VEGFR2 Trafficking, Signaling and Proteolysis Is Regulated by The

doi:10.1111/tra.12341 VEGFR2 Trafficking, Signaling and Proteolysis is Regulated by the Ubiquitin Isopeptidase USP8 Gina A. Smith1, Gareth W. Fearnley1, Izma Abdul-Zani1, Stephen B. Wheatcroft2,DarrenC.Tomlinson3, Michael A. Harrison4 and Sreenivasan Ponnambalam1∗ 1Endothelial Cell Biology Unit, School of Molecular & Cellular Biology, University of Leeds, LS2 9JT Leeds, UK 2Leeds Institute of Cardiovascular & Metabolic Medicine, University of Leeds, LS2 9JT Leeds, UK 3Biomedical Health Research Centre & Astbury Centre for Structural Molecular Biology, University of Leeds, LS2 9JT Leeds, UK 4School of Biomedical Sciences, University of Leeds, LS2 9JT Leeds, UK ∗Corresponding author: Sreenivasan Ponnambalam, [email protected] Abstract Vascular endothelial growth factor A (VEGF-A) regulates many aspects VEGFR2 trafficking impaired VEGF-A-stimulated signal transduction in of vascular function. VEGF-A binding to vascular endothelial growth USP8-depleted cells. Thus, regulation of VEGFR2 ubiquitination and factor receptor 2 (VEGFR2) stimulates endothelial signal transduction de-ubiquitination has important consequences for the endothelial cell and regulates multiple cellular responses. Activated VEGFR2 under- response and vascular physiology. goes ubiquitination but the enzymes that regulate this post-translational modification are unclear. In this study, the de-ubiquitinating enzyme, Keywords de-ubiquitination, proteolysis, signal transduction, traffick- USP8, is shown to regulate VEGFR2 trafficking, de-ubiquitination, prote- ing, USP8, VEGF-A, VEGFR2 olysis and signal transduction. USP8-depleted endothelial cells displayed altered VEGFR2 ubiquitination and production of a unique VEGFR2 Received 27 February 2015, revised and accepted for publication 8 extracellular domain proteolytic fragment caused by VEGFR2 accu- October 2015, uncorrected manuscript published online 13 October mulation in the endosome–lysosome system. In addition, perturbed 2015, published online 2 December 2015 Vascular endothelial growth factor-A (VEGF-A) is a Ubiquitination of VEGFR2 acts as an endosomal sort- potent pro-angiogenic growth factor involved in regulating signal by binding to the ubiquitin-interacting motif of ing angiogenesis (1). VEGF exerts its effects by binding ESCRT-0 components, Hrs and STAM (4–6). Internalized andactivatingafamilyofthreevascularendothelial VEGFR2 can recycle back to the plasma membrane or be growth factor receptor tyrosine kinases (VEGFRs), namely committed for lysosomal degradation (6,7). Ubiquitination VEGFR1, VEGFR2 and VEGFR3. VEGFR2 (KDR) is the is a dynamic protein modification that coordinates receptor principal receptor through which VEGF-A transmits its trafficking, recycling and degradation (8). Reversibilty of ubiquitination is credited to the action of de-ubiquitinating pro-angiogenic signals in vascular endothelial cells (2,3). enzymes (DUBs) (8). These enzymes thus play a distinct VEGF-A binding to VEGFR2 promotes dimerization and but crucial role in receptor tyrosine kinase trafficking and trans-autophosphorylation of several key tyrosine residues turnover (8). present within its cytoplasmic kinase domain (1). Upon activation, VEGFR2 enters the endosome–lysosome DUBs are a superfamily of 91 enzymes that can be sub- system through incorporation into clathrin-coated divided into five distinct subfamilies with differing speci- vesicles and trafficking to early endosomal vesicular ficities for the isopeptide bond that links ubiquitin chains compartments (4). (9). De-ubiquitination of plasma membrane receptors © 2015 The Authors. Traffic published by John Wiley & Sons Ltd. www.traffic.dk 53 This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. Smith et al. facilitates recycling and is essential for maintaining the downstream degradation due to general defects in endoso- free ubiquitin pool upon which receptor trafficking is mal sorting (11). USP8 thus seemed a likely candidate for dependent. Similar to the co-ordinated but opposing regulating VEGFR2 trafficking. To test this, we used siRNA effects of kinase and phosphatase activity, ubiquitination duplexes to deplete USP8 in primary human endothelial is kept in balance by the activity of DUBs (10). cells prior to VEGF-A stimulation and immunofluorescence analysis (Figure 1A). In control cells treated Although it is known that VEGFR2 is recycled from endo- with non-targeting siRNA, internalized VEGFR2 was somes back to the plasma membrane, it is unknown detectedinpunctatestructuresatearly(0–15min)stages which DUBs prevent its lysosomal degradation. of VEGF-A stimulation (Figure 1A). After VEGF-A stim- Ubiquitin-specific protease Y (UBPY or USP8) is a DUB ulation for 60 