Ubiquitination of the epidermal growth factor receptor (EGFR) that occurs when Cbl and Grb2 bind to three phosphotyrosine residues (pY1045, pY1068 and pY1086) on the receptor displays a sharp threshold effect as a function of EGF concentration (Figure 1).


We used an ODE based modelling approach together with experiments to show that the establishment of the threshold requires both the multiplicity of binding sites and cooperative binding of Cbl and Grb2 to the EGFR (Figure 2).


While the threshold is remarkably robust, we confirmed experimentally that the system has evolved to perform optimally at physiological levels of EGFR. As a consequence, this system displays an intrinsic weakness that causes - at the supraphysiological levels of receptor and/or ligand associated with cancer - uncoupling of the mechanisms leading to signaling through phosphorylation and attenuation through ubiquitination. Under these conditions, the model predicted, and experiments confirmed, that the relative ubiquitination of the receptor decreases much more abruptly than receptor phosphorylation (Figure 3), which would translate in a faster relative attenuation of NCE-mediated EGFR degradation with respect to signaling.


These findings are highly relevant to human cancers, because in tumors EGFR is frequently overexpressed. Our data show that, both in an isogenic model and in tumor cell lines, there is a progressive uncoupling of EGFR phosphorylation and ubiquitination at the supraphysiological EGFR level. The effect is evident for EGF concentrations above 1 ng/ml, which is a frequent condition in human tumors (where overexpression of EGFR is frequently accompanied with overproduction of its ligands). In other terms, the optimization of the system for physiological EGFR levels also harbors an intrinsic weakness, which is exploited by cancer cells to obtain a proliferative advantage. In turn, this point of weakness, now identified, might constitute a suitable point of intervention for therapeutic purposes.



Figure 1 Comparison of experimental (dashed lines) and modelled (solid lines) phosphorylation (pY) and ubiquitination (Ub) dose-response curves for EGFR. Experimental data are expressed for each condition as normalized to the maximum value for that condition; simulations are normalized to optimized maxima. Inset shows the ratio of ubiquitination to phosphorylation as a function of EGF concentration for experimental and modelled data. Average results and error bars have been calculated from at least three independent experiments.


Figure 2 Different EGFR states that have been considered in the ODE model. Starting from left: (i) EGFR with close extracellular domain; (ii) EGFR with open extracellular domain; (iii) EGFR with EGF bound; (iv) EGFR dimer; (v) phosphorylated EGFR dimer (only phosphorylation sites pY1045, pY1068 and pY1086 are represented); (vi) ubiquitinated EGFR dimer.



Figure 3 Relative EGFR phosphorylation (pY/total EGFR, black lines) and ubiquitination (Ub/total EGFR, red lines) levels, as given by the ODE model, for the indicated EGF concentrations. The grey area represents the physiological range of EGFR levels. Dashed lines indicate the maximal phosphorylation and ubiquitination. Data are normalized to the maximum phosphorylation/ubiquitination at 100 ng/ml of EGF. Red squares and black circles represent experimental measurements of EGFR Ub and pY, respectively, obtained by the ELISA-based assay in NIH-EGFR clones with increasing numbers of EGFRs.


References

F. Capuani, A. Conte, E. Argenzio, L. Marchetti, C. Priami, S. Polo, P. P. Di Fiore, S. Sigismund, A. Ciliberto. Quantitative analysis reveals how EGFR activation and downregulation are coupled in normal but not in cancer cells. NATURE COMMUNICATIONS 6:7999, 2015.

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