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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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Related in: MedlinePlus

Effects of GAB1 depletion on ERK activation induced by EGF, insulin, or their combination. (A) Computational analysis of ERK activation kinetics in response to 0.1 nM EGF in the absence (left panel) or presence (right panel) of 100 nM insulin at the indicated levels of GAB1 protein. Time courses are shown for 225 nM (control; black—solid line), 150 nM (red—long dash line), 115 nM (dark yellow—short–long–short dash line), 95 nM (dark pink—long–short–short dash line), 75 nM (green—short dash line), 37.5 nM (blue—dash–dot line), 15.0 nM (cyan—dash–dot–dot line), 1.5 nM (gray—long–short dash line) and 0 nM (dark red—dotted line) GAB1 concentrations. (B) Simulated dependences of phospho-ERK level at 1.8 min (left panel) and maximal phospho-ERK level (right panel) on the GAB1 abundance for cells stimulated with 0.1 nM EGF in the presence (red solid line) or absence (blue dashed line) of 100 nM insulin. (C) HEK293 cells transfected with specific siRNA against GAB1 (+) or non-targeting siRNA (−) were stimulated with 100 nM insulin and/or 1 nM EGF for 1.5 min. Immunoblots were analyzed for phosphorylated ERK1/2 (T202/Y204) or GAB1 (Y627). GAB1 protein levels demonstrate the efficacy of GAB1 suppression. GAPDH was used as a loading control. Representative blot (left panel) and the bar graph of respective numerical values (right panel) are shown.
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f5: Effects of GAB1 depletion on ERK activation induced by EGF, insulin, or their combination. (A) Computational analysis of ERK activation kinetics in response to 0.1 nM EGF in the absence (left panel) or presence (right panel) of 100 nM insulin at the indicated levels of GAB1 protein. Time courses are shown for 225 nM (control; black—solid line), 150 nM (red—long dash line), 115 nM (dark yellow—short–long–short dash line), 95 nM (dark pink—long–short–short dash line), 75 nM (green—short dash line), 37.5 nM (blue—dash–dot line), 15.0 nM (cyan—dash–dot–dot line), 1.5 nM (gray—long–short dash line) and 0 nM (dark red—dotted line) GAB1 concentrations. (B) Simulated dependences of phospho-ERK level at 1.8 min (left panel) and maximal phospho-ERK level (right panel) on the GAB1 abundance for cells stimulated with 0.1 nM EGF in the presence (red solid line) or absence (blue dashed line) of 100 nM insulin. (C) HEK293 cells transfected with specific siRNA against GAB1 (+) or non-targeting siRNA (−) were stimulated with 100 nM insulin and/or 1 nM EGF for 1.5 min. Immunoblots were analyzed for phosphorylated ERK1/2 (T202/Y204) or GAB1 (Y627). GAB1 protein levels demonstrate the efficacy of GAB1 suppression. GAPDH was used as a loading control. Representative blot (left panel) and the bar graph of respective numerical values (right panel) are shown.

Mentions: We have recently shown that RNA interference (RNAi)-mediated GAB1 depletion can reduce the peak amplitude and decrease the duration of ERK activation (Kiyatkin et al, 2006). These findings are also supported by independent data (Meng et al, 2005). To get further insight into the mechanisms of crosstalk, we simulated the dynamics of ERK responses to EGF versus EGF plus insulin in cells where the GAB1 protein level was held at different levels with respect to control (Figure 5A). As expected, GAB1 suppression reduces the phospho-ERK level, and its decline is more pronounced for EGF (Figure 5A, left panel) than for EGF plus insulin (Figure 5A, right panel). To test the model, HEK293 cells were transfected with siRNA against human GAB1, resulting in ∼75% reduced GAB1 protein level relative to control (non-targeting siRNA-transfected cells) (Figure 5C, quantitation of blots is shown in Supplementary Figure S11D). The phospho-ERK levels measured at 1.5 min following stimulation (Figure 5C) are consistent with the simulations (Figure 5B, left). These results confirm the in silico prediction of the larger influence of GAB1 depletion on EGF- rather than on EGF plus insulin-induced ERK phosphorylation.


