Research ArticleCOMPUTATIONAL BIOLOGY

Cell Type–Specific Importance of Ras–c-Raf Complex Association Rate Constants for MAPK Signaling

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Science Signaling  28 Jul 2009:
Vol. 2, Issue 81, pp. ra38
DOI: 10.1126/scisignal.2000397
  • Fig. 1

    Flow chart representation of the EGF-Ras-MAP kinase pathway as used for model generation. Activating processes are shown in black, inhibitory processes, in blue. EGFRi, internalized EGFR; RasGAP, here, p120GAP; TF, transcription factors phosphorylated by active ERK; PPN, nuclear phosphatase; SHP, protein-tyrosine phosphatase, phosphatase for EGFR; PP, Ser-Thr phosphatase PP, phosphatase for c-Raf; XPP, Ser-Thr phosphatase for MEK; MKP, MAP kinase phosphatase.

  • Fig. 2

    Amino acid residues in the Raf-RBD that were mutated to change affinities and kinetic rate constants regulating Ras and Raf-RBD interactions. (A) Mutations introduced to increase koff (R89L) and increase kon (N71K, D80K, and E125K). D38 of Ras is shown to depict its short-range interactions with R89 of c-Raf. (B) Mutations introduced to decrease koff (A85K) and decrease kon (T57D, K87D, and R73D). D38 of Ras is shown to depict its short-range interactions with R89 and the mutated K85 (blue) of c-Raf. Abbreviations for the amino acid residues are as follows: A, Ala; D, Asp; E, Glu; K, Lys; N, Asn; R, Arg; and T, Thr.

  • Fig. 3

    ERK and EGFR phosphorylation kinetics after EGF stimulation in HEK293 and RK13 cells. HEK293 (A and C) or RK13 (B and D) cells were serum-starved for 24 hours and then stimulated with EGF (50 ng/ml), lysed at indicated times, and analyzed by immunoblot. (A and B) Time course of ERK phosphorylation. (C and D) Time course of EGFR phosphorylation. Quantification of Western blot bands was done with ImageJ software.

  • Fig. 4

    Effect of ERK inhibition on Sos1 phosphorylation kinetics and luciferase activity. HEK293 (A) or RK13 (B) cells were serum-starved and pretreated with ERK inhibitor II (see Materials and Methods) for 10 min and then stimulated with EGF (50 ng/ml). Cells were lysed at indicated time points and Sos1 was detected by immunoblot. The arrows in (A) indicate the upshift due to Sos1 phosphorylation. HEK293 (C) and RK13 (D) were transfected with luciferase-encoding reporter plasmids and the indicated amounts of ERK inhibitor II were added. The luciferase assay was performed as described. ERK phosphorylation kinetics in HEK293 cells (E and G) was assayed by preincubating cells with 25 μM ERK inhibitor II and then stimulating with EGF (50 ng/ml). Cells were lysed at indicated time points, and ERK 1/2-P immunoblots were performed. (E) ERK-P and total ERK kinetics on immunoblots; (G) ERK-P quantification with the ImageJ software. ERK phosphorylation kinetics in RK13 cells (F and H) was assayed by preincubating cells with 25 μM ERK inhibitor II and then stimulating with EGF (50 ng/ml). Cells were lysed at indicated time points, and ERK 1/2-P immunoblots were performed. (F) ERK-P kinetics on immunoblots; (H) total ERK.

  • Fig. 5

    Comparison between predicted FoldX affinities (ΔGint) and FoldX kon values (ΔGkon) with experimental and simulated luciferase activity. (A) Correlation of luciferase activity and FoldX affinity (R = 0.34). (B) Correlation of luciferase activity and FoldX kon values (R = 0.74). (C) Luciferase activities from three to four repetitions measured in RK13 cells expressing the 17 different c-Raf mutants. The mutants on the left side all contain A85K, which decreases koff, plus compensating mutations that decrease kon. The mutants on the right side all contain R89L, which increases koff, plus compensating mutations that increase kon. The two mutants marked with an arrow are those with affinities closest to the WT, but also the greatest changes in both kon and koff. The error bars for luciferase activities and FoldX ΔG and ΔGkon values represent the SD. (D) Simulated luciferase activities of mutants with different association and dissociation rate constants for the Ras–c-Raf interaction (reaction 19 of the model). The color codes for (A), (B), and (C) are the same so that mutants can be identified in the correlation plots. The color code in (D) follows the same trend as that in (A), (B), and (C) in terms of changes in kon and koff: decreasing kon on the left side and increasing kon on the right side. The heavy black arrows point to mutants in which the Kd is predicted to be equivalent to that of the wild type, but with compensating changes in kon and koff.

