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 [18
]. In Drosophila
, despite a few notes that Myc transcriptional activity acts downstream of insulin and TOR pathways [23
], no clear molecular mechanisms linking these pathways to Myc have been elucidated yet.
We previously demonstrated that inhibition of GSK3β prevents Myc degradation by the proteasome pathway [10
]. In this report, we further unravel the pathways that control Myc protein stability and show 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 [8
]. With our biochemical data we propose that GSK3β acts as a common point where insulin and TOR signaling converge to regulate Myc protein stability (Figure ). In particular, we showed that activation of insulin signaling induces activation of Akt, an event that is accompanied by GSK3β phosphorylation on Ser 9 that causes its inactivation and Myc protein to stabilize (Figure ). 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 we showed that activation of insulin signaling leads to GSK3β inhibition and to an increase in Myc protein, if insulin and GSK3β signaling were acting independently, we would expect 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. Our results instead showed a similar level of Myc protein accumulation with insulin in the presence of GSK3β inhibitors as compared to insulin alone (Figure and , compare lane 2 and 4), supporting the hypothesis that GSK3β and insulin signaling, at least in our experimental condition, depend on each other in the mechanism that regulates Myc protein stability.
Figure 5 Model showing the proposed relationship between Myc and the insulin and TOR signaling pathways. AA: amino acids; DILPs: Drosophila insulin-like peptides; IRS: insulin-receptor substrate; PI3K: phosphatidylinositol-3 kinase; Rheb: Ras homolog enriched (more ...)
In a similar biochemical approach, we analyzed the effect of AAs 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 we were able to demonstrate that AAs increased Myc protein stability, and we also showed 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 (Figure ). 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 [14
]. Our 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 (Figure ).
Reducing GSK3β activity with LiCl, in medium lacking AAs, resulted in a slight increase in Myc protein levels (Figure , lane 1 and 2). 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 (Figure , lane 3 and 4). These events were accompanied by phosphorylation of S6K on Thr 398, which correlated with phosphorylation of GSK3β on Ser 9. From these experiments we conclude that TOR signaling also converges to inhibit GSK3β activity to regulate Myc protein stability (Figure ). However, we need to point out that since AAs alone increased Myc protein levels to a higher extent than that observed with LiCl alone (Figure , compare lane 2 and 3), our 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, our biochemical experiments demonstrate that GSK3β acts downstream of insulin and TOR pathways to control Myc stability, however we do not exclude that other pathways may control Myc protein stability upon insulin and amino acids stimulation in S2 cells.
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 [29
]. Our 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
(Figure ). 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.
During these experiments we 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 we also observed it in cells surrounding the clones mutant for components of the Hippo pathway [52
] or for the tumor suppressor lethal giant larvae (lgl)
, which upregulates Myc protein cell-autonomously [53
]. We suspect that this non-autonomous regulation of Myc may be induced by a novel mechanism that controls proliferation of cells when 'growth' is unbalanced. We can speculate 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.
In order to distinguish if Myc activity was required downstream of insulin and TOR signaling to induce growth, we performed a genetic analysis. 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
). Our 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 [2
], and our 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.
Recent genomic analysis showed a strong correlation between the targets of Myc and those of the TOR pathway [24
], implying that they may share common targets. In support of this observation our 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 (Additional file 8
), further supporting the idea that Myc acts downstream of TOR to activate targets that control growth in these clones.
Our genetic interaction revealed a stronger dependence on Myc expression when Rheb was used as opposed to S6K (Table ). 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 [54
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 led us to speculate 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 [55
] that may result in a stress response and induces cell death. Further analysis is required to delineate the mechanisms underlying this process.