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)

Effects of GAB1 depletion on ERK activation induced by EGF, insulin, or their combination. (A) Computational analysis of ERK activation kinetics in response to 0.1 nM EGF in the absence (left panel) or presence (right panel) of 100 nM insulin at the indicated levels of GAB1 protein. Time courses are shown for 225 nM (control; black—solid line), 150 nM (red—long dash line), 115 nM (dark yellow—short–long–short dash line), 95 nM (dark pink—long–short–short dash line), 75 nM (green—short dash line), 37.5 nM (blue—dash–dot line), 15.0 nM (cyan—dash–dot–dot line), 1.5 nM (gray—long–short dash line) and 0 nM (dark red—dotted line) GAB1 concentrations. (B) Simulated dependences of phospho-ERK level at 1.8 min (left panel) and maximal phospho-ERK level (right panel) on the GAB1 abundance for cells stimulated with 0.1 nM EGF in the presence (red solid line) or absence (blue dashed line) of 100 nM insulin. (C) HEK293 cells transfected with specific siRNA against GAB1 (+) or non-targeting siRNA (−) were stimulated with 100 nM insulin and/or 1 nM EGF for 1.5 min. Immunoblots were analyzed for phosphorylated ERK1/2 (T202/Y204) or GAB1 (Y627). GAB1 protein levels demonstrate the efficacy of GAB1 suppression. GAPDH was used as a loading control. Representative blot (left panel) and the bar graph of respective numerical values (right panel) are shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f5: Effects of GAB1 depletion on ERK activation induced by EGF, insulin, or their combination. (A) Computational analysis of ERK activation kinetics in response to 0.1 nM EGF in the absence (left panel) or presence (right panel) of 100 nM insulin at the indicated levels of GAB1 protein. Time courses are shown for 225 nM (control; black—solid line), 150 nM (red—long dash line), 115 nM (dark yellow—short–long–short dash line), 95 nM (dark pink—long–short–short dash line), 75 nM (green—short dash line), 37.5 nM (blue—dash–dot line), 15.0 nM (cyan—dash–dot–dot line), 1.5 nM (gray—long–short dash line) and 0 nM (dark red—dotted line) GAB1 concentrations. (B) Simulated dependences of phospho-ERK level at 1.8 min (left panel) and maximal phospho-ERK level (right panel) on the GAB1 abundance for cells stimulated with 0.1 nM EGF in the presence (red solid line) or absence (blue dashed line) of 100 nM insulin. (C) HEK293 cells transfected with specific siRNA against GAB1 (+) or non-targeting siRNA (−) were stimulated with 100 nM insulin and/or 1 nM EGF for 1.5 min. Immunoblots were analyzed for phosphorylated ERK1/2 (T202/Y204) or GAB1 (Y627). GAB1 protein levels demonstrate the efficacy of GAB1 suppression. GAPDH was used as a loading control. Representative blot (left panel) and the bar graph of respective numerical values (right panel) are shown.
Mentions: We have recently shown that RNA interference (RNAi)-mediated GAB1 depletion can reduce the peak amplitude and decrease the duration of ERK activation (Kiyatkin et al, 2006). These findings are also supported by independent data (Meng et al, 2005). To get further insight into the mechanisms of crosstalk, we simulated the dynamics of ERK responses to EGF versus EGF plus insulin in cells where the GAB1 protein level was held at different levels with respect to control (Figure 5A). As expected, GAB1 suppression reduces the phospho-ERK level, and its decline is more pronounced for EGF (Figure 5A, left panel) than for EGF plus insulin (Figure 5A, right panel). To test the model, HEK293 cells were transfected with siRNA against human GAB1, resulting in ∼75% reduced GAB1 protein level relative to control (non-targeting siRNA-transfected cells) (Figure 5C, quantitation of blots is shown in Supplementary Figure S11D). The phospho-ERK levels measured at 1.5 min following stimulation (Figure 5C) are consistent with the simulations (Figure 5B, left). These results confirm the in silico prediction of the larger influence of GAB1 depletion on EGF- rather than on EGF plus insulin-induced ERK phosphorylation.

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