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

Borisov N, Aksamitiene E, Kiyatkin A, Legewie S, Berkhout J, Maiwald T, Kaimachnikov NP, Timmer J, Hoek JB, Kholodenko BN - Mol. Syst. Biol. (2009)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Anatomy and Cell Biology, Thomas Jefferson University, Philadelphia, PA 19107, USA.

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.

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Insulin amplifies EGF-induced Ras/MAPK pathway activation at low EGF doses. Comparison of the calculated in silico dynamics of Ras-GTP (A), phospho-MEK (B), phospho-ERK1/2 (C), and phospho-GAB1 (D) stimulated with EGF (0.1 or 1 nM) or EGF plus insulin (EGF+Ins) in the absence or presence of PI3K inhibitor wortmannin (WT) with the corresponding kinetic measurements (shown in bottom (A, B) or right (C, D) panels) carried out in HEK293 cells stimulated with EGF (0.1, 1 or 20 nM) or co-stimulated with insulin (100 nM) plus EGF (+ or − indicate the presence or absence of the ligand). Grb2 levels serve as a loading control to show that equal amounts of protein were loaded per lane. Representative blots are shown (n=3). (E) HEK293 cells were pretreated with 100 nM WT (+) or equivalent amounts of solvent DMSO (−) for 30 min and stimulated with 0.1 nM EGF or 100 nM insulin or both ligands simultaneously for 1.5 min (left panel), 5 min (middle panel) or 15 min (right panel). Immunoblots were analyzed for phosphorylated MEK (S217/221), ERK1/2 (T202/Y204), or AKT (S473) (representative blots on the upper part of each panel). The ligand-induced ERK responses are expressed in arbitrary units (AU) (mean±s.d., n=7).
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f3: Insulin amplifies EGF-induced Ras/MAPK pathway activation at low EGF doses. Comparison of the calculated in silico dynamics of Ras-GTP (A), phospho-MEK (B), phospho-ERK1/2 (C), and phospho-GAB1 (D) stimulated with EGF (0.1 or 1 nM) or EGF plus insulin (EGF+Ins) in the absence or presence of PI3K inhibitor wortmannin (WT) with the corresponding kinetic measurements (shown in bottom (A, B) or right (C, D) panels) carried out in HEK293 cells stimulated with EGF (0.1, 1 or 20 nM) or co-stimulated with insulin (100 nM) plus EGF (+ or − indicate the presence or absence of the ligand). Grb2 levels serve as a loading control to show that equal amounts of protein were loaded per lane. Representative blots are shown (n=3). (E) HEK293 cells were pretreated with 100 nM WT (+) or equivalent amounts of solvent DMSO (−) for 30 min and stimulated with 0.1 nM EGF or 100 nM insulin or both ligands simultaneously for 1.5 min (left panel), 5 min (middle panel) or 15 min (right panel). Immunoblots were analyzed for phosphorylated MEK (S217/221), ERK1/2 (T202/Y204), or AKT (S473) (representative blots on the upper part of each panel). The ligand-induced ERK responses are expressed in arbitrary units (AU) (mean±s.d., n=7).

Mentions: The model also incorporates and analyzes complex feedback circuitry of the EGFR and IR networks. For instance, PIP3-dependent positive feedback circuits in the model involve GAB1–PI3K and IRS–PI3K interactions (Rodrigues et al, 2000; Johnston et al, 2003; Mattoon et al, 2004). Activated ERK inhibits SOS (Dong et al, 1996; Fucini et al, 1999), GAB1 (Lehr et al, 2004) and IRS (De Fea and Roth, 1997) by direct phosphorylation. Activated mTOR mediates multiple modes of feedback, including positive feedback to AKT and negative feedback loops to IRS (Gual et al, 2003; Sarbassov et al, 2005). Although AKT-induced inhibitory phosphorylation of Raf (Zimmermann and Moelling, 1999; Wellbrock et al, 2004) is included in the model, we assume this inhibition to be weak in HEK293 cells, as no noticeable MEK or ERK activation was detected experimentally, following inhibition of AKT activity (see Supplementary Figure S4). The current model involves many parameters that have no analogs in our previously published models. We used the experimental data that are shown in Figures 2 and 3 (excluding experiments with PI3K inhibitor) as a training data set to obtain reasonable fit between the model simulations and data by manually varying the parameter values (see Supplementary Table S1). However, when parameters were fitted, their upper and lower bounds were in agreement with experimental observations for similar reaction types. In addition, reaction rates were always constrained not to be faster than the diffusion limit.


