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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.

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Time-course activity of signaling molecules with 10 nM EGF in ErbB signaling.The responses of the signaling molecules during 30 min are plotted with the corresponding experimental data listed in Table 1 (nos. 4 and 8–10). (A) Phosphorylated EGFR in E1 and E1/4 cells, respectively. (B) Phosphorylated ErbB4 receptor in E1/4 cells. (C) Phosphorylated Shc in E1 and E1/4 cells, respectively. (D) Ras-GTP in E1 and E1/4 cells, respectively. Blue and red lines indicate simulation data for E1 and E1/4 cells, respectively, and filled squares and circles indicate experimental data E1 and E1/4 cells, respectively. Graphs represent activity (% of total protein).
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pone-0001782-g007: Time-course activity of signaling molecules with 10 nM EGF in ErbB signaling.The responses of the signaling molecules during 30 min are plotted with the corresponding experimental data listed in Table 1 (nos. 4 and 8–10). (A) Phosphorylated EGFR in E1 and E1/4 cells, respectively. (B) Phosphorylated ErbB4 receptor in E1/4 cells. (C) Phosphorylated Shc in E1 and E1/4 cells, respectively. (D) Ras-GTP in E1 and E1/4 cells, respectively. Blue and red lines indicate simulation data for E1 and E1/4 cells, respectively, and filled squares and circles indicate experimental data E1 and E1/4 cells, respectively. Graphs represent activity (% of total protein).

Mentions: Many studies revealed that ErbB receptor-mediated Ras signaling is deeply involved in ERK activation upon EGF stimulation [31]. Interestingly, it was reported that Shc activity, which results in Ras activation [30], [32], might be regulated by mechanisms that differ between E1 and E1/4 cells [33]. However, the detailed mechanism concerning EGFR/ErbB4 heterodimer-mediated signaling is still unclear. In the previous section we demonstrated that differences in amplitude of ERK activity between the two cell lines might be caused predominantly by different Ras activities independently of Rap1 activation dynamics, where we assumed that the pathway structure and kinetic parameters of the central Raf-MEK-ERK cascade were the same. We therefore constructed a detailed upstream model of EGFR/ErbB4 heterodimer-mediated Ras activation to predict how differences in amplitude of Ras activity between E1 and E1/4 cells can be caused (Figure 6). We assumed that one of the crucial factors is the presence of the ErbB4 receptor in the pathways, implying that homodimer-mediated pathways (steps 1–12, the left box in Figure 6) are common in the two cell lines and that heterodimer-mediated pathways (steps 14–22, the right box in Figure 6) are E1/4 cell-specific. For a simulation of E1 cells, the concentration of the ErbB4 receptor was set to zero. Then, steps 14–22 were inactive and Ras activity could be controlled by only E11P_ShcP_GS (step 12), otherwise control was achieved through E11P_ShcP_GS and E14P_ShcP_GS (steps 12 and 22). Steps 1–11, which include Shc recruitment to the receptor, binding to the Grb2-SOS complex and receptor internalization, were adopted from the earlier study [26]. However, those were re-simplified to maintain the essential dynamics of the pathway [29]. Our experimental data indicated that the signal amplitude of EGFR phosphorylation was similar in E1 and E1/4 cells although the dynamics seemed to differ (Figure 7A), while the amplitude and dynamics of Shc phosphorylation were very different (Figure 7C). Therefore, we hypothetically introduced the steps 14–22 in order to take into account the effect of ErbB4 receptor-mediated signaling on Shc and Ras regulation. Kinetic parameters were also estimated using GLSDC (Genetic Local Search with distance independent Diversity Control) by comparing the experimental data (Table 1, nos. 4, 8–10) with simulated values (See Materials and Methods for parameter estimation of the upstream model). The model description and kinetic parameters are provided in Tables S7 and S8. When the simulation model was used to examine the effect of a 10 nM EGF perturbation on E1 and E1/4 cells, the model reasonably reproduced a similar signal amplitude of EGFR phosphorylation, different amplitude and dynamics of Shc phosphorylation, different amplitude of Ras activation, and transient dynamics of ErbB4 phosphorylation in E1/4 cells (Figure 7).


