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

Abstract

This study shows that MAPK signalling is robust against protein level changes due to a strong negative feedback from Erk to Raf. Surprisingly, robustness is provided through a fast post-translational mechanism although variation of Erk levels occurs on a timescale of days.

Abstract

• MAPK signalling is robust against variation in protein level.

• Robustness is mediated by a negative feedback to Raf.

• Loss of negative feedback due to mutation in B-Raf opens the door for targeted intervention.

Abstract

Protein levels within signal transduction pathways vary strongly from cell to cell. For example, it has been reported that concentrations of the last kinase within the MAPK signalling module, Erk, varies about four-fold between clonal cells under the same conditions. In the present study, we analysed how signalling pathways can still process information quantitatively despite strong heterogeneity in protein levels. Mathematical analysis of isolated phosphorylation–dephosphorylation cycles predicts that phosphorylation of a signalling molecule is proportional to the protein concentration. We systematically perturbed the protein levels of Erk in human cell lines by siRNA. We found that the steady-state phosphorylation of Erk is very robust against perturbations of Erk protein level, suggesting that there are mechanisms that provide robustness to the pathway against protein fluctuations. Using mathematical modelling, we identified three potential mechanisms that may provide robustness against fluctuating protein levels:

1. Kinetic effects (saturation of the activating kinase Mek),

2. Transcriptional negative feedbacks,

3. Negative feedbacks on the post-translational level.

By experimental analysis of the systems, which included analysis of Erk phosphorylation under Mek overexpression, measuring transcript levels of negative feedback regulators, and application of generic inhibitors of transcription and translation, we could exclude kinetic effects and transcriptional negative feedback as mechanisms of robustness.

By analysing a panel of cell lines, we found that cells are robust as long as the signal passes through Raf-1. In contrast, cells where the pathway is activated by a mutation in B-Raf lose robustness. Detailed molecular analysis of the system shows that a single post-translational feedback to Raf mediates robustness. Thus, robustness is provided through a fast post-translational mechanism although variation of Erk levels occurs on a timescale of days.

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.