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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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Association of Grb2, PI3K, or SHP2 with GAB1 in response to EGF, insulin, or their combination. (A) The left and middle panels show simulated dynamics of GAB1-containing complexes (scales on the Y axis are different). The right panel shows quantitation of p85–PI3K in GAB1-IP (see Materials and methods). (B) HEK293 cells were stimulated with 100 nM insulin and/or 1 nM EGF for 1.5 min. p85–PI3K–IP was analyzed for IRS4, GAB1, or p85–PI3K proteins. (C) Modeling predictions of relative SHP2 levels in GAB1-IP at 1.5 min after stimulation with 100 nM insulin and/or 0.1 and 1 nM EGF. (D) HEK293 cells were stimulated with 100 nM insulin and/or 0.1, 1, and 20 nM EGF for 1.5 min. GAB1-IP was analyzed for total phosphotyrosines (pY20), Grb2, SHP2, or GAB1 proteins. (E) HEK293 cells were pretreated with 100 nM WT (+) or equivalent amounts of solvent DMSO (−) for 30 min and stimulated with 1 nM EGF for 3 min. GAB1-IP was analyzed for total phosphotyrosines (pY20), Grb2, SHP2, p85–PI3K, or GAB1 proteins. Representative blots are shown in each panel (n=3).
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f4: Association of Grb2, PI3K, or SHP2 with GAB1 in response to EGF, insulin, or their combination. (A) The left and middle panels show simulated dynamics of GAB1-containing complexes (scales on the Y axis are different). The right panel shows quantitation of p85–PI3K in GAB1-IP (see Materials and methods). (B) HEK293 cells were stimulated with 100 nM insulin and/or 1 nM EGF for 1.5 min. p85–PI3K–IP was analyzed for IRS4, GAB1, or p85–PI3K proteins. (C) Modeling predictions of relative SHP2 levels in GAB1-IP at 1.5 min after stimulation with 100 nM insulin and/or 0.1 and 1 nM EGF. (D) HEK293 cells were stimulated with 100 nM insulin and/or 0.1, 1, and 20 nM EGF for 1.5 min. GAB1-IP was analyzed for total phosphotyrosines (pY20), Grb2, SHP2, or GAB1 proteins. (E) HEK293 cells were pretreated with 100 nM WT (+) or equivalent amounts of solvent DMSO (−) for 30 min and stimulated with 1 nM EGF for 3 min. GAB1-IP was analyzed for total phosphotyrosines (pY20), Grb2, SHP2, p85–PI3K, or GAB1 proteins. Representative blots are shown in each panel (n=3).

Mentions: Quantitative data on the dynamics of protein–protein complexes can further validate the model and provide insight into mechanisms of integrative cellular responses to insulin–EGF co-stimulation. As the data (Figure 3D and Supplementary Figure S8) suggest that GAB1 is one of the key mediators of insulin–EGF crosstalk, we determined GAB1 interactions with other network nodes. Although it is not immediately obvious, the simulations predict that following co-stimulation with insulin and EGF, the amount of GAB1-bound PI3K will increase (compared with EGF stimulation) only on a short time scale (Figure 4A, blue dashed lines versus green dotted lines), whereas the post-peak concentration of GAB1-bound PI3K will decrease owing to dephosphorylation of GAB1 phosphotyrosines by GAB1-bound SHP2 and the competitive recruitment of PI3K by phosphorylated IRS. To test this prediction, the binding of PI3K and SHP2 to GAB1 and of PI3K to IRS4 (the major IRS protein in these cells (Fantin et al, 1998)) was measured by immunoprecipitation analysis following stimulation of HEK293 cells with EGF, insulin and their combination. The data confirm that insulin–EGF co-stimulation increased the PI3K–GAB1 complex concentration at 30 s and 1.5 min, whereas the post-peak levels were decreased compared with EGF only stimulation, in agreement with in silico predictions (Figure 4A, right panel). At 3 min of stimulation with insulin and EGF, the data show robust PI3K–IRS binding response and decreased PI3K–GAB1 binding, compared with EGF alone (Figure 4B). The predicted increase in GAB1-bound Grb2 (Figure 4A, solid versus dash–dotted lines) and SHP2 (Figure 4C) correlates with an increase in the total level of GAB1 tyrosine phosphorylation (Figures 3D and 4E) and is supported by the data shown in Figure 4D for 1.5-min stimulation (quantitation of the blots shown in Figure 4B, D and E is presented in Supplementary Figure S11A–C).


