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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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Dynamics of EGF or insulin-induced Ras-GTP, ERK and AKT activation, and GAB1 phosphorylation. The left panels show the time courses calculated in silico and right panels show the corresponding time courses measured in HEK293 cells stimulated with insulin (Ins, 100 nM) or EGF (0.1, 1 or 20 nM) for the indicated time intervals (min). Active GTP-bound Ras was immunoprecipitated (IP) from total cell lysates (TCL) by the agarose-conjugated Ras-binding domain (RBD) of Raf as described in Materials and methods. Proteins from Ras-IP or TCL were separated on LDS-PAGE (4–12%), transferred to nitrocellulose membrane, and immunoblotted (IB) with anti-Ras (A) or anti-phospho-ERK1/2 (T202/Y204), anti-phospho-AKT (S473) or anti-phospho-GAB1 (Y627) antibodies (B–D), respectively. The signal intensities of phosphorylated ERK1/2, AKT, or GAB1 normalized against the appropriate signal of α-tubulin protein level are expressed in arbitrary units (AU). Data shown are the mean of normalized signal intensities±s.d. from five independent experiments each performed in triplicates.
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f2: Dynamics of EGF or insulin-induced Ras-GTP, ERK and AKT activation, and GAB1 phosphorylation. The left panels show the time courses calculated in silico and right panels show the corresponding time courses measured in HEK293 cells stimulated with insulin (Ins, 100 nM) or EGF (0.1, 1 or 20 nM) for the indicated time intervals (min). Active GTP-bound Ras was immunoprecipitated (IP) from total cell lysates (TCL) by the agarose-conjugated Ras-binding domain (RBD) of Raf as described in Materials and methods. Proteins from Ras-IP or TCL were separated on LDS-PAGE (4–12%), transferred to nitrocellulose membrane, and immunoblotted (IB) with anti-Ras (A) or anti-phospho-ERK1/2 (T202/Y204), anti-phospho-AKT (S473) or anti-phospho-GAB1 (Y627) antibodies (B–D), respectively. The signal intensities of phosphorylated ERK1/2, AKT, or GAB1 normalized against the appropriate signal of α-tubulin protein level are expressed in arbitrary units (AU). Data shown are the mean of normalized signal intensities±s.d. from five independent experiments each performed in triplicates.

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)

Dynamics of EGF or insulin-induced Ras-GTP, ERK and AKT activation, and GAB1 phosphorylation. The left panels show the time courses calculated in silico and right panels show the corresponding time courses measured in HEK293 cells stimulated with insulin (Ins, 100 nM) or EGF (0.1, 1 or 20 nM) for the indicated time intervals (min). Active GTP-bound Ras was immunoprecipitated (IP) from total cell lysates (TCL) by the agarose-conjugated Ras-binding domain (RBD) of Raf as described in Materials and methods. Proteins from Ras-IP or TCL were separated on LDS-PAGE (4–12%), transferred to nitrocellulose membrane, and immunoblotted (IB) with anti-Ras (A) or anti-phospho-ERK1/2 (T202/Y204), anti-phospho-AKT (S473) or anti-phospho-GAB1 (Y627) antibodies (B–D), respectively. The signal intensities of phosphorylated ERK1/2, AKT, or GAB1 normalized against the appropriate signal of α-tubulin protein level are expressed in arbitrary units (AU). Data shown are the mean of normalized signal intensities±s.d. from five independent experiments each performed in triplicates.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: Dynamics of EGF or insulin-induced Ras-GTP, ERK and AKT activation, and GAB1 phosphorylation. The left panels show the time courses calculated in silico and right panels show the corresponding time courses measured in HEK293 cells stimulated with insulin (Ins, 100 nM) or EGF (0.1, 1 or 20 nM) for the indicated time intervals (min). Active GTP-bound Ras was immunoprecipitated (IP) from total cell lysates (TCL) by the agarose-conjugated Ras-binding domain (RBD) of Raf as described in Materials and methods. Proteins from Ras-IP or TCL were separated on LDS-PAGE (4–12%), transferred to nitrocellulose membrane, and immunoblotted (IB) with anti-Ras (A) or anti-phospho-ERK1/2 (T202/Y204), anti-phospho-AKT (S473) or anti-phospho-GAB1 (Y627) antibodies (B–D), respectively. The signal intensities of phosphorylated ERK1/2, AKT, or GAB1 normalized against the appropriate signal of α-tubulin protein level are expressed in arbitrary units (AU). Data shown are the mean of normalized signal intensities±s.d. from five independent experiments each performed in triplicates.
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