Limits...
Topological analysis of MAPK cascade for kinetic ErbB signaling.

Nakakuki T, Yumoto N, Naka T, Shirouzu M, Yokoyama S, Hatakeyama M - PLoS ONE (2008)

Bottom Line: Ligand-induced homo- and hetero-dimer formation of ErbB receptors results in different biological outcomes irrespective of recruitment and activation of similar effector proteins.We found that the pathway structure is characterized by ERK-mediated positive feedback regulation of B-Raf and B-Raf-mediated negative regulation of Raf-1.From a sensitivity analysis of the detailed upstream model for Ras activation, we concluded that Ras activation dynamics is dominated by heterodimer-mediated signaling coordination with a large initial speed of dimerization when the concentration of the ErbB4 receptor is considerably high.

View Article: PubMed Central - PubMed

Affiliation: Cellular Systems Biology Team, Computational and Experimental Systems Biology Group, RIKEN Genomic Sciences Center, Tsurumi-ku, Yokohama, Kanagawa, Japan.

ABSTRACT
Ligand-induced homo- and hetero-dimer formation of ErbB receptors results in different biological outcomes irrespective of recruitment and activation of similar effector proteins. Earlier experimental research indicated that cells expressing both EGFR (epidermal growth factor receptor) and the ErbB4 receptor (E1/4 cells) induced E1/4 cell-specific B-Raf activation and higher extracellular signal-regulated kinase (ERK) activation, followed by cellular transformation, than cells solely expressing EGFR (E1 cells) in Chinese hamster ovary (CHO) cells. Since our experimental data revealed the presence of positive feedback by ERK on upstream pathways, it was estimated that the cross-talk/feedback pathway structure of the Raf-MEK-ERK cascade might affect ERK activation dynamics in our cell system. To uncover the regulatory mechanism concerning the ERK dynamics, we used topological models and performed parameter estimation for all candidate structures that possessed ERK-mediated positive feedback regulation of Raf. The structure that reliably reproduced a series of experimental data regarding signal amplitude and duration of the signaling molecules was selected as a solution. We found that the pathway structure is characterized by ERK-mediated positive feedback regulation of B-Raf and B-Raf-mediated negative regulation of Raf-1. Steady-state analysis of the estimated structure indicated that the amplitude of Ras activity might critically affect ERK activity through ERK-B-Raf positive feedback coordination with sustained B-Raf activation in E1/4 cells. However, Rap1 that positively regulates B-Raf activity might be less effective concerning ERK and B-Raf activity. Furthermore, we investigated how such Ras activity in E1/4 cells can be regulated by EGFR/ErbB4 heterodimer-mediated signaling. From a sensitivity analysis of the detailed upstream model for Ras activation, we concluded that Ras activation dynamics is dominated by heterodimer-mediated signaling coordination with a large initial speed of dimerization when the concentration of the ErbB4 receptor is considerably high. Such characteristics of the signaling cause the preferential binding of the Grb2-SOS complex to heterodimer-mediated signaling molecules.

Show MeSH

Related in: MedlinePlus

The effect of MEK inhibitor U0126 on MEK phosphorylation in E1 and E1/4 cells.Serum-starved E1 and E1/4 cells were incubated with 10 nM EGF for the indicated time period with or without pretreatment of 200 nM U0126. MEK phosphorylation was analyzed by Western blot with the corresponding anti-phospho-specific MEK antibodies (upper panel), and then reblotted with an anti-MEK antibody (lower panel). (A) Western blot for E1 cells. (B) Western blot for E1/4 cells. Data show a representative figure of three independent experiments. Quantified data are available in Table S1 (no. 3).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2262155&req=5

pone-0001782-g001: The effect of MEK inhibitor U0126 on MEK phosphorylation in E1 and E1/4 cells.Serum-starved E1 and E1/4 cells were incubated with 10 nM EGF for the indicated time period with or without pretreatment of 200 nM U0126. MEK phosphorylation was analyzed by Western blot with the corresponding anti-phospho-specific MEK antibodies (upper panel), and then reblotted with an anti-MEK antibody (lower panel). (A) Western blot for E1 cells. (B) Western blot for E1/4 cells. Data show a representative figure of three independent experiments. Quantified data are available in Table S1 (no. 3).

