Background: Insulin and insulin-like growth factors (IGFs) signal through a highly conserved pathway and control growth and metabolism in both vertebrates and invertebrates. In mammals, insulin-like growth factor binding proteins (IGFBPs) bind IGFs with high affinity and modulate their mitogenic, anti-apoptotic and metabolic actions, but no functional homologs have been identified in invertebrates so far. Results: Here, we show that the secreted Imaginal morphogenesis protein-Late 2 (Imp-L2) binds Drosophila insulin-like peptide 2 (Dilp2) and inhibits growth non-autonomously. Whereas over-expressing Imp-L2 strongly reduces size, loss of Imp-L2 function results in an increased body size. Imp-L2 is both necessary and sufficient to compensate Dilp2-induced hyperinsulinemia in vivo. Under starvation conditions, Imp-L2 is essential for proper dampening of insulin signaling and larval survival. Conclusion: Imp-L2, the first functionally characterized insulin-binding protein in invertebrates, serves as a nutritionally controlled suppressor of insulin-mediated growth in Drosophila. Given that Imp-L2 and the human tumor suppressor IGFBP-7 show sequence homology in their carboxy-terminal immunoglobulin-like domains, we suggest that their common precursor was an ancestral insulin-binding protein.

Background: Genetic studies in Drosophila melanogaster 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 we tried to identify novel mechanisms that control Myc activity upon activation of insulin and TOR signaling pathways. Results: Our 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-beta (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. Our 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. Conclusions: Our study presents novel evidence that Myc activity acts downstream of insulin and TOR pathways to control growth in Drosophila. At the biochemical level we found that both these pathways converge at GSK3β to control Myc protein stability, while our 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.

Insulin/insulin-like growth factor signaling (IIS) plays a pivotal role in the regulation of growth at the cellular and the organismal level during animal development. Flies with impaired IIS are developmentally delayed and small due to fewer and smaller cells. In the search for new growth-promoting genes, we identified mutations in the gene encoding Lnk, the single fly member of the SH2B family of adaptor molecules. Flies lacking lnk function are viable but severely reduced in size. Furthermore, lnk mutants display phenotypes reminiscent of reduced IIS, such as developmental delay, female sterility, and accumulation of lipids. Genetic epistasis analysis places lnk downstream of the insulin receptor (InR) and upstream of phosphoinositide 3-kinase (PI3K) in the IIS cascade, at the same level as chico (encoding the single fly insulin receptor substrate [IRS] homolog). Both chico and lnk mutant larvae display a similar reduction in IIS activity as judged by the localization of a PIP₃ reporter and the phosphorylation of protein kinase B (PKB). Furthermore, chico; lnk double mutants are synthetically lethal, suggesting that Chico and Lnk fulfill independent but partially redundant functions in the activation of PI3K upon InR stimulation. The regulation of growth is among the most fundamental processes during development of multicellular organisms. Research over the past years has established a key function of the insulin/insulin-like growth factor signaling (IIS) pathway in ensuring proper growth at the cellular and the organismal level. Impaired IIS has been associated with diseases such as type 2 diabetes, leprechaunism, and heart disease; and deregulated IIS often contributes to the development of cancer. Here, we describe the characterization of the Drosophila SH2B family adaptor protein Lnk. Mutants in lnk are viable but unable to reach the normal size due to a reduction in cell size and cell number. Our characterization of lnk mutant flies has revealed phenotypes associated with impaired IIS, such as developmental delay, female sterility, and increased lipid levels in adults. Using a combination of genetic interaction experiments and molecular readouts for IIS activity, we demonstrate that Lnk acts in parallel to the IRS homolog Chico downstream of the insulin receptor to regulate cellular growth.

The kinase TOR is found in two complexes, TORC1, which is involved in growth control, and TORC2, whose roles are less well defined. Here, we asked whether TORC2 has a role in sustaining cellular stress. We show that TORC2 inhibition in Drosophila melanogaster leads to a reduced tolerance to heat stress, whereas sensitivity to other stresses is not affected. Accordingly, we show that upon heat stress, both in the animal and Drosophila cultured S2 cells, TORC2 is activated and is required for maintaining the level of its known target, Akt1 (also known as PKB). We show that the phosphorylation of the stress-activated protein kinases is not modulated by TORC2 nor is the heat-induced upregulation of heat-shock proteins. Instead, we show, both in vivo and in cultured cells, that TORC2 is required for the assembly of heat-induced cytoprotective ribonucleoprotein particles, the pro-survival stress granules. These granules are formed in response to protein translation inhibition imposed by heat stress that appears to be less efficient in the absence of TORC2 function. We propose that TORC2 mediates heat resistance in Drosophila by promoting the cell autonomous formation of stress granules.

The integrity of the intestinal epithelium is crucial for the barrier function of the gut. Replenishment of the gut epithelium by intestinal stem cells contributes to gut homeostasis, but how the differentiated enterocytes are protected against stressors is less well understood. Here we use the Drosophila larval hindgut as a model system in which damaged enterocytes are not replaced by stem cell descendants. By performing a thorough genetic analysis, we demonstrate that a signalling complex consisting of p38b and MK2 forms a branch of SAPK signalling that is required in the larval hindgut to prevent stress-dependent damage to the enterocytes. Impaired p38b/MK2 signalling leads to apoptosis of the enterocytes and a subsequent loss of hindgut epithelial integrity, as manifested by the deterioration of the overlaying muscle layer. Damaged hindguts show increased JNK activity, and removing upstream activators of JNK suppresses the loss of hindgut homeostasis. Thus, the p38/MK2 complex ensures homeostasis of the hindgut epithelium by counteracting JNK-mediated apoptosis of the enterocytes upon chronic stress. The gut epithelium forms the first barrier against pathogens and stressors in the gut lumen, and a loss of this defence function can result in intestinal diseases. Damage in the gut epithelium triggers the proliferation of intestinal stem cells to replenish the epithelium. However, little is known about how the enterocytes are protecting themselves against stressors. We addressed the function of stress-activated protein kinase (SAPK) signal transduction pathways in the larval gut of Drosophila. Our study revealed that a particular module of the p38 SAPK signal cascade is required to protect the larval hindgut epithelium against chronic salt stress. We identified the two kinases, p38b and MK2, as key components of this protective signal. In the absence of p38b or MK2, the stress-inducible JNK cascade is locally upregulated and eventually induces apoptosis. Although the function of the p38b/MK2 module is only required in the enterocytes, the elimination of the affected cells results in atrophy of the overlaying muscle layer and subsequent systemic defects in the larvae (e.g., induction of antimicrobial peptides). We hope that our findings will contribute to a better understanding of early (i.e., pre-inflammatory) events in the development of human intestinal diseases.

How single cells in a mitotic tissue progressively acquire hallmarks of cancer is poorly understood. We exploited mitotic recombination in developing Drosophila imaginal tissues to analyze the behavior of cells devoid of the tumor suppressor PTEN, a negative regulator of PI3K signaling, under varying nutritional conditions. Cells lacking PTEN strongly overproliferated specifically in nutrient restricted larvae. Although the PTEN mutant cells were sensitive to starvation, they successfully competed with neighboring cells by autonomous and non-autonomous mechanisms distinct from cell competition. The overgrowth was strictly dependent on the activity of the downstream components Akt/PKB and TORC1, and a reduction in amino acid uptake by reducing the levels of the amino acid transporter Slimfast caused clones of PTEN mutant cells to collapse. Our findings demonstrate how limiting nutritional conditions impact on cells lacking the tumor suppressor PTEN to cause hyperplastic overgrowth.