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

Borisov N, Aksamitiene E, Kiyatkin A, Legewie S, Berkhout J, Maiwald T, Kaimachnikov NP, Timmer J, Hoek JB, Kholodenko BN - Mol. Syst. Biol. (2009)

Insulin amplifies EGF-induced Ras/MAPK pathway activation at low EGF doses. Comparison of the calculated in silico dynamics of Ras-GTP (A), phospho-MEK (B), phospho-ERK1/2 (C), and phospho-GAB1 (D) stimulated with EGF (0.1 or 1 nM) or EGF plus insulin (EGF+Ins) in the absence or presence of PI3K inhibitor wortmannin (WT) with the corresponding kinetic measurements (shown in bottom (A, B) or right (C, D) panels) carried out in HEK293 cells stimulated with EGF (0.1, 1 or 20 nM) or co-stimulated with insulin (100 nM) plus EGF (+ or − indicate the presence or absence of the ligand). Grb2 levels serve as a loading control to show that equal amounts of protein were loaded per lane. Representative blots are shown (n=3). (E) HEK293 cells were pretreated with 100 nM WT (+) or equivalent amounts of solvent DMSO (−) for 30 min and stimulated with 0.1 nM EGF or 100 nM insulin or both ligands simultaneously for 1.5 min (left panel), 5 min (middle panel) or 15 min (right panel). Immunoblots were analyzed for phosphorylated MEK (S217/221), ERK1/2 (T202/Y204), or AKT (S473) (representative blots on the upper part of each panel). The ligand-induced ERK responses are expressed in arbitrary units (AU) (mean±s.d., n=7).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2683723&req=5

f3: Insulin amplifies EGF-induced Ras/MAPK pathway activation at low EGF doses. Comparison of the calculated in silico dynamics of Ras-GTP (A), phospho-MEK (B), phospho-ERK1/2 (C), and phospho-GAB1 (D) stimulated with EGF (0.1 or 1 nM) or EGF plus insulin (EGF+Ins) in the absence or presence of PI3K inhibitor wortmannin (WT) with the corresponding kinetic measurements (shown in bottom (A, B) or right (C, D) panels) carried out in HEK293 cells stimulated with EGF (0.1, 1 or 20 nM) or co-stimulated with insulin (100 nM) plus EGF (+ or − indicate the presence or absence of the ligand). Grb2 levels serve as a loading control to show that equal amounts of protein were loaded per lane. Representative blots are shown (n=3). (E) HEK293 cells were pretreated with 100 nM WT (+) or equivalent amounts of solvent DMSO (−) for 30 min and stimulated with 0.1 nM EGF or 100 nM insulin or both ligands simultaneously for 1.5 min (left panel), 5 min (middle panel) or 15 min (right panel). Immunoblots were analyzed for phosphorylated MEK (S217/221), ERK1/2 (T202/Y204), or AKT (S473) (representative blots on the upper part of each panel). The ligand-induced ERK responses are expressed in arbitrary units (AU) (mean±s.d., n=7).
Mentions: The model also incorporates and analyzes complex feedback circuitry of the EGFR and IR networks. For instance, PIP3-dependent positive feedback circuits in the model involve GAB1–PI3K and IRS–PI3K interactions (Rodrigues et al, 2000; Johnston et al, 2003; Mattoon et al, 2004). Activated ERK inhibits SOS (Dong et al, 1996; Fucini et al, 1999), GAB1 (Lehr et al, 2004) and IRS (De Fea and Roth, 1997) by direct phosphorylation. Activated mTOR mediates multiple modes of feedback, including positive feedback to AKT and negative feedback loops to IRS (Gual et al, 2003; Sarbassov et al, 2005). Although AKT-induced inhibitory phosphorylation of Raf (Zimmermann and Moelling, 1999; Wellbrock et al, 2004) is included in the model, we assume this inhibition to be weak in HEK293 cells, as no noticeable MEK or ERK activation was detected experimentally, following inhibition of AKT activity (see Supplementary Figure S4). The current model involves many parameters that have no analogs in our previously published models. We used the experimental data that are shown in Figures 2 and 3 (excluding experiments with PI3K inhibitor) as a training data set to obtain reasonable fit between the model simulations and data by manually varying the parameter values (see Supplementary Table S1). However, when parameters were fitted, their upper and lower bounds were in agreement with experimental observations for similar reaction types. In addition, reaction rates were always constrained not to be faster than the diffusion limit.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Anatomy and Cell Biology, Thomas Jefferson University, Philadelphia, PA 19107, USA.

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.

Show MeSH
Related in: MedlinePlus