin and TOR signaling to induce growth, a genetic analysis was performed. The ability to induce growth and proliferation was measured in the eye by measuring the size and number of the ommatidia from animals expressing members of the insulin and TOR pathways in different dm genetic background (dm+, dmP0 and dm4). The data showed that Dp110 increased the size and number of the ommatidia, however only the alteration in the total number was dependent on dm levels. These data suggest that Myc is required downstream of insulin pathway to achieve the proper number of ommatidia. However, when insulin signaling was reduced by PTEN, a significant decrease in the size of ommatidia was seen and it was dependent on dm expression levels, suggesting that Myc activity is limiting for ommatidial size and number. Activation of TOR signaling induces growth, and the genetic analysis showed that Myc significantly contributes to the size of the ommatidial cells thus suggesting that Myc acts downstream of TOR pathway to control growth (Parisi, 2011).

Recent genomic analysis showed a strong correlation between the targets of Myc and those of the TOR pathway, implying that they may share common targets. In support of this observation, mosaic analysis with a repressible cell marker (MARCM) experiments in the developing wing disc showed that overexpression of Myc partially rescues the growth disadvantage of clones mutant for the hypomorphic Rheb7A1 allele, further supporting the idea that Myc acts downstream of TOR to activate targets that control growth in these clones (Parisi, 2011).

The genetic interaction revealed a stronger dependence on Myc expression when Rheb was used as opposed to S6K. A possible explanation for this difference could lie in the fact that S6K is not capable of auto-activation of its kinase domain unless stimulated by TOR kinase. TOR activity is dependent on its upstream activator Rheb; consequently the enzymatic activity of the Rheb/GTPase is the limiting factor that influences S6K phosphorylation and therefore capable of maximizing its activity (Parisi, 2011).

Interestingly, these experiments also showed that activation of TOR signaling has a negative effect on the number of ommatidia, and this correlates with the ability of RhebAV4 to induce cell death during the development of the eye imaginal disc. Rheb-induced cell death was rescued in a dmP0 mutant background, which leading to the speculation that 'excessive' protein synthesis, triggered by overexpression of TOR signaling, could elicit a Myc-dependent stress response, which induces apoptosis. Alternatively, high protein synthesis could result in an enrichment of misfolded proteins that may result in a stress response and induces cell death. Further analysis is required to delineate the mechanisms underlying this process (Parisi, 2011).

This analyses provide novel genetic and biochemical evidences supporting a role for Myc in the integration of the insulin and TOR pathway during the control of growth, and highlights the role of GSK3β in this signaling. It was found that insulin signaling inactivates GSK3β to control Myc protein stability, and a similar biochemical regulation is also shared by activation of the TOR pathways. In support of this data, a recent genomic analysis in whole larvae showed a strong correlation between the targets of Myc and those of the TOR pathway; however, less overlap was found between the targets of Myc and those of PI3K signaling (Parisi, 2011).

Statistical analysis applied to the genetic interaction experiments revealed that, in the Drosophila eye, proliferation induced by activation of the insulin pathway is sensitive to variations in Myc levels, while a significant interaction was seen mostly when TOR increased cell size. The data therefore suggests that there is a correlation between Myc and the InR signaling and it is expected that the InR pathway also shares some transcriptional targets with Myc. Indeed, an overlap was found between the targets induced by insulin and Myc in Drosophila S2 cells and these targets have also been reported in transcriptome analyses in the fat body upon nutritional stress, suggesting that Myc acts downstream of InR/PI3K and TOR signaling and that this interaction might be specific to some tissues or in a particular metabolic state of the cell (Parisi, 2011).


dmax is expressed as a single 1.2 kb transcript at constant levels throughout development. In contrast, a dmyc transcript of 6 kb is expressed at the highest levels during early embryogenesis and in adult females. It is barely detectable during the larval and pupal stages. Early embryos and adult females contain smaller dmyc transcripts that appear to differ primarily in their untranslated regions (Gallant, 1996).

The dMyc protein is only 26% identical to human c-Myc over its entire amino acid sequence. The N-terminal region shows a 57% identity to human c-Myc box II, which contains the conserved sequence DCMW, mutations that abrogate myc activity. The centrally located acidic region shows a 57% identity to vertebrate c-Myc, and the C-terminal bHLH leucine zipper motif retains a 40% identity. The Myc-box I may also be present, but its sequence is less conserved. All but one of the basic region residues predicted to contact DNA are identical to those in human c-Myc. The dmyc gene is comprised of three exons with the protein-coding region spanning exons 2 and 3, as in mammalian myc (Gallant, 1996).


Exons - 3


Amino Acids - 717

Structural Domains

Myc proteins consist of two N-terminal segments (referred to as Myc boxes 1 and 2), a central block of acidic residues, and a highly charged carboxyl-terminal region. Myc box 1 contains two of the four phosphorylation sites identified to date in mammalian myc: one for p34cdc2 (see Drosophila Cyclin B, along with its dimerization partner cdc2) or a related kinase and a second for an unidentified kinase. The C-terminal region contains three structural motifs: the leucine zipper, helix-loop-helix, and basic regions. The HLH and leucine zipper regions have been shown to mediate protein oligomerization, while the basic region, when oligomerized, allows sequence specific DNA binding (Pendergast, 1992).

Myc: Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

date revised: 30 October 2015 

Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.