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Systems-level interactions between insulin-EGF networks amplify mitogenic signaling.

ABSTRACT: Crosstalk mechanisms have not been studied as thoroughly as individual signaling pathways. We exploit experimental and computational approaches to reveal how a concordant interplay between the insulin and epidermal growth factor (EGF) signaling networks can potentiate mitogenic signaling. In HEK293 cells, insulin is a poor activator of the Ras/ERK (extracellular signal-regulated kinase) cascade, yet it enhances ERK activation by low EGF doses. We find that major crosstalk mechanisms that amplify ERK signaling are localized upstream of Ras and at the Ras/Raf level. Computational modeling unveils how critical network nodes, the adaptor proteins GAB1 and insulin receptor substrate (IRS), Src kinase, and phosphatase SHP2, convert insulin-induced increase in the phosphatidylinositol-3,4,5-triphosphate (PIP(3)) concentration into enhanced Ras/ERK activity. The model predicts and experiments confirm that insulin-induced amplification of mitogenic signaling is abolished by disrupting PIP(3)-mediated positive feedback via GAB1 and IRS. We demonstrate that GAB1 behaves as a non-linear amplifier of mitogenic responses and insulin endows EGF signaling with robustness to GAB1 suppression. Our results show the feasibility of using computational models to identify key target combinations and predict complex cellular responses to a mixture of external cues.

SUBMITTER: Borisov N

PROVIDER: S-EPMC2683723 | biostudies-literature | 2009

REPOSITORIES: biostudies-literature

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Systems-level interactions between insulin-EGF networks amplify mitogenic signaling.

Borisov Nikolay N Aksamitiene Edita E Kiyatkin Anatoly A Legewie Stefan S Berkhout Jan J Maiwald Thomas T Kaimachnikov Nikolai P NP Timmer Jens J Hoek Jan B JB Kholodenko Boris N BN

Molecular systems biology 20090407

Crosstalk mechanisms have not been studied as thoroughly as individual signaling pathways. We exploit experimental and computational approaches to reveal how a concordant interplay between the insulin and epidermal growth factor (EGF) signaling networks can potentiate mitogenic signaling. In HEK293 cells, insulin is a poor activator of the Ras/ERK (extracellular signal-regulated kinase) cascade, yet it enhances ERK activation by low EGF doses. We find that major crosstalk mechanisms that amplify ...[more]

PMID: 19357636

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Project description:BackgroundCellular response to external stimuli requires propagation of corresponding signals through molecular signaling pathways. However, signaling pathways are not isolated information highways, but rather interact in a number of ways forming sophisticated signaling networks. Since defects in signaling pathways are associated with many serious diseases, understanding of the crosstalk between them is fundamental for designing molecularly targeted therapy. Unfortunately, we still lack technology that would allow high throughput detailed measurement of activity of individual signaling molecules and their interactions. This necessitates developing methods to prioritize selection of the molecules such that measuring their activity would be most informative for understanding the crosstalk. Furthermore, absence of the reaction coefficients necessary for detailed modeling of signal propagation raises the question whether simple parameter-free models could provide useful information about such pathways.ResultsWe study the combined signaling network of three major pro-survival signaling pathways: Epidermal Growth Factor Receptor (EGFR), Insulin-like Growth Factor-1 Receptor (IGF-1R), and Insulin Receptor (IR). Our study involves static analysis and dynamic modeling of this network, as well as an experimental verification of the model by measuring the response of selected signaling molecules to differential stimulation of EGF, IGF and insulin receptors. We introduced two novel measures of the importance of a node in the context of such crosstalk. Based on these measures several molecules, namely Erk1/2, Akt1, Jnk, p70S6K, were selected for monitoring in the network simulation and for experimental studies. Our simulation method relies on the Boolean network model combined with stochastic propagation of the signal. Most (although not all) trends suggested by the simulations have been confirmed by experiments.ConclusionThe simple model implemented in this paper provides a valuable first step in modeling signaling networks. However, to obtain a fully predictive model, a more detailed knowledge regarding parameters of individual interactions might be necessary.

Project description:Building mechanistic models of kinase-driven signaling pathways requires quantitative measurements of protein phosphorylation across physiologically relevant conditions, but this is rarely done because of the insensitivity of traditional technologies. By using a multiplexed deep phosphoproteome profiling workflow, we were able to generate a deep phosphoproteomics dataset of the EGFR-MAPK pathway in non-transformed MCF10A cells across physiological ligand concentrations with a time resolution of <12 min and in the presence and absence of multiple kinase inhibitors. An improved phosphosite mapping technique allowed us to reliably identify >46,000 phosphorylation sites on >6600 proteins, of which >4500 sites from 2110 proteins displayed a >2-fold increase in phosphorylation in response to EGF. This data was then placed into a cellular context by linking it to 15 previously published protein databases. We found that our results were consistent with much, but not all previously reported data regarding the activation and negative feedback phosphorylation of core EGFR-ERK pathway proteins. We also found that EGFR signaling is biphasic with substrates downstream of RAS/MAPK activation showing a maximum response at <3ng/ml EGF while direct substrates, such as HGS and STAT5B, showing no saturation. We found that RAS activation is mediated by at least 3 parallel pathways, two of which depend on PTPN11. There appears to be an approximately 4-minute delay in pathway activation at the step between RAS and RAF, but subsequent pathway phosphorylation was extremely rapid. Approximately 80 proteins showed a >2-fold increase in phosphorylation across all experiments and these proteins had a significantly higher median number of phosphorylation sites (~18) relative to total cellular phosphoproteins (~4). Over 60% of EGF-stimulated phosphoproteins were downstream of MAPK and included mediators of cellular processes such as gene transcription, transport, signal transduction and cytoskeletal arrangement. Their phosphorylation was either linear with respect to MAPK activation or biphasic, corresponding to the biphasic signaling seen at the level of the EGFR. This deep, integrated phosphoproteomics data resource should be useful in building mechanistic models of EGFR and MAPK signaling and for understanding how downstream responses are regulated.

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Systems-level interactions between insulin-EGF networks amplify mitogenic signaling.

Publications

Systems-level interactions between insulin-EGF networks amplify mitogenic signaling.

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