min, VEGFR2 staining was substantially be involved in the trafficking of epidermal growth receptor reduced consistent with ligand-induced degradation tyrosine kinase (EGFR) (11–13). USP8 is a cysteine pro- (Figure 1A). tease and member of the ubiquitin-specific protease (UBP) family of DUB enzymes capable of catalyzing complete However, in USP8-depleted endothelial cells resting breakdown of both K48- and K63-linked polyubiquitin VEGFR2 was already accumulated in enlarged punc- into its component monomers (11,14,15). USP8 has diverse tate structures (Figure 1A). This pattern of VEGFR2 roles in membrane trafficking ranging from endosomal distribution continued after VEGF-A stimulation for regulation to retrograde transport (11,13). The early endo- 60 min suggesting accumulation of VEGFR2 within some ESCRT-0 subunit, STAM, is a USP8-binding partner the endosome-lysosome system (Figure 1A). Persis- (16,17). This interaction occurs via the SH3 domain of tence of these enlarged, VEGFR2-enriched punctate STAM and the proline-rich STAM-binding motif in USP8 structures following VEGF-A stimulation indicated per- (17). USP8 depletion inhibits EGFR degradation and turbed VEGFR2 trafficking and degradation. VEGFR2 causes accumulation of ubiquitinated proteins on enlarged accumulation also occurred when cells were treated endosomes (11,12). with individual USP8 siRNAs to limit off-target effects (Figure S1, Supporting Information). To quantify distri- Once internalized cargo has been committed for degrada- bution of mature VEGFR2 in the endosomal pathway, tion, conjugated ubiquitin must be recycled and removed USP8-depleted endothelial cells were pre-treated with by endosomal DUBs such as USP8, which also asso- cycloheximide (CHX) to block protein synthesis fol- ciate with the ESCRT-III complex on late endosomes lowed by VEGF-A stimulation (Figure 1A). Although the (8,18). A model was proposed in which USP8 acts further biosynthetic pool of Golgi-localized VEGFR2 was absent downstream of early endosomes to recycle ubiquitin after after CHX treatment, mature VEGFR2 still accumulated endosomal sorting and prior to lysosomal sequestra- in USP8-depleted cells (Figure 1A). Quantification of tion, suggesting a role in facilitating membrane receptor VEGFR2 residing in the endosome–lysosome system degradation(14).USP8thusfunctionsattwostagesof upon CHX treatment revealed VEGFR2 levels were 30% plasma membrane receptor trafficking: in early endo- higher in non-stimulated, USP8-depleted cells (Figure 1B). somes via ESCRT-0 interaction or in late endosomes In addition, VEGFR2 underwent 20% VEGF-A-stimulated via ESCRT-III interaction. In this study, we show that degradation in control cells. Contrastingly, high levels of regulation of VEGFR2 trafficking and de-ubiquitination accumulated VEGFR2 persisted in USP8-depleted cells, by USP8 impacts on downstream signal transduction and undergoing only 5% VEGF-A-stimulated degradation proteolysis. (Figure 1B). Results To ascertain the nature of these VEGFR2-enriched structures, USP8-depleted endothelial cells were stained for USP8 regulates VEGFR2 trafficking VEGFR2 and an early endosome marker, early endosome Previous studies have shown that USP8 depletion causes antigen-1 (EEA-1) (Figure 1C). Quantification revealed EGFR accumulation in early endosomes and inhibits increased co-distribution between VEGFR2 and EEA1 54 Traffic 2016; 17: 53–65 Regulation of VEGFR2 by USP8 Figure 1: USP8 is essential for VEGFR2 trafficking. A) Endothelial cells transfected with non-targeting or USP8 siRNA, pre-treated with CHX and stimulated with 25 ng/mL VEGF-A were fixed and processed for immunofluorescence microscopy using antibodies to VEGFR2 followed by fluorescent species-specific secondary antibodies (green). Nuclei were stained with DNA-binding dye, DAPI (blue). Scale bar represents 200 μm. B) Quantification of VEGFR2 levels residing in the endosome-lysosome system in endothelial cells pre-treated with CHX and stimulated with VEGF-A prior to immunofluorescence analysis. C) Endothelial cells transfected with USP8 siRNA and stimulated with 25 ng/mL VEGF-A for 15 min were fixed and processed for immunofluorescence microscopy using antibodies to VEGFR2 (green) and EEA1 (red) followed by fluorescent species-specific secondary antibodies. Nuclei were stained with DNA-binding dye, DAPI (blue). Scale bar represents 70 μm. D) Quantification of co-distribution between VEGFR2 and EEA1 in endothelial cells treated with non-targeting or USP8 siRNA and stimulated with VEGF-A prior to immunofluorescence analysis. Errors bars indicated ±SEM (n ≥ 3); p < 0.05 (*), p < 0.0001 (****). Traffic 2016; 17: 53–65 55 Smith et al. before and after VEGF-A stimulation in

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