Mentions: We first experimentally investigated the presence of feedback regulation by ERK on upstream signal transduction pathways using the MEK inhibitor. The MEK-mediated activation of ERK through phosphorylation of ERK threonine/tyrosine residues has been well established. The inhibition of kinase activity of MEK on ERK and subsequent measurement of MEK phosphorylation represents one approach that could be used to evaluate the effect of ERK-mediated feedback on the upstream pathways. U0126 is a selective inhibitor of MEK and can therefore be used to block the phosphorylation of ERK by MEK [27]. Results indicated that EGF-induced MEK activity decreased when ERK activation was inhibited through the use of U0126 in both E1 and E1/4 cells (Figure 1) (The Western blot data relating to ERK activity following U0126 use is provided in Figure S1). This fact implied positive feedback regulation by ERK on upstream molecules, although the feedback point was not clear. Additionally, there is a possibility of cross-talk between Raf isoforms, which has been reported in many cell lines [20], [22], [23]. In order to determine the structure of feedback/cross-talk in CHO cells, we carried out topological modeling of the central Raf-MEK-ERK cascade (Figure 2). We began by using the main structure of the MAPK cascade (steps 1–11) originally given by Heinrich et al. [24], and then included feedback (steps 13, 15, 16, 18), cross-talk (steps 12, 14, 17, 19), and dual phosphorylation of ERK (steps 8–11) components [28]–[30]. Detailed information pertaining to model construction is summarized in Materials and Methods. We assumed that the structure and kinetic parameters are identical for E1 and E1/4 cells, whereas time courses relating to Ras- and Rap1-GTPs, which represent inputs of the cascade, can differ. Moreover, we assumed that the effect of feedback regulation by ERK on upstream pathways can be approximated by the effect on Raf activity. For instance, suppose that SOS (Son of Sevenless homologue protein) is negatively regulated by ERK [16], and the down-regulation of SOS activity reduces Raf-1 activity via the down-regulation of Ras. Raf-1 activation is then modeled with ERK negative feedback in our topological model. Similarly, we assumed cross-talk regulation between Raf-1 and B-Raf.


Topological analysis of MAPK cascade for kinetic ErbB signaling.

Nakakuki T, Yumoto N, Naka T, Shirouzu M, Yokoyama S, Hatakeyama M - PLoS ONE (2008)

The effect of MEK inhibitor U0126 on MEK phosphorylation in E1 and E1/4 cells.Serum-starved E1 and E1/4 cells were incubated with 10 nM EGF for the indicated time period with or without pretreatment of 200 nM U0126. MEK phosphorylation was analyzed by Western blot with the corresponding anti-phospho-specific MEK antibodies (upper panel), and then reblotted with an anti-MEK antibody (lower panel). (A) Western blot for E1 cells. (B) Western blot for E1/4 cells. Data show a representative figure of three independent experiments. Quantified data are available in Table S1 (no. 3).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2262155&req=5

