First systematic analysis of the evolutionary conserved InR/TOR pathway interaction proteome in Drosophila.Quantitative mass spectrometry revealed that 22% of identified protein interactions are regulated by the growth hormone insulin affecting membrane proximal as well as intracellular signaling complexes.Systematic RNA interference linked a significant fraction of network components to the control of dTOR kinase activity.Combined biochemical and genetic data suggest dTTT, a dTOR-containing complex required for cell growth control by dTORC1 and dTORC2 in vivo.
Cellular growth is a fundamental process that requires constant adaptations to changing environmental conditions, like growth factor and nutrient availability, energy levels and more. Over the years, the insulin receptor/target of rapamycin pathway (InR/TOR) emerged as a key signaling system for the control of metazoan cell growth. Genetic screens carried out in the fruit fly Drosophila melanogaster identified key InR/TOR pathway components and their relationships. Phenotypes such as altered cell growth are likely to emerge from perturbed dynamic networks containing InR/TOR pathway components, which stably or transiently interact with other cellular proteins to form complexes and networks thereof. Systematic studies on the topology and dynamics of protein interaction networks become therefore highly relevant to gain systems level understanding of deregulated cell growth. Despite much progress in genetic analysis only few systematic protein interaction studies have been reported for Drosophila, which in most cases lack quantitative information representing the dynamic nature of such networks. Here, we present the first quantitative affinity purification mass spectrometry (AP–MS/MS) analysis on the evolutionary conserved InR/TOR signaling network in Drosophila. Systematic RNAi-based functional analysis of identified network components revealed key components linked to the regulation of the central effector kinase dTOR. This includes also dTTT, a novel dTOR-containing complex required for the control of dTORC1 and dTORC2 in vivo.
For systematic AP–MS analysis, we generated Drosophila Kc167 cell lines inducibly expressing affinity-tagged bait proteins previously linked to InR/TOR signaling. Bait expressing Kc167 cell lines were harvested before and after insulin stimulation for subsequent affinity purification. Following LC–MS/MS analysis and probabilistic data filtering using SAINT (Choi et al, 2010), we generated a quantitative network model from 97 high confidence protein–protein interactions and 58 network components (Figure 2). The presented network displayed a high degree of orthologous interactions conserved also in human cells and identified a number of novel molecular interactions with InR/TOR signaling components for future hypothesis driven analysis.
To measure insulin-induced changes within the InR/TOR interaction proteome, we applied a recently introduced label-free quantitative MS approach (Rinner et al, 2007). The obtained quantitative data suggest that 22% of all interactions in the network are regulated by insulin. Major changes could be observed within the membrane proximal InR/chico/PI3K signaling complexes, and also in 14-3-3 protein containing signaling complexes and dTORC1, a complex that contains besides dTOR all major orthologous proteins found also in human mTORC1 including the two dTORC1 substrates d4E-BP (Thor) and S6 Kinase (S6K). Insulin triggered both, dissociation and association of dTORC1 proteins. Among the proteins that showed enhanced binding to dTORC1 upon insulin stimulation we found Unkempt, a RING-finger protein with a proposed role in ubiquitin-mediated protein degradation (Lores et al, 2010). Besides dTORC1 our systematic AP–MS analysis also revealed the presence of dTORC2, the second major TOR complex in Drosophila. dTORC2 contains the Drosophila orthologous of human mTORC2 proteins, but in contrast to dTORC1 was not affected upon insulin stimulation. Interestingly, we also found a specific set of proteins that were not linked to the canonical TOR complexes TORC1 and TORC2 in dTOR purifications. These include LqfR (liquid facets related), Pontin, Reptin, Spaghetti and the gene product of CG16908. We found the same set of proteins when we used CG16908 as a bait, suggesting complex formation among the identified proteins. None of the dTORC1/2 components besides dTOR was identified in CG16908 purifications, indicating that these proteins form dTOR complexes distinct from dTORC1 and dTORC2. Based on known interaction information from other species and data obtained from this study we refer to this complex as dTTT (Drosophila
TOR, TELO2, TTI1) (Horejsi et al, 2010; Hurov et al, 2010; Kaizuka et al, 2010). A directed quantitative MS analysis of dTOR complex components suggests that dTORC1 is the most abundant dTOR complex we identified in Kc167 cells.
We next studied the potential roles of the identified network components for controlling the activity of the dInR/TOR pathway using systematic RNAi depletion and quantitative western blotting to measure the changes in abundance of phosphorylated substrates of dTORC1 (Thor/d4E-BP, dS6K) and dTORC2 (dPKB) in RNAi-treated cells (Figure 5). Overall, we could identify 16 proteins (out of 58) whose depletion caused an at least 50% increase or decrease in the levels of phosphorylated d4E-BP, S6K and/or PKB compared with control GFP RNAi. Besides established pathway components, we found several novel regulators within the dInR/TOR interaction network. For example, RNAi against the novel insulin-regulated dTORC1 component Unkempt resulted in enhanced phosphorylation of the dTORC1 substrate d4E-BP, which suggests a negative role for Unkempt on dTORC1 activity. In contrast, depletion of CG16908 and LqfR caused hypo-phosphorylation of all dTOR substrates similar to dTOR itself, suggesting a positive role for the dTTT complex on dTOR activity. Subsequently, we tested whether dTTT components also plays a role in dTOR-mediated cell growth in vivo. Depletion of both dTTT components, CG16908 and LqfR, in the Drosophila eye resulted in a substantial decrease in eye size. Likewise, FLP-FRT-mediated mitotic recombination resulted in CG16908 and LqfR mutant clones with a similar reduced growth phenotype as observed in dTOR mutant clones. Hence, the combined biochemical and genetic analysis revealed dTTT as a dTOR-containing complex required for the activity of both dTORC1 and dTORC2 and thus plays a critical role in controlling cell growth.
Taken together, these results illustrate how a systematic quantitative AP–MS approach when combined with systematic functional analysis in Drosophila can reveal novel insights into the dynamic organization of regulatory networks for cell growth control in metazoans.
Using quantitative mass spectrometry, this study reports how insulin affects the modularity of the interaction proteome of the Drosophila InR/TOR pathway, an evolutionary conserved signaling system for the control of metazoan cell growth. Systematic functional analysis linked a significant number of identified network components to the control of dTOR activity and revealed dTTT, a dTOR complex required for in vivo cell growth control by dTORC1 and dTORC2.
Genetic analysis in Drosophila melanogaster has been widely used to identify a system of genes that control cell growth in response to insulin and nutrients. Many of these genes encode components of the insulin receptor/target of rapamycin (InR/TOR) pathway. However, the biochemical context of this regulatory system is still poorly characterized in Drosophila. Here, we present the first quantitative study that systematically characterizes the modularity and hormone sensitivity of the interaction proteome underlying growth control by the dInR/TOR pathway. Applying quantitative affinity purification and mass spectrometry, we identified 97 high confidence protein interactions among 58 network components. In all, 22% of the detected interactions were regulated by insulin affecting membrane proximal as well as intracellular signaling complexes. Systematic functional analysis linked a subset of network components to the control of dTORC1 and dTORC2 activity. Furthermore, our data suggest the presence of three distinct dTOR kinase complexes, including the evolutionary conserved dTTT complex (Drosophila TOR, TELO2, TTI1). Subsequent genetic studies in flies suggest a role for dTTT in controlling cell growth via a dTORC1- and dTORC2-dependent mechanism.