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Capuani et al., (2015). Quantitative analysis reveals how EGFR activation and downregulation are coupled in normal but not in cancer cell.

September 2016, model of the month by Lu Li
Original model: BIOMD0000000594, BIOMD0000000595


The epidermal growth factor (EGF) binding to its receptor (EGFR) promotes receptor dimerization, inter-molecular tyrosine phosphorylation, and activation of down-stream signalling molecules. Tyrosine phosphorylation facilitates the binding of ubiquitin ligase (Cbl) to EGFR, to mediate internalization of the receptor [1].

Although correlated, tyrosine phosphorylation and ubiquitination of EGF receptors display different cooperativities towards EGF concentration [2]. The steep increase in the number of receptors undergoing ubiquitination when EGF concentration reaches above a certain threshold, highlights the two qualitatively different internalization mechanisms. At low EGF concentrations, EGFR is primarily internalized via clathrin-mediated mechanism, recycling the receptor and maintaining signalling; whereas high EGF concentrations promote a significant proportion of EGFR undergoes ubiquitination, therefore attenuating the EGFR signalling [2, 3]

Cbl binds to Tyr1045phospho-EGFR directly, or to Tyr1068phospho- or Tyr1086phospho-EGFR indirectly via forming Cbl-Grb2 (Growth factor receptor-bound protein 2) complex1⁠. Crucially, to maintain high cooperativity between EGFR ubiquitination and EGF concentration, therefore establishing the threshold, both direct and indirect binding to EGFR are required. Capuani et al., (2015) [4] applied modelling approaches to explore how EGF threshold is established for EGF receptor ubiquitination

Figure 1

Figure 1. Model simulations reproduce EGFR phosphorylation and ubiquitination does-response curves. Comparison between model simulation results (solid line) and experimental observations (dash line, solid dots show mean, error bars demonstrate s.e.m based on 3 duplicates.) (a) EGFR phosphorylation (black), ubiquitination (red) and the ratio of ubiquitination to phosphorylation (inset) are shown as functions of EGF concentrations. (b) EGFR ubiquitination with over-expressed (Cbl OE, red) or down-regulated Cbl (Cbl 70Z, blue). Figure taken from [4].

Figure 2

Figure 2. Cooperativity in Cbl binding is required for establishing EGFR ubiquitination threshold. Model simulation results of EGFR ubiquitination with cooperative binding of Cbl (red line) or without (blue dash line), in comparison with EGFR phosphorylation dose-response curves (black line). Figure taken from [4].

The authors first developed a minimum model to simulate EGFR early phosphorylation (2min) after EGF binding. Previous experiments demonstrated, within this time interval, all tyrosine sites are phosphorylated independently. Therefore, this model adopted a “free-enzyme” regime to apply first-order sequential reactions for 9 tyrosine phosphorylations and dephosphorylations regardless of the specific identity of the site. All phosphorylation or dephosophrylation reactions are simulated at the same rate, kkin and kptp correspondingly. In addition, kkin is dynamically regulated by EGF concentration via a hill equation. This model is capable of reproducing EGF dose-response curve of EGFR tyrosine phosphorylation and demonstrated that phosphorylations of wild-type EGFR and EGFR-3Y (3 key tyrosine phosphorylation) respond to EGF in quantitatively similar manner (Figure 1a).

To add EGFR ubiquitination, the authors expended the above model and explicitly modelled the three key tyrosine phosphorylation/dephosphorylation reactions, that are Tyr1045, Try1068, and Tyr1086. The authors also implemented Cbl and Crb2 binding to corresponding tyrosine phosphorylated EGFR, and dynamically increased their binding rates when EGFR is pre-bound with one of them. Finally, they used the quantity of Cbl-EGFR complex to represent the amount of EGFRs undergoing ubiquitination. With the measured quantities of receptors, Grb2 and Cbl in Hela cells, simulation results reproduced EGFR ubiquitination dose response curve to EGF as observed experimentally (Figure 1a). Interestingly, despite being crucial in EGFR ubiquitination, Cbl concentration seems to not affect the EGF threshold for EGFR ubiquitination as demonstrated in both simulation results and experimental observations (Figure 1b). However, the cooperative binding between Cbl and Grb2 to EGFR is essential for establishing EGF threshold for receptor internalization (Figure 2).

