Dataset Information

Comparative proteomics and transcriptomics study of signaling network proteins across multiple normal and cancer cell lines

ABSTRACT: It is not known whether cancer cells generally show quantitative differences in the expression of signaling pathway proteins that could dysregulate signal transduction. To explore this issue, we first defined the primary components of the EGFR-MAPK pathway in normal human mammary epithelial cells, identifying 16 core proteins and 10 feedback regulators. We then quantified their absolute abundance at both the protein and mRNA level across a panel of normal and breast cancer cell lines. We found that core pathway proteins were present at very similar levels across all cell types. In contrast, the EGFR and transcriptionally controlled feedback regulators were present at highly variable levels. The absolute abundance of most core proteins was between 50,000-70,000 copies per cell, but the adaptors SOS1, SOS2, and GAB1 were found at far lower levels (2,000-5,000 per cell). MAPK signaling showed saturation in all cells between 3,000-10,000 occupied EGFR, consistent with the idea that adaptors limit signaling. Our results suggest that the relative stoichiometry of core MAPK pathway proteins is very similar across different cell types, with cell-specific differences mostly restricted to variable levels of feedback regulators. The low abundance of adaptors relative to the EGFR could be responsible for previous observation of saturable signaling, endocytosis, and high affinity EGFR.

ORGANISM(S): Homo sapiens

PROVIDER: GSE81032 | GEO | 2016/05/03

SECONDARY ACCESSION(S): PRJNA320318

REPOSITORIES: GEO

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Project description:Abstract: RAS-like (RAL) GTPases function in Wnt signalling-dependent intestinal stem cell proliferation and regeneration. Whether RAL proteins work as canonical RAS effectors in the intestine, and the mechanisms of how they contribute to tumorigenesis remain unclear. Here, we show that RAL GTPases are necessary and sufficient to activate EGFR/MAPK signalling in the intestine. We identify non-canonical roles of RAL GTPases not as RAS effectors, but rather by acting upstream of RAS activation via induction of EGFR internalisation . Knocking down Drosophila RalA from intestinal stem and progenitor cells leads to increased levels of plasma membrane-associated EGFR and decreased MAPK pathway activation. Importantly, in addition to impacting stem cell proliferation and damage-induced intestinal regeneration, this function of RAL GTPases drives EGFR-dependent tumorigenic growth in the intestine and in human mammary epithelium. Altogether, our results reveal previously unrecognised cellular and molecular contexts where RAL GTPases become essential mediators of EGFR-driven tissue homeostasis and malignant transformation. Results: RalA is required within ISCs to induce midgut adult midgut regeneration following damage by oral infection with Erwinia carotovora carotovora 15 (Ecc15) (Johansson et al., 2019). To achieve a global view of intestinal pathways affected by RalA, we performed a transcriptomic analysis by RNAseq of whole midguts from vehicle treated (Mock) or damaged (Ecc15 fed) control animals or following RalA knockdown in intestinal stem and progenitor cells using the escargot-gal4 driver (ISC/EB>) (Micchelli and Perrimon, 2006). Consistent with its effect on ISC proliferation (Johansson et al., 2019), RalA knockdown significantly impaired damage-induced upregulation of cell cycle genes in the midgut. Additionally, levels of multiple transcriptional targets of the EGFR/MAPK pathway (Golembo et al., 1996; Hsu et al., 2001; Jin et al., 2015; Meng and Biteau, 2015), such as argos (aos), rhomboid (rho), Sox21a and string (stg) were increased following Ecc15 infection in control midguts. The upregulation of these target genes was significantly impaired upon RalA knockdown.

Project description:The highly conserved Epidermal Growth Factor-receptor (Egfr) pathway is required in all animals for normal development and homeostasis; consequently, aberrant Egfr signaling is implicated in a number of diseases. Genetic analysis of Drosophila melanogaster Egfr has contributed significantly to understanding this conserved pathway and has led to the discovery of new components and targets. Here we used microarray analysis of Drosophila third instar wing discs, in which Egfr signaling was perturbed, to identify new Egfr-responsive genes. Upregulated transcripts included five known targets suggesting the approach was valid. We investigated the function of 29 previously uncharacterized genes, which had pronounced responses. The Egfr pathway is important for wing-vein patterning and using reverse genetic analysis we identified five genes that showed venation defects. Three of these genes are expressed in vein primordia and all showed transcriptional changes in response to altered Egfr activity consistent with being targets of the pathway. Genetic interactions with Egfr further linked two of the genes, Sulfated (Sulf1), an endosulfatase gene, and CG4096, an ADAMTS (A Disintegrin And Metalloproteinase with ThromboSpondin motifs) gene, to the pathway. Sulf1 showed a strong genetic interaction with the neuregulin-like ligand vein (vn) and may influence binding of Vn to heparan-sulfated proteoglycans (HSPGs). Genetic evidence also shows that CG4096 functions by modulating activity of the Egfr ligands. The substrate(s) and how ligand activity is affected are unknown, but interestingly vertebrate EGF ligands are regulated by a related ADAMTS protein. We conclude Sulf1 and CG4096 are negative feedback regulators of Egfr signaling that function in the extracellular space to influence ligand activity. 3 replicates each of wing disc samples in which EGFR dominant negative (EGFR-DN) or EGFR activated (EGFR-TOP) transgenes were expressed with the 71B-Gal4 driver.

Project description:Drosophila Lnk is the single ancestral orthologue of a highly conserved family of structurally-related intracellular adaptor proteins, the SH2B proteins. As adaptors, they lack catalytic activity but contain several protein-protein interaction domains, thus playing a critical role in signal transduction from receptor tyrosine kinases to form protein networks. Physiological studies of SH2B function in mammals have produced conflicting data. However, a recent study in Drosophila has shown that Lnk is an important regulator of the insulin/insulin-like growth factor (IGF)-1 signaling (IIS) pathway during growth, functioning in parallel to the insulin receptor substrate, Chico. As this pathway also has an evolutionary conserved role in the determination of organism lifespan, we investigated whether Lnk is required for normal lifespan in Drosophila. Phenotypic analysis of mutants for Lnk revealed that loss of Lnk function results in increased lifespan and improved survival under conditions of oxidative stress and starvation. Starvation resistance was found to be associated with increased metabolic stores of carbohydrates and lipids indicative of impaired metabolism. Biochemical and genetic data suggest that Lnk functions in both the IIS and Ras/Mitogen activated protein Kinase (MapK) signaling pathways. Microarray studies support this model, showing transcriptional feedback onto genes in both pathways as well as indicating global changes in both lipid and carbohydrate metabolism. Finally, our data also suggest that Lnk itself may be a direct target of the IIS responsive transcription factor, dFoxo, and that dFoxo may repress Lnk expression. We therefore describe novel functions for a member of the SH2B protein family and provide the first evidence for potential mechanisms of SH2B regulation. Our findings suggest that IIS signaling in Drosophila may require the activity of a second intracellular adaptor, thereby yielding fundamental new insights into the functioning and role of the IIS pathway in ageing and metabolism.

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Comparative proteomics and transcriptomics study of signaling network proteins across multiple normal and cancer cell lines

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