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)

Association of Grb2, PI3K, or SHP2 with GAB1 in response to EGF, insulin, or their combination. (A) The left and middle panels show simulated dynamics of GAB1-containing complexes (scales on the Y axis are different). The right panel shows quantitation of p85–PI3K in GAB1-IP (see Materials and methods). (B) HEK293 cells were stimulated with 100 nM insulin and/or 1 nM EGF for 1.5 min. p85–PI3K–IP was analyzed for IRS4, GAB1, or p85–PI3K proteins. (C) Modeling predictions of relative SHP2 levels in GAB1-IP at 1.5 min after stimulation with 100 nM insulin and/or 0.1 and 1 nM EGF. (D) HEK293 cells were stimulated with 100 nM insulin and/or 0.1, 1, and 20 nM EGF for 1.5 min. GAB1-IP was analyzed for total phosphotyrosines (pY20), Grb2, SHP2, or GAB1 proteins. (E) HEK293 cells were pretreated with 100 nM WT (+) or equivalent amounts of solvent DMSO (−) for 30 min and stimulated with 1 nM EGF for 3 min. GAB1-IP was analyzed for total phosphotyrosines (pY20), Grb2, SHP2, p85–PI3K, or GAB1 proteins. Representative blots are shown in each panel (n=3).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: Association of Grb2, PI3K, or SHP2 with GAB1 in response to EGF, insulin, or their combination. (A) The left and middle panels show simulated dynamics of GAB1-containing complexes (scales on the Y axis are different). The right panel shows quantitation of p85–PI3K in GAB1-IP (see Materials and methods). (B) HEK293 cells were stimulated with 100 nM insulin and/or 1 nM EGF for 1.5 min. p85–PI3K–IP was analyzed for IRS4, GAB1, or p85–PI3K proteins. (C) Modeling predictions of relative SHP2 levels in GAB1-IP at 1.5 min after stimulation with 100 nM insulin and/or 0.1 and 1 nM EGF. (D) HEK293 cells were stimulated with 100 nM insulin and/or 0.1, 1, and 20 nM EGF for 1.5 min. GAB1-IP was analyzed for total phosphotyrosines (pY20), Grb2, SHP2, or GAB1 proteins. (E) HEK293 cells were pretreated with 100 nM WT (+) or equivalent amounts of solvent DMSO (−) for 30 min and stimulated with 1 nM EGF for 3 min. GAB1-IP was analyzed for total phosphotyrosines (pY20), Grb2, SHP2, p85–PI3K, or GAB1 proteins. Representative blots are shown in each panel (n=3).
Mentions: Quantitative data on the dynamics of protein–protein complexes can further validate the model and provide insight into mechanisms of integrative cellular responses to insulin–EGF co-stimulation. As the data (Figure 3D and Supplementary Figure S8) suggest that GAB1 is one of the key mediators of insulin–EGF crosstalk, we determined GAB1 interactions with other network nodes. Although it is not immediately obvious, the simulations predict that following co-stimulation with insulin and EGF, the amount of GAB1-bound PI3K will increase (compared with EGF stimulation) only on a short time scale (Figure 4A, blue dashed lines versus green dotted lines), whereas the post-peak concentration of GAB1-bound PI3K will decrease owing to dephosphorylation of GAB1 phosphotyrosines by GAB1-bound SHP2 and the competitive recruitment of PI3K by phosphorylated IRS. To test this prediction, the binding of PI3K and SHP2 to GAB1 and of PI3K to IRS4 (the major IRS protein in these cells (Fantin et al, 1998)) was measured by immunoprecipitation analysis following stimulation of HEK293 cells with EGF, insulin and their combination. The data confirm that insulin–EGF co-stimulation increased the PI3K–GAB1 complex concentration at 30 s and 1.5 min, whereas the post-peak levels were decreased compared with EGF only stimulation, in agreement with in silico predictions (Figure 4A, right panel). At 3 min of stimulation with insulin and EGF, the data show robust PI3K–IRS binding response and decreased PI3K–GAB1 binding, compared with EGF alone (Figure 4B). The predicted increase in GAB1-bound Grb2 (Figure 4A, solid versus dash–dotted lines) and SHP2 (Figure 4C) correlates with an increase in the total level of GAB1 tyrosine phosphorylation (Figures 3D and 4E) and is supported by the data shown in Figure 4D for 1.5-min stimulation (quantitation of the blots shown in Figure 4B, D and E is presented in Supplementary Figure S11A–C).

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