Endosomal binding kinetics of Eps15 and Hrs specifically regulate the degradation of RTKs

Linda Hofstad Haugen,Oddmund Bakke,Trygve Bergeland,Frode Miltzow Skjeldal

doi:10.1038/s41598-017-17320-2

Linda Hofstad Haugen, Oddmund Bakke + Show 2 more

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https://doi.org/10.1038/s41598-017-17320-2

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Journal: Scientific Reports	Publication Date: Dec 1, 2017
Citations: 9	License type: open-access

Affiliation: University of Oslo, Kappa Bioscience (Norway)

Abstract

Activation of EGF-R and PDGF-R triggers autophosphorylation and the recruitment of Eps15 and Hrs. These two endosomal proteins are important for specific receptor sorting. Hrs is recruiting ubiquitinated receptors to early endosomes to further facilitate degradation through the ESCRT complex. Upon receptor activation Hrs becomes phosphorylated and is relocated to the cytosol, important for receptor degradation. In this work we have studied the endosomal binding dynamics of Eps15 and Hrs upon EGF-R and PDGF-R stimulation. By analysing the fluorescence intensity on single endosomes after ligand stimulation we measured a time-specific decrease in the endosomal fluorescence level of Eps15-GFP and Hrs-YFP. Through FRAP experiments we could further register a specific change in the endosomal-membrane to cytosol binding properties of Eps15-GFP and Hrs-YFP. This specific change in membrane fractions proved to be a redistribution of the immobile fraction, which was not shown for the phosphorylation deficient mutants. We here describe a mechanism that can explain the previously observed relocation of Hrs from the endosomes to cytosol after EGF stimulation and show that Eps15 follows a similar mechanism. Moreover, this specific redistribution of the endosomal protein binding dynamics proved to be of major importance for receptor degradation.

Highlights

Receptor tyrosine kinases (RTK) play an important role in the control of fundamental cellular processes, including the cell cycle, cell migration, cell metabolism and survival, cell proliferation and differentiation[1,2]
Hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) recognizes ubiquitinated receptors through the ubiquitin interacting motif (UIM), and together with signal-transduction adaptor-molecule (STAM) it acts as part of the sorting machinery for degradation via the ESCRT machinery[12,13]
M1 cells stably transfected with invariant chain (Ii) were co-transfected with either Epidermal growth factor receptor substrate 15 (Eps15)-GFP or Hrs-mRFP

Summary

Introduction

Receptor tyrosine kinases (RTK) play an important role in the control of fundamental cellular processes, including the cell cycle, cell migration, cell metabolism and survival, cell proliferation and differentiation[1,2]. Binding of ligand is the activation signal for all the RTKs, which triggers trans-autophosphorylation of the receptor. Binding of ligand leads to ubiquitination of the receptor and recruitment of Hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) and Epidermal growth factor receptor pathway substrate 15 (Eps[15]). This process targets RTKs to the lumen of multivesicular bodies (MVBs) for lysosomal degradation[3]. In this study we have described a downstream effect of EGF-R ligand binding on the phosphorylation and membrane binding kinetics of Hrs and Eps[15]. Expression of Ii in model cell lines has been found to increase the early endosomal fusion rate and resulting in an enlargement of the endosomes[16,17,18,19,20]

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