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EGFR activation coupled to inhibition of tyrosine phosphatases causes lateral signal propagation

Abstract

The epidermal growth factor receptor (EGFR) belongs to the receptor tyrosine kinase (RTK) superfamily and is involved in regulating cell proliferation, differentiation and motility1. Growth factor binding induces receptor oligomerization at the plasma membrane2,3,4,5, which leads to activation of the intrinsic RTK activity and trans-phosphorylation of tyrosine residues in the intracellular part of the receptor6,7. These residues are docking sites for proteins containing Src homology domain 2 and phosphotyrosine-binding domains that relay the signal inside the cell8,9,10. In response to EGF attached to beads, lateral propagation of EGFR phosphorylation occurs at the plasma membrane11, representing an early amplification step in EGFR signalling. Here we have investigated an underlying reaction network that couples RTK activity to protein tyrosine phosphatase (PTP) inhibition by reactive oxygen species. Mathematical analysis of the chemical kinetic equations of the minimal reaction network detects general properties of this system that can be observed experimentally by imaging EGFR phosphorylation in cells. The existence of a bistable state in this reaction network explains a threshold response and how a high proportion of phosphorylated receptors can be maintained in plasma membrane regions that are not exposed to ligand.

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Figure 1: Hydrogen peroxide enables EGFR lateral phosphorylation propogation.
Figure 2: Analysis of the RTK and PTP reaction network.
Figure 3: Threshold response of phosphorylation propagation.
Figure 4: EGF dose–response of EGFR-expressing MCF7 cells.

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Acknowledgements

We thank K. Jalink for critically reading the manuscript; M. Offterdinger for the PTB–YFP construct; T. Zimmerman for technical help with movies; I. Yudushkin for DPI control experiments and E.-L. Florin for comments on the modelling. P.J.V. was supported by a Marie Curie Fellowship of the European Community.

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Correspondence to Philippe I. H. Bastiaens.

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Supplementary Figures

Figure S1 a, PAO (50 µM) induces ligand-independent EGFR-GFP phosphorylation in MCF7 cells. b, Kinetics of Cy3-EGF binding and Grb2-GFP membrane translocation in EGFR-CFP transfected MCF7 cells. (PDF 246 kb)

Figure S2 MCF7 cells co-expressing EGFR-CFP and protein tyrosine binding domain fused to YFP (PTB-YFP) stimulated for ten minutes with either 10 mM hydrogen peroxide or 100 ng/ml soluble EGF.

Figure S3 HRas activation monitored by imaging FRET between CFP-HRas and RafRBD-YFP using FLIM measurements.

Figure S4 a, DPI attenuates EGF-bead induced lateral EGFR-GFP phosphorylation. b, Histograms of phosphorylated EGFR-GFP populations.

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Reynolds, A., Tischer, C., Verveer, P. et al. EGFR activation coupled to inhibition of tyrosine phosphatases causes lateral signal propagation. Nat Cell Biol 5, 447–453 (2003). https://doi.org/10.1038/ncb981

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