In order to identify other key model parameters for regulating the EGF threshold of EGFR ubiquitination. The authors further expanded the above model to include a detailed mechanical model of EGFR activation. In this model, each EGFR monomer can be in open or close state, phosphorylated at any combinations of the three phosphorylation sites, and forming a dimmer with another open-state monomer (model structure as shown in Figure 3). Based on this model, the authors unveiled the total EGFR number (Rt) is a crucial regulator on EGF threshold to trigger EGFR ubiquitination. By manipulating the EGFR expression level in Hella cells, they demonstrated that a four-fold decrease in EGFR level shifted the dose-response ubiquitination curve to the right, increasing the EGF threshold required (Figure 4).

Figure 3

Figure 3.The model structure of EGFR activation, phosphorylation, and dimerization. Each EGFR entity can be with (black) or without (grey) ligand, in the closed (oval) or open (circle) state, or phosphorylated at any combinations of the three tyrosine sites. Figure taken from [4].

Figure 4

Figure 4.Modification of EGFR expression level shifts its ubiquitination does-response curve. Comparison between model simulation results (solid lines) and experimental assessments (dash lines) in down-regulation of EGFR level (red lines, KD) and wild type (black lines, WT). Experiments are shown as mean∓s.d from at least three experiments. Figure taken from [4].

The authors simulated this model with a wider range of EGFR quantities. As shown in Figure 5a, at physiological range of EGFR expression (grey area), the more EGF stimulating cells, the more receptors are ubiquitinated. However, to reach the peak ubiquitination level, the more EGF is involved the less EGFR is required (as indicated by the dash lines). This is in contrast with the increasing EGFR quantity required for reaching peak phosphorylation level. Beyond physiological ranges of EGFR, the gap between EGFR phosphorylation and ubiquitination grow wider at every EGF strengths, as phosphorylation level continues to increase while the ubiquitination starts to decrease. This dissociation between EGFR phosphorylation and ubiquitination has often been observed in cancer cells, as EGFR is often overexpressed.

The authors investigated the negative correlation between EGFR concentration and peak EGFR ubiquitination and concluded that this is due to the rate limiting expression level of Cbl. As when Cbl is overexpressed, in silico and experimentally, EGFR concentration corresponding to the highest level of ubiquitination increases (Figure 5b).

Figure 5

Figure 5.EGFR phosphorylation and ubiquitination as a function of EGF and EGFR concentrations (a) Relative EGFR phosphorylation (black) and ubiquitination (red), obtained experimentally (dots) and in silico (solid lines), are plotted as a function of EGFR concentration, for the indicated EGF levels. The grey area represents physiological range of EGFR concentration and the dashed lines indicate the highest phosphorylation and ubiquitination of EGFR. (b) Relative EGFR ubiquitination, under control (sold line) or Cbl over-expressing (dash line) conditions. Figure taken from [4].

By combining modelling approaches with experiments, Capuani et al. demonstrated that to establish the EGF threshold for EGFR ubiquitination, two requirements are necessary. One is the increased probability of EGFR multi-tyrosine phosphorylation in relation to EGF concentration rises; the other is the cooperative binding of Crb2 and Cbl to EGFR. And most importantly, this model demonstrated that the expression level of EGFR produces a non-linear regulation on its own ubiquitination.


References

  1. Levkowitz et al. Ubiquitin ligase activity and tyrosine phosphorylation underlie suppression of growth factor signaling by c-Cbl/Sli-1.. Mol Cell. 1999 Dec;4(6):1029-40.
  2. Sigismund et al. Threshold-controlled ubiquitination of the EGFR directs receptor fate.. EMBO J. 2013 Jul 31;32(15):2140-57.
  3. Sigismund et al. Clathrin-mediated internalization is essential for sustained EGFR signaling but dispensable for degradation.. Dev Cell. 2008 Aug;15(2):209-19.
  4. Capuani et al. Quantitative analysis reveals how EGFR activation and downregulation are coupled in normal but not in cancer cells.. Nat Commun. 2015 Aug 12;6:7999.
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