VEGFR2 Trafficking, Signaling and Proteolysis is Regulated by the Ubiquitin Isopeptidase USP8

Gina A Smith,Gareth W Fearnley,Sreenivasan Ponnambalam,Darren C Tomlinson,Michael A Harrison,Stephen B Wheatcroft,Izma Abdul‐Zani

doi:10.1111/tra.12341

Gina A Smith, Gareth W Fearnley + Show 5 more

Open Access

https://doi.org/10.1111/tra.12341

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Journal: Traffic	Publication Date: Dec 2, 2015
Citations: 30	License type: CC BY 4.0

Affiliation: University of Leeds

Abstract

Vascular endothelial growth factor A (VEGF‐A) regulates many aspects of vascular function. VEGF‐A binding to vascular endothelial growth factor receptor 2 (VEGFR2) stimulates endothelial signal transduction and regulates multiple cellular responses. Activated VEGFR2 undergoes ubiquitination but the enzymes that regulate this post‐translational modification are unclear. In this study, the de‐ubiquitinating enzyme, USP8, is shown to regulate VEGFR2 trafficking, de‐ubiquitination, proteolysis and signal transduction. USP8‐depleted endothelial cells displayed altered VEGFR2 ubiquitination and production of a unique VEGFR2 extracellular domain proteolytic fragment caused by VEGFR2 accumulation in the endosome–lysosome system. In addition, perturbed VEGFR2 trafficking impaired VEGF‐A‐stimulated signal transduction in USP8‐depleted cells. Thus, regulation of VEGFR2 ubiquitination and de‐ubiquitination has important consequences for the endothelial cell response and vascular physiology.

Highlights

vascular endothelial growth factor receptor 2 (VEGFR2) trafficking impaired Vascular endothelial growth factor-A (VEGF-A)-stimulated signal transduction in USP8-depleted cells
In control cells treated with non-targeting siRNA, internalized VEGFR2 was detected in punctate structures at early (0–15 min) stages of VEGF-A stimulation (Figure 1A)
To quantify distribution of mature VEGFR2 in the endosomal pathway, USP8-depleted endothelial cells were pre-treated with cycloheximide (CHX) to block protein synthesis followed by VEGF-A stimulation (Figure 1A)

Summary

Introduction

VEGFR2 trafficking impaired VEGF-A-stimulated signal transduction in USP8-depleted cells. VEGF exerts its effects by binding and activating a family of three vascular endothelial growth factor receptor tyrosine kinases (VEGFRs), namely VEGFR1, VEGFR2 and VEGFR3. VEGFR2 (KDR) is the principal receptor through which VEGF-A transmits its pro-angiogenic signals in vascular endothelial cells [2,3]. Reversibilty of ubiquitination is credited to the action of de-ubiquitinating enzymes (DUBs) [8] These enzymes play a distinct but crucial role in receptor tyrosine kinase trafficking and turnover [8]. De-ubiquitination of plasma membrane receptors www.traffic.dk 53.

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