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Regulation of EGFR signal transduction by analogue-to-digital conversion in endosomes.

Villaseñor R, Nonaka H, Del Conte-Zerial P, Kalaidzidis Y, Zerial M - Elife (2015)

Bottom Line: By mathematical modelling, we found that this mechanism confers both robustness and regulation to signalling output.Different growth factors caused specific changes in endosome number and size in various cell systems and changing the distribution of p-EGFR between endosomes was sufficient to reprogram cell-fate decision upon EGF stimulation.We propose that the packaging of p-RTKs in endosomes is a general mechanism to ensure the fidelity and specificity of the signalling response.

View Article: PubMed Central - PubMed

Affiliation: Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany.

ABSTRACT
An outstanding question is how receptor tyrosine kinases (RTKs) determine different cell-fate decisions despite sharing the same signalling cascades. Here, we uncovered an unexpected mechanism of RTK trafficking in this process. By quantitative high-resolution FRET microscopy, we found that phosphorylated epidermal growth factor receptor (p-EGFR) is not randomly distributed but packaged at constant mean amounts in endosomes. Cells respond to higher EGF concentrations by increasing the number of endosomes but keeping the mean p-EGFR content per endosome almost constant. By mathematical modelling, we found that this mechanism confers both robustness and regulation to signalling output. Different growth factors caused specific changes in endosome number and size in various cell systems and changing the distribution of p-EGFR between endosomes was sufficient to reprogram cell-fate decision upon EGF stimulation. We propose that the packaging of p-RTKs in endosomes is a general mechanism to ensure the fidelity and specificity of the signalling response.

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Phosphatases can control the p-EGFR packaging in endosomes.(A) Total p-EGFR integral intensity after continuousstimulation with 10 ng/ml EGF in HeLa EGFR-GFP BAC cells and down-regulationof the indicated phosphatase or treatment with transfection reagent only.(B) Mean p-EGFR integral intensity per endosome aftercontinuous stimulation with 10 ng/ml EGF in HeLa EGFR-GFP BAC cells anddown-regulation of the indicated phosphatase or treatment with transfectionreagent only. In both cases, bars are colour coded according to theirrespective phosphatase. All phosphatases were down-regulated with at leastthree oligos. Bars show mean ± SEM of all images for each oligo.DOI:http://dx.doi.org/10.7554/eLife.06156.022
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fig2s8: Phosphatases can control the p-EGFR packaging in endosomes.(A) Total p-EGFR integral intensity after continuousstimulation with 10 ng/ml EGF in HeLa EGFR-GFP BAC cells and down-regulationof the indicated phosphatase or treatment with transfection reagent only.(B) Mean p-EGFR integral intensity per endosome aftercontinuous stimulation with 10 ng/ml EGF in HeLa EGFR-GFP BAC cells anddown-regulation of the indicated phosphatase or treatment with transfectionreagent only. In both cases, bars are colour coded according to theirrespective phosphatase. All phosphatases were down-regulated with at leastthree oligos. Bars show mean ± SEM of all images for each oligo.DOI:http://dx.doi.org/10.7554/eLife.06156.022

Mentions: Which phosphatases are responsible for controlling p-EGFR packaging in endosomes? Toidentify them, we performed a focused RNAi screen against 21 protein tyrosinephosphatases (PTP) expressed in HeLa cells (Tarcic etal., 2009). Hits were defined if silencing satisfied three conditions: (1) itincreased the total amount of p-EGFR in endosomes and (2) increased the mean amount ofp-EGFR per endosome, and (3) the phenotype was observed with at least two siRNAs pergene. Five phosphatases, PTP4A1, PTPN11, PTPN9, PTPN18, and PTPRK, increased the amountof p-EGFR in individual endosomes (Figure 2F andFigure 2—figure supplement 8).Interestingly, PTPN11 is an EGFR interactor (Deribe etal., 2009) whose activity is enhanced upon tyrosine phosphorylation (Agazie and Hayman, 2003), suggesting a molecularmechanism whereby p-EGFR could regulate its own de-phosphorylation in endosomes.


