Strong negative feedback from Erk to Raf confers robustness to MAPK signalling

Abstract

Protein levels within signal transduction pathways vary strongly from cell to cell. Here, we analysed how signalling pathways can still process information quantitatively despite strong heterogeneity in protein levels. We systematically perturbed the protein levels of Erk, the terminal kinase in the MAPK signalling pathway in a panel of human cell lines. We found that the steady-state phosphorylation of Erk is very robust against perturbations of Erk protein level. Although a multitude of mechanisms exist that may provide robustness against fluctuating protein levels, we found that one single feedback from Erk to Raf-1 accounts for the observed robustness. Surprisingly, robustness is provided through a fast post-translational mechanism although variation of Erk levels occurs on a timescale of days.

Figure 1

Robustness of MAPK signalling. (A) Mathematical analysis of Erk phosphorylation kinetics suggests that the phospho-Erk level depends linearly on Erk protein concentration (green line, no robustness). The red line shows hypothetical partial robustness, where phosho-Erk level depends sublinearly on Erk. The blue line corresponds to a fully robust system, where phosho-Erk can fully compensate loss of Erk. (B) The consequences of variability in Erk expression (grey) on phospo-Erk expression for a non-robust, partially robust and fully robust system are shown. (C) Steady-state phospho-Erk level of LIM1215 cells depends only weakly on Erk concentration. Each dot shows quantified pan-isoform phospho-Erk and Erk levels from western blots of cells treated with siRNA against Erk1 or Erk2 alone, Erk1 and Erk2 in combination in percent of the scrambled control. (D) Possible mechanisms providing robustness illustrated for knockdown of Erk2: competition for upstream kinase Mek, where loss of Erk2 results in higher access of Erk1 to Mek; post-translational negative feedback, where loss of Erk2 results in relieve of negative feedback and therefore stronger upstream signalling; and transcriptional negative feedback, where knockdown of Erk2 results in decreased concentrations of deactivating phosphatases. Source data is available for this figure at www.nature.com/msb.

Figure 2

Transcriptional negative feedback is present but not utilised to compensate variations in Erk protein level. The bar graphs show the expression of several classical dual-specificity phosphatases (DUSPs) measured using q-RT–PCR in comparison with the level in cells treated with control (DMSO or scrambled, in (A) or (B), respectively). (A) DUSP5, 6, 7 are negative feedback regulators in LIM1215 cells, as treatment with high doses of the Mek inhibitor U0126 resulted in strong downregulation of DUSP5, 6, 7. (B) None of the DUSPs shows significant downregulation after knockdown of Erk1 and/or Erk2 in LIM1215 cells. Source data is available for this figure at www.nature.com/msb.

Figure 3

Detailed analysis of post-translational compensation of varying Erk concentration. (A) Position of mutations of the analysed cells in the pathway: five colon carcinoma cell lines were analysed, LIM1215 has no mutation in the MAPK signalling pathway, HT29 and RKO express constitutively active B-Raf (V600E), and SW480 and HCT116 harbour an activating mutation in K-Ras. (B) Changes in expression of Erk1 (left) and Erk2 (right) 48 h after treating the cells with scrambled control siRNA or siRNA targeting Erk1, Erk2 or both isoforms in the five cell lines. If one isoform is knocked down, no significant change in the other isoform can be observed. (C) Pan-isoform Erk and phospho-Erk levels after knockdown of Erk1 and/or Erk2 were calculated as fraction of the unperturbed scrambled controls. Cells with B-Raf mutation show a linear relation between Erk concentration and phospho-Erk level that is predicted by a mathematical model for a system without feedback (shown as line). Cells with B-Raf wild type show strong robustness in phospho-Erk level corresponding to response coefficients of 0.36 and 0.20 for HCT116 and SW480, respectively. (D) Representative western blot images of knockdown experiments in SW480 and HT29. (E) Changes in Mek phosphorylation 48 h after treating the cells with scrambled control siRNA or siRNA against Erk1, Erk2 or both isoforms. While B-Raf wild-type cells show a strong increase in phospho-Mek, B-Raf-mutated cells (HT29 and RKO) show no change in phospho-Mek after knockdown. Source data is available for this figure at www.nature.com/msb.

Figure 4

Feedback acts on c-Raf. (A) Phosphorylation of Erk-specific phosphorylation sites in Raf-1 changes strongly after Mek inhibition. HT29 and HCT116 cells were treated with MEK inhibitor U0126 or vehicle controls (DMSO) for 24 h, and subsequently analysed using western blotting. Raf-1 displays a prominent phosphorylation at the cluster of ERK phosphorylation sites within the hinge region of Raf-1 in vehicle, but not in U0126-treated cells. Total Raf-1 is accelerated in its electrophoretic mobility after application of U0126. (B) Ras activity does not increase in HCT116 and SW480 cells after treating the cells with Mek inhibitor AZD6244, when compared with untreated or vehicle controls. Ras activity was monitored by affinity precipitation using a GST fusion protein containing the Ras-binding domain of Raf-1 and subsequent western blots. GTP and GDP were added to the lysates as positive and negative control for the assay. Representative western blots are shown and quantification relative to DMSO for three assays. (C) Analysis of isogenic cells shows that signalling from the catalytic domain of Raf-1 or the V600E mutation in B-Raf diminishes feedback regulation. Left: HEK cells expressing a fusion protein of the catalytic domain of Raf-1 with oestrogen receptor-binding domain show no feedback regulation post-Mek inhibition when stimulated with tamoxifen (4OHT), but show feedback regulation when stimulated with FGF. Right: CaCo2 cells expressing B-Raf V600E show no feedback post-Mek inhibition, while cells with control constructs show strong feedback regulation. Source data is available for this figure at www.nature.com/msb.

Figure 5

Mathematical analysis shows that the observed phosphorylation of Erk and Mek after knockdown of Erk isoforms can be fully attributed to post-translational feedback to Raf-1. (A) Pan-isoform Erk and phospho-Erk levels after knockdown of Erk1 and/or Erk2 were calculated as fraction of the scrambled controls. A mathematical model that includes negative feedback can fit the data. (B) Transient expression of Mek1 WT vector in SW480 cells. Even strong overexpression of Mek1 (left panel) does not change the phosphorylation of Erk (normalised to empty expression vector, right panel). Source data is available for this figure at www.nature.com/msb.

Figure 6

Efficiency of Mek inhibitors is strongly reduced by negative feedback to Raf-1. (A) Reduction of phospho-Erk by Mek inhibitors was simulated by a mathematical model for the two cell lines SW480 and HCT116, and compared with the situation in B-Raf-mutated cells, where efficiency of the inhibitors is predicted to be strongly enhanced. In B-Raf wild-type cells, the model predicts a dose-dependent rise of pMek when the inhibitor is applied. (B) Experiments show that inhibition of Mek with Mek inhibitor U0126 results in strong increase in Mek phosphorylation in B-Raf wild-type cell lines, while B-Raf V600E cells show no increase. (C) A time series experiment after inhibition of Mek with inhibitor AZD6244 shows a rise of pMek within 1 h. At t=0, controls of untreated and DMSO-treated samples are shown. (D) Cells are treated with Mek inhibitor AZD6244 (filled symbols) or not (open symbols) for indicated times, and phosphorylation of Mek was measured for four cell lines. In addition, translation inhibitor cycloheximide (circles) or transcription inhibitor actinomycin D was applied. No significant difference between feedback action in cells with non-inhibited gene expression (squares) and cells with inhibited expression can be observed. Shown are mean and s.d. of three replicates relative to untreated samples at 0 time point. Source data is available for this figure at www.nature.com/msb.