pone-0001782-g001: The effect of MEK inhibitor U0126 on MEK phosphorylation in E1 and E1/4 cells.Serum-starved E1 and E1/4 cells were incubated with 10 nM EGF for the indicated time period with or without pretreatment of 200 nM U0126. MEK phosphorylation was analyzed by Western blot with the corresponding anti-phospho-specific MEK antibodies (upper panel), and then reblotted with an anti-MEK antibody (lower panel). (A) Western blot for E1 cells. (B) Western blot for E1/4 cells. Data show a representative figure of three independent experiments. Quantified data are available in Table S1 (no. 3).
Mentions: We first experimentally investigated the presence of feedback regulation by ERK on upstream signal transduction pathways using the MEK inhibitor. The MEK-mediated activation of ERK through phosphorylation of ERK threonine/tyrosine residues has been well established. The inhibition of kinase activity of MEK on ERK and subsequent measurement of MEK phosphorylation represents one approach that could be used to evaluate the effect of ERK-mediated feedback on the upstream pathways. U0126 is a selective inhibitor of MEK and can therefore be used to block the phosphorylation of ERK by MEK [27]. Results indicated that EGF-induced MEK activity decreased when ERK activation was inhibited through the use of U0126 in both E1 and E1/4 cells (Figure 1) (The Western blot data relating to ERK activity following U0126 use is provided in Figure S1). This fact implied positive feedback regulation by ERK on upstream molecules, although the feedback point was not clear. Additionally, there is a possibility of cross-talk between Raf isoforms, which has been reported in many cell lines [20], [22], [23]. In order to determine the structure of feedback/cross-talk in CHO cells, we carried out topological modeling of the central Raf-MEK-ERK cascade (Figure 2). We began by using the main structure of the MAPK cascade (steps 1–11) originally given by Heinrich et al. [24], and then included feedback (steps 13, 15, 16, 18), cross-talk (steps 12, 14, 17, 19), and dual phosphorylation of ERK (steps 8–11) components [28]–[30]. Detailed information pertaining to model construction is summarized in Materials and Methods. We assumed that the structure and kinetic parameters are identical for E1 and E1/4 cells, whereas time courses relating to Ras- and Rap1-GTPs, which represent inputs of the cascade, can differ. Moreover, we assumed that the effect of feedback regulation by ERK on upstream pathways can be approximated by the effect on Raf activity. For instance, suppose that SOS (Son of Sevenless homologue protein) is negatively regulated by ERK [16], and the down-regulation of SOS activity reduces Raf-1 activity via the down-regulation of Ras. Raf-1 activation is then modeled with ERK negative feedback in our topological model. Similarly, we assumed cross-talk regulation between Raf-1 and B-Raf.

Bottom Line: Ligand-induced homo- and hetero-dimer formation of ErbB receptors results in different biological outcomes irrespective of recruitment and activation of similar effector proteins.We found that the pathway structure is characterized by ERK-mediated positive feedback regulation of B-Raf and B-Raf-mediated negative regulation of Raf-1.From a sensitivity analysis of the detailed upstream model for Ras activation, we concluded that Ras activation dynamics is dominated by heterodimer-mediated signaling coordination with a large initial speed of dimerization when the concentration of the ErbB4 receptor is considerably high.

View Article: PubMed Central - PubMed

Affiliation: Cellular Systems Biology Team, Computational and Experimental Systems Biology Group, RIKEN Genomic Sciences Center, Tsurumi-ku, Yokohama, Kanagawa, Japan.

ABSTRACT
Ligand-induced homo- and hetero-dimer formation of ErbB receptors results in different biological outcomes irrespective of recruitment and activation of similar effector proteins. Earlier experimental research indicated that cells expressing both EGFR (epidermal growth factor receptor) and the ErbB4 receptor (E1/4 cells) induced E1/4 cell-specific B-Raf activation and higher extracellular signal-regulated kinase (ERK) activation, followed by cellular transformation, than cells solely expressing EGFR (E1 cells) in Chinese hamster ovary (CHO) cells. Since our experimental data revealed the presence of positive feedback by ERK on upstream pathways, it was estimated that the cross-talk/feedback pathway structure of the Raf-MEK-ERK cascade might affect ERK activation dynamics in our cell system. To uncover the regulatory mechanism concerning the ERK dynamics, we used topological models and performed parameter estimation for all candidate structures that possessed ERK-mediated positive feedback regulation of Raf. The structure that reliably reproduced a series of experimental data regarding signal amplitude and duration of the signaling molecules was selected as a solution. We found that the pathway structure is characterized by ERK-mediated positive feedback regulation of B-Raf and B-Raf-mediated negative regulation of Raf-1. Steady-state analysis of the estimated structure indicated that the amplitude of Ras activity might critically affect ERK activity through ERK-B-Raf positive feedback coordination with sustained B-Raf activation in E1/4 cells. However, Rap1 that positively regulates B-Raf activity might be less effective concerning ERK and B-Raf activity. Furthermore, we investigated how such Ras activity in E1/4 cells can be regulated by EGFR/ErbB4 heterodimer-mediated signaling. From a sensitivity analysis of the detailed upstream model for Ras activation, we concluded that Ras activation dynamics is dominated by heterodimer-mediated signaling coordination with a large initial speed of dimerization when the concentration of the ErbB4 receptor is considerably high. Such characteristics of the signaling cause the preferential binding of the Grb2-SOS complex to heterodimer-mediated signaling molecules.

Show MeSH
Related in: MedlinePlus