Paladin is a phosphoinositide phosphatase regulating endosomal VEGFR2 signalling and angiogenesis
2020; Springer Nature; Volume: 22; Issue: 2 Linguagem: Inglês
10.15252/embr.202050218
ISSN1469-3178
AutoresAnja Nitzsche, Riikka Pietilä, Dominic Love, Chiara Testini, Takeshi Ninchoji, Ross Smith, Elisabet Ekvärn, Jimmy Larsson, Francis P. Roche, Isabel Egaña, Suvi Jauhiainen, Philipp Berger, Lena Claesson‐Welsh, Mats Hellström,
Tópico(s)melanin and skin pigmentation
ResumoArticle28 December 2020Open Access Source DataTransparent process Paladin is a phosphoinositide phosphatase regulating endosomal VEGFR2 signalling and angiogenesis Anja Nitzsche Anja Nitzsche orcid.org/0000-0003-0567-6790 Science for Life Laboratory, The Rudbeck Laboratory, Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, SwedenThese authors contributed equally to this work Search for more papers by this author Riikka Pietilä Riikka Pietilä Science for Life Laboratory, The Rudbeck Laboratory, Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, SwedenThese authors contributed equally to this work Search for more papers by this author Dominic T Love Dominic T Love Science for Life Laboratory, The Rudbeck Laboratory, Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, SwedenThese authors contributed equally to this work Search for more papers by this author Chiara Testini Chiara Testini Science for Life Laboratory, The Rudbeck Laboratory, Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden Search for more papers by this author Takeshi Ninchoji Takeshi Ninchoji Science for Life Laboratory, The Rudbeck Laboratory, Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden Search for more papers by this author Ross O Smith Ross O Smith Science for Life Laboratory, The Rudbeck Laboratory, Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden Search for more papers by this author Elisabet Ekvärn Elisabet Ekvärn Science for Life Laboratory, The Rudbeck Laboratory, Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden Search for more papers by this author Jimmy Larsson Jimmy Larsson Science for Life Laboratory, The Rudbeck Laboratory, Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden Search for more papers by this author Francis P Roche Francis P Roche Science for Life Laboratory, The Rudbeck Laboratory, Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden Search for more papers by this author Isabel Egaña Isabel Egaña Science for Life Laboratory, The Rudbeck Laboratory, Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden Search for more papers by this author Suvi Jauhiainen Suvi Jauhiainen Science for Life Laboratory, The Rudbeck Laboratory, Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden Search for more papers by this author Philipp Berger Philipp Berger Laboratory of Nanoscale Biology, Paul-Scherrer Institute, Villigen, Switzerland Search for more papers by this author Lena Claesson-Welsh Lena Claesson-Welsh orcid.org/0000-0003-4275-2000 Science for Life Laboratory, The Rudbeck Laboratory, Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden Search for more papers by this author Mats Hellström Corresponding Author Mats Hellström [email protected] orcid.org/0000-0002-7088-9533 Science for Life Laboratory, The Rudbeck Laboratory, Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden Search for more papers by this author Anja Nitzsche Anja Nitzsche orcid.org/0000-0003-0567-6790 Science for Life Laboratory, The Rudbeck Laboratory, Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, SwedenThese authors contributed equally to this work Search for more papers by this author Riikka Pietilä Riikka Pietilä Science for Life Laboratory, The Rudbeck Laboratory, Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, SwedenThese authors contributed equally to this work Search for more papers by this author Dominic T Love Dominic T Love Science for Life Laboratory, The Rudbeck Laboratory, Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, SwedenThese authors contributed equally to this work Search for more papers by this author Chiara Testini Chiara Testini Science for Life Laboratory, The Rudbeck Laboratory, Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden Search for more papers by this author Takeshi Ninchoji Takeshi Ninchoji Science for Life Laboratory, The Rudbeck Laboratory, Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden Search for more papers by this author Ross O Smith Ross O Smith Science for Life Laboratory, The Rudbeck Laboratory, Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden Search for more papers by this author Elisabet Ekvärn Elisabet Ekvärn Science for Life Laboratory, The Rudbeck Laboratory, Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden Search for more papers by this author Jimmy Larsson Jimmy Larsson Science for Life Laboratory, The Rudbeck Laboratory, Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden Search for more papers by this author Francis P Roche Francis P Roche Science for Life Laboratory, The Rudbeck Laboratory, Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden Search for more papers by this author Isabel Egaña Isabel Egaña Science for Life Laboratory, The Rudbeck Laboratory, Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden Search for more papers by this author Suvi Jauhiainen Suvi Jauhiainen Science for Life Laboratory, The Rudbeck Laboratory, Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden Search for more papers by this author Philipp Berger Philipp Berger Laboratory of Nanoscale Biology, Paul-Scherrer Institute, Villigen, Switzerland Search for more papers by this author Lena Claesson-Welsh Lena Claesson-Welsh orcid.org/0000-0003-4275-2000 Science for Life Laboratory, The Rudbeck Laboratory, Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden Search for more papers by this author Mats Hellström Corresponding Author Mats Hellström [email protected] orcid.org/0000-0002-7088-9533 Science for Life Laboratory, The