  • Fig. 6

    Experimental analysis in RK13 and HEK293 cells. Effect of dominant-negative Ras (S17N) and a c-Raf mutant with impaired kinase activity on ERK phosphorylation. HEK293 (A) or RK13 (B) cells were transiently transfected with HRas S17N (a mutant that cannot be activated by Sos1) or c-RafK375M (a mutant with impaired kinase activity), starved for 24 hours, and stimulated with EGF (50 ng/ml).

  • Fig. 7

    Computer modeling results for ERK-P in HEK-like and RK13-like model. Simulation results of the effect of changing Ras-Raf kinetic rate constants (reaction R19) on simulated ERK phosphorylation changes after EGF stimulation in the HEK-like (A) and the RK13-like (B) model. The inset in (A) shows the end levels of the simulation (from 120 to 180 min) with a different y scale, to demonstrate that the relative end levels of ERK-P in the HEK293 and the RK13-like models are similar for all mutants, but the absolute values differ substantially.

Additional Files

  • Supplementary Materials for:

    Cell Type–Specific Importance of Ras–c-Raf Complex Association Rate Constants for MAPK Signaling

    Christina Kiel* and Luis Serrano

    *To whom correspondence should be addressed. E-mail: christina.kiel{at}crg.es

    This PDF file includes:

    • Materials and Methods
    • Fig. S1. Overview of EGF signal transduction.
    • Fig. S2. Comparing experimental and FoldX predicted kon and ΔG values.
    • Fig. S3. Predicted ΔGint values for Ras-Raf WT and mutant complexes as predicted by FoldX.
    • Fig. S4. ERK phosphorylation kinetics and Sos1 upshift in PC12 cells and mouse embryo fibroblasts.
    • Fig. S5. Dose-response curve of ERK-P 5 min after EGF stimulation over a concentration range of 0.075 to 50 ng/ml.
    • Fig. S6. Correlation of ERK phosphorylation and luciferase activity in RK13 cells.
    • Fig. S7. Effect of ERK inhibitor on luciferase activity, as a measure of ERK target gene�dependent gene activation, in RK13 cells.
    • Fig. S8. Transient expression of TAP (Flag-StrepII)-c-Raf plasmids.
    • Fig. S9. Luciferase assay and ERK-P kinetics for c-Raf WT, A85K, and R89L in RK13 and HEK293 cells.
    • Fig. S10. Abundance of c-Raf WT and mutants in RK13 and HEK293 cells.
    • Fig. S11. PI3K contribution to ERK phosphorylation at different EGF concentrations in RK13 cells.
    • Fig. S12. PI3K contribution to ERK phosphorylation in HEK293 cells.
    • Fig. S13. Luciferase activities for c-Raf WT, A85K, and R89L in HEK293 and RK13 cells treated with the PI3K inhibitors LY294002 or wortmannin.
    • Fig. S14. Comparing experimental and simulated time courses for Sos1, EGFR-P, and ERK-P in HEK293 cells and HEK293-like model.
    • Fig. S15. Comparing experimental and simulated time courses for EGFR-P and ERK-P in RK13 cells and RK13-like model.
    • Table S1. Comparing FoldX mutagenesis results and ionic strength dependence of WT with in vitro experiments using stopped-flow and isothermal titration calorimetry.
    • Table S2. Design of c-Raf mutants using FoldX.
    • Table S3. Modeling parameters.
    • Appendix: Sensitivity Analysis

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    Citation: C. Kiel, L. Serrano, Cell Type�Specific Importance of Ras�c-Raf Complex Association Rate Constants for MAPK Signaling. Sci. Signal.2, ra38 (2009).

    © 2009 American Association for the Advancement of Science

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