Figure 1.
A model for Ras/ERK signaling pathway regulated by scaffolds and modulators.
More detailed biochemical model of scaffolding actions of KSR and MP1 in the dashed rectangular boxes are shown in Figures 2 and 3, respectively. Molecules highlighted in blue, green and orange boxes represent the separate modules of Ras/ERK signaling operating from the conventional mode (no-scaffolds; membrane), KSR-supported mode (membrane) and MP1-supported mode (late endosome), respectively.
Figure 2.
A detailed biochemical model of scaffolding action of KSR.
Please refer to “Methods” for the considerations and assumptions used and Supplementary Table S1 for detailed descriptions of the kinetics parameters. “P” denotes protein phosphorylation.
Figure 3.
A detailed biochemical model of scaffolding action of MP1.
Please refer to “Methods” for the considerations and assumptions used and Supplementary Table S1 for detailed descriptions of their kinetics parameters. “P” denotes protein phosphorylation.
Figure 4.
The biphasic effect of KSR on ERK activation.
(A) Percentage of active ERK was plotted over the period indicated by varying the concentrations of KSR from 0 to 2 µM. (B) Existence of an optimal scaffold concentration of KSR (0.3–0.5 µM) by plotting the time integral of ppERK/ERK in the first 1000 seconds and KSR's initial concentrations.
Figure 5.
Profile of ERK activation before and after p14 knockout.
The amount of active ERK produced in the later phase (>600 seconds) is reduced.
Figure 6.
The biphasic effect of MP1 on ERK activation.
(A) Percentage of active ERK was plotted over the period indicated by varying the concentrations of MP1 from 0 to 2 µM. (B) Existence of an optimal scaffold concentration of MP1 (0.1 µM) by plotting the time integral of ppERK/ERK in the first 1000 seconds and MP1's initial concentrations.
Figure 7.
The collective effect of scaffold proteins KSR and MP1 on ERK activation.
(A) Overall signaling and contribution from individual modules. (B) Signaling profile under knockout conditions.
Figure 8.
The synergistic effect of the two membrane associated signaling components, the conventional EGFR–Ras–Raf–MEK–ERK signaling module (Convent) and the KSR-mediated module (KSR) on ERK activation.
Figure 9.
ERK activation regulated by membrane- and endosome-based modules.
The contribution of the two membrane and one endosome components, the conventional EGFR–Ras–Raf–MEK–ERK signaling module (Convent), the KSR-mediated module (KSR), and the MP1 module (MP1), on ERK activation at various EGF concentrations: (A) 100 ng/ml, (B) 60 ng/ml, (C) 40 ng/ml, (D) 25 ng/ml.
Figure 10.
Differential sensitivity of ppERK from (A) membrane and (B) endosomal subpathways for EGF under various Cbl-CIN85 and Endophilin A1 concentrations when both scaffold proteins KSR and MP1 are expressed at sub-optimal “low” levels.
For clarity, the sensitivity levels are denoted by colors in the scale bars. Arrows indicate response curves under the conditions specified and detailed in Figure 12, Panels A, E, I, M.
Figure 11.
Differential sensitivity of ppERK from (A) membrane and (B) endosomal subpathways for EGF under various Cbl-CIN85 and Endophilin A1 concentrations when scaffold proteins KSR and MP1 are highly expressed – optimal for signaling.
For clarity, the sensitivity levels are denoted by colors in the scale bars. Arrows indicate response curves under the conditions specified and detailed in Figure 12, Panels B, F, J, N.
Figure 12.
Detailed analysis on the sensitivities of activated ERK mediated by scaffold proteins KSR and MP1 and modulators Cbl-CIN85 and Endophilin A1 under various situations.
Sensitivities of first and third panel (A, B, C, D, I, J, K, L) KSR-mediated and conventional; second and fourth panel (E, F, G, H, M, N, O, P) MP1-mediated subpathways toward EGF variation regulated by (A - H) Endophilin A1 concentration variation when Cbl-CIN85 is low (0.0001 µM) or high (0.8 µM); (I - P) Cbl-CIN85 concentration variation when Endophilin A1 is low or high at four conditions: (A, E, I, M) when both scaffolds are present in suboptimal “low” levels [KSR = 0.02 µM, MP1 = 0.02 µM]; (B, F, J, N) when both scaffolds are present in optimally “high” concentrations as determined earlier [KSR = 0.3 µM, MP1 = 0.3 µM]; (C, G, K, O) when KSR is present at “high” level [0.3 µM] and MP1 at “low” level [0.02 µM]; (D, H, L, P) when MP1 is present at “high” level [0.3 µM] and KSR is at “low” level [0.02 µM].
Figure 13.
Detailed analysis on the sensitivities of EGFR endocytosis mediated by scaffold proteins KSR and MP1 coregulated by Cbl-CIN85 and Endophilin A1.
Sensitivities of endocytosed EGFR toward EGF variation (A to D) regulated by Endophilin A1 concentration variation when Cbl-CIN85 is low (0.0001 µM) or high (0.8 µM); (E to H) Cbl-CIN85 concentration variation when Endophilin A1 is low (0.0001 µM) or high (0.8 µM) at four conditions: (A, E) when both scaffolds are present in suboptimal “low” levels [KSR = 0.02 µM, MP1 = 0.02 µM]; (B, F) when both scaffolds are present in optimally “high” concentrations as determined earlier [KSR = 0.3 µM, MP1 = 0.3 µM]; (C, G) when KSR is present at “high” level [0.3 µM] and MP1 at “low” level [0.02 µM]; (D, H) when MP1 is present at “high” level [0.3 µM] and KSR is at “low” level [0.02 µM].
Figure 14.
Distinct dynamics of EGFR endocytosis and ERK activation sensitivities mediated by scaffolds KSR and MP1, co-regulated by Cbl-CIN85 and Endophilin A1.
EGF-induced ERK activation are collectively regulated at two different compartments (subpathways): near the plasma membrane (the conventional EGFR-Ras-Raf-MEK-ERK module and KSR-mediated signaling), and at late endosomes (MP1-mediated signaling). In this model, (A) when scaffold proteins are present at low levels, the two subpathways show distinctive sensitivities toward growth factor stimulation. MP1 exerts a more robust response to both endocytosed EGFR and ERK activation (lower sensitivity, denoted by blue lines) which could be influenced further by both Cbl-CIN85 and Endophilin (denoted by red lines). However, KSR has little effect on endocytosed EGFR but it affects sensitivity of ERK activation, co-regulated mainly by Cbl-CIN85 (denoted by red line) and not by Endophilin A1. (B) When both scaffold proteins are present at high levels, the sensitivity of endocytosed EGFR becomes milder and “analog-like” for the endosomal ERK activation (denoted by blue line) which is also co-regulated by both Cbl-CIN85 and Endophilin (denoted by red lines). However, high levels of KSR could now exert greater influence on the sensitivity of endocytosed EGFR (denoted by red line) to become more “digital” while the sensitivity of ERK mediated by KSR is further regulated more profoundly by Endophilin A1 (denoted by red line) but less by Cbl-CIN85. For further clarify, light green boxes denote components contributing towards lower sensitivity, high green boxes denote components contributing towards higher sensitivity.