Regulation of EGFR signal transduction by analogue-to-digital conversion in endosomes.

Villaseñor R, Nonaka H, Del Conte-Zerial P, Kalaidzidis Y, Zerial M - Elife (2015)

Phosphatases can control the p-EGFR packaging in endosomes.(A) Total p-EGFR integral intensity after continuousstimulation with 10 ng/ml EGF in HeLa EGFR-GFP BAC cells and down-regulationof the indicated phosphatase or treatment with transfection reagent only.(B) Mean p-EGFR integral intensity per endosome aftercontinuous stimulation with 10 ng/ml EGF in HeLa EGFR-GFP BAC cells anddown-regulation of the indicated phosphatase or treatment with transfectionreagent only. In both cases, bars are colour coded according to theirrespective phosphatase. All phosphatases were down-regulated with at leastthree oligos. Bars show mean ± SEM of all images for each oligo.DOI:http://dx.doi.org/10.7554/eLife.06156.022
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4384751&req=5

fig2s8: Phosphatases can control the p-EGFR packaging in endosomes.(A) Total p-EGFR integral intensity after continuousstimulation with 10 ng/ml EGF in HeLa EGFR-GFP BAC cells and down-regulationof the indicated phosphatase or treatment with transfection reagent only.(B) Mean p-EGFR integral intensity per endosome aftercontinuous stimulation with 10 ng/ml EGF in HeLa EGFR-GFP BAC cells anddown-regulation of the indicated phosphatase or treatment with transfectionreagent only. In both cases, bars are colour coded according to theirrespective phosphatase. All phosphatases were down-regulated with at leastthree oligos. Bars show mean ± SEM of all images for each oligo.DOI:http://dx.doi.org/10.7554/eLife.06156.022
Mentions: Which phosphatases are responsible for controlling p-EGFR packaging in endosomes? Toidentify them, we performed a focused RNAi screen against 21 protein tyrosinephosphatases (PTP) expressed in HeLa cells (Tarcic etal., 2009). Hits were defined if silencing satisfied three conditions: (1) itincreased the total amount of p-EGFR in endosomes and (2) increased the mean amount ofp-EGFR per endosome, and (3) the phenotype was observed with at least two siRNAs pergene. Five phosphatases, PTP4A1, PTPN11, PTPN9, PTPN18, and PTPRK, increased the amountof p-EGFR in individual endosomes (Figure 2F andFigure 2—figure supplement 8).Interestingly, PTPN11 is an EGFR interactor (Deribe etal., 2009) whose activity is enhanced upon tyrosine phosphorylation (Agazie and Hayman, 2003), suggesting a molecularmechanism whereby p-EGFR could regulate its own de-phosphorylation in endosomes.

Bottom Line: By mathematical modelling, we found that this mechanism confers both robustness and regulation to signalling output.Different growth factors caused specific changes in endosome number and size in various cell systems and changing the distribution of p-EGFR between endosomes was sufficient to reprogram cell-fate decision upon EGF stimulation.We propose that the packaging of p-RTKs in endosomes is a general mechanism to ensure the fidelity and specificity of the signalling response.

View Article: PubMed Central - PubMed

Affiliation: Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany.

ABSTRACT
An outstanding question is how receptor tyrosine kinases (RTKs) determine different cell-fate decisions despite sharing the same signalling cascades. Here, we uncovered an unexpected mechanism of RTK trafficking in this process. By quantitative high-resolution FRET microscopy, we found that phosphorylated epidermal growth factor receptor (p-EGFR) is not randomly distributed but packaged at constant mean amounts in endosomes. Cells respond to higher EGF concentrations by increasing the number of endosomes but keeping the mean p-EGFR content per endosome almost constant. By mathematical modelling, we found that this mechanism confers both robustness and regulation to signalling output. Different growth factors caused specific changes in endosome number and size in various cell systems and changing the distribution of p-EGFR between endosomes was sufficient to reprogram cell-fate decision upon EGF stimulation. We propose that the packaging of p-RTKs in endosomes is a general mechanism to ensure the fidelity and specificity of the signalling response.

Show MeSH
Related in: MedlinePlus