Topological analysis of MAPK cascade for kinetic ErbB signaling.

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

Time-course activity of signaling molecules with 10 nM EGF in ErbB signaling.The responses of the signaling molecules during 30 min are plotted with the corresponding experimental data listed in Table 1 (nos. 4 and 8–10). (A) Phosphorylated EGFR in E1 and E1/4 cells, respectively. (B) Phosphorylated ErbB4 receptor in E1/4 cells. (C) Phosphorylated Shc in E1 and E1/4 cells, respectively. (D) Ras-GTP in E1 and E1/4 cells, respectively. Blue and red lines indicate simulation data for E1 and E1/4 cells, respectively, and filled squares and circles indicate experimental data E1 and E1/4 cells, respectively. Graphs represent activity (% of total protein).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0001782-g007: Time-course activity of signaling molecules with 10 nM EGF in ErbB signaling.The responses of the signaling molecules during 30 min are plotted with the corresponding experimental data listed in Table 1 (nos. 4 and 8–10). (A) Phosphorylated EGFR in E1 and E1/4 cells, respectively. (B) Phosphorylated ErbB4 receptor in E1/4 cells. (C) Phosphorylated Shc in E1 and E1/4 cells, respectively. (D) Ras-GTP in E1 and E1/4 cells, respectively. Blue and red lines indicate simulation data for E1 and E1/4 cells, respectively, and filled squares and circles indicate experimental data E1 and E1/4 cells, respectively. Graphs represent activity (% of total protein).
Mentions: Many studies revealed that ErbB receptor-mediated Ras signaling is deeply involved in ERK activation upon EGF stimulation [31]. Interestingly, it was reported that Shc activity, which results in Ras activation [30], [32], might be regulated by mechanisms that differ between E1 and E1/4 cells [33]. However, the detailed mechanism concerning EGFR/ErbB4 heterodimer-mediated signaling is still unclear. In the previous section we demonstrated that differences in amplitude of ERK activity between the two cell lines might be caused predominantly by different Ras activities independently of Rap1 activation dynamics, where we assumed that the pathway structure and kinetic parameters of the central Raf-MEK-ERK cascade were the same. We therefore constructed a detailed upstream model of EGFR/ErbB4 heterodimer-mediated Ras activation to predict how differences in amplitude of Ras activity between E1 and E1/4 cells can be caused (Figure 6). We assumed that one of the crucial factors is the presence of the ErbB4 receptor in the pathways, implying that homodimer-mediated pathways (steps 1–12, the left box in Figure 6) are common in the two cell lines and that heterodimer-mediated pathways (steps 14–22, the right box in Figure 6) are E1/4 cell-specific. For a simulation of E1 cells, the concentration of the ErbB4 receptor was set to zero. Then, steps 14–22 were inactive and Ras activity could be controlled by only E11P_ShcP_GS (step 12), otherwise control was achieved through E11P_ShcP_GS and E14P_ShcP_GS (steps 12 and 22). Steps 1–11, which include Shc recruitment to the receptor, binding to the Grb2-SOS complex and receptor internalization, were adopted from the earlier study [26]. However, those were re-simplified to maintain the essential dynamics of the pathway [29]. Our experimental data indicated that the signal amplitude of EGFR phosphorylation was similar in E1 and E1/4 cells although the dynamics seemed to differ (Figure 7A), while the amplitude and dynamics of Shc phosphorylation were very different (Figure 7C). Therefore, we hypothetically introduced the steps 14–22 in order to take into account the effect of ErbB4 receptor-mediated signaling on Shc and Ras regulation. Kinetic parameters were also estimated using GLSDC (Genetic Local Search with distance independent Diversity Control) by comparing the experimental data (Table 1, nos. 4, 8–10) with simulated values (See Materials and Methods for parameter estimation of the upstream model). The model description and kinetic parameters are provided in Tables S7 and S8. When the simulation model was used to examine the effect of a 10 nM EGF perturbation on E1 and E1/4 cells, the model reasonably reproduced a similar signal amplitude of EGFR phosphorylation, different amplitude and dynamics of Shc phosphorylation, different amplitude of Ras activation, and transient dynamics of ErbB4 phosphorylation in E1/4 cells (Figure 7).

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