Rudbeck Laboratory, Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden Search for more papers by this author Author Information Anja Nitzsche1,3, Riikka Pietilä1, Dominic T Love1, Chiara Testini1,4, Takeshi Ninchoji1, Ross O Smith1, Elisabet Ekvärn1,5, Jimmy Larsson1,6, Francis P Roche1, Isabel Egaña1, Suvi Jauhiainen1, Philipp Berger2, Lena Claesson-Welsh1 and Mats Hellström *,1 1Science for Life Laboratory, The Rudbeck Laboratory, Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden 2Laboratory of Nanoscale Biology, Paul-Scherrer Institute, Villigen, Switzerland 3Present address: Université de Paris, Paris Cardiovascular Research Center, INSERM U970, Paris, France 4Present address: Division of Nephrology, Department of Medicine, Boston Children's Hospital, Boston, MA, USA 5Present address: Cepheid AB, Solna, Sweden 6Present address: Department of Cell and Molecular Biology, Uppsala University, Uppsala, Sweden *Corresponding author. Tel: +46 708 717001; E-mail: [email protected] EMBO Reports (2021)22:e50218https://doi.org/10.15252/embr.202050218 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Cell signalling governs cellular behaviour and is therefore subject to tight spatiotemporal regulation. Signalling output is modulated by specialized cell membranes and vesicles which contain unique combinations of lipids and proteins. The phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), an important component of the plasma membrane as well as other subcellular membranes, is involved in multiple processes, including signalling. However, which enzymes control the turnover of non-plasma membrane PI(4,5)P2, and their impact on cell signalling and function at the organismal level are unknown. Here, we identify Paladin as a vascular PI(4,5)P2 phosphatase regulating VEGFR2 endosomal signalling and angiogenesis. Paladin is localized to endosomal and Golgi compartments and interacts with vascular endothelial growth factor receptor 2 (VEGFR2) in vitro and in vivo. Loss of Paladin results in increased internalization of VEGFR2, over-activation of extracellular regulated kinase 1/2, and hypersprouting of endothelial cells in the developing retina of mice. These findings suggest that inhibition of Paladin, or other endosomal PI(4,5)P2 phosphatases, could be exploited to modulate VEGFR2 signalling and angiogenesis, when direct and full inhibition of the receptor is undesirable. SYNOPSIS This study identifies Paladin as a vascular PI(4,5)P2 phosphatase, which restricts VEGFR2 internalization and activation of downstream signaling, thereby dampening angiogenesis. Paladin is a phosphoinositide phosphatase with preference for PI(4,5)P2. VEGF-A induces co-localization of Paladin with VEGFR2 in EEA1+ early endosomes. Paladin deficiency promotes increased VEGFR2 phosphorylation, internalization, and ERK1/2 signalling. Increased ERK1/2 signalling in Paladin knockout mice leads to endothelial cell hypersprouting in physiological and pathological retinal angiogenesis. Introduction In the eukaryotic cell, membranes in different subcellular compartments play distinct roles in cell signalling. Growth factor receptor signalling is initiated at the cell surface and continues after internalization and during endosome trafficking (Lampugnani et al, 2006; Simons et al, 2016). However, signalling is quantitatively and qualitatively distinct depending on the specialized membrane compartment (Di Paolo & De Camilli, 2006). Key to the maintenance of membrane specialization are lipid kinases and phosphatases that phosphorylate/dephosphorylate distinct phospholipids with an inositol head group, i.e., phosphoinositides (PI). PIs are specifically distributed to generate "membrane codes" on intracellular vesicles and the plasma membrane (Di Paolo & De Camilli, 2006; Lemmon, 2008). These PIs together with Rab GTPases are required for the maintenance and coordination of endocytosis and membrane trafficking (Jean & Kiger, 2012) through recruitment of effector proteins to assemble specific endocytic complexes (Botelho et al, 2008; Jin et al, 2008; Lemmon, 2008; Chagpar et al, 2010; Mizuno-Yamasaki et al, 2010). Consequently, as lipid kinases and phosphatases are key regulators of membrane identity and function, they are also regulators of cell signalling. However, the kinases and phosphatases involved in the generation of the specific PIs at distinct subcellular localizations are still not fully identified and their roles at the organismal level are only partially known. PIs can become phosphorylated at the 3′, 4′, and 5′ position of the inositol ring, giving rise to seven different PI species. The main PIs in the plasma membrane, early endosomes, late endosomes, and the Golgi apparatus are PI(4,5)P2, PI(3)P, PI(3,5)P2, and PI(4)P, respectively (Tan et al, 2015). These PI pools, present in microdomains of membrane vesicles, provide a unique environment for signalling and sorting (Tan et al, 2015). Growth factor signalling is initiated at the plasma membrane and involves activation of enzymes that use PIs as substrates. PI(4,5)P2 at the plasma membrane is a substrate for phosphoinositide-3′ kinase (PI 3-kinase) resulting in generation of the second messenger PI(3,4,5)P3, while hydrolysis of PI(4,5)P2 by phospholipase C (PLC) generates inositol-1,4,5-trisphosphate and diacylglycerol (Katan & Cockcroft, 2020). Growth factor stimulation moreover leads to clathrin-mediated endocytosis whereby the PI(4,5)P2 membrane pool is metabolized to PI(3)P via PI(4)P and PI(3,4)P2 intermediates (He et al, 2017). PI(4,5)P2 is also present to a lower extent in intracellular membranes, as demonstrated by immuno-electron microscopy and further suggested by the presence of lipid kinases and phosphatases for which PI(4,5)P2 is a substrate or product. An important role for PI(4,5)P2 dephosphorylation has been identified in growth factor receptor internalization and sorting in early endosomes. For example, PI(4,5)P2 generated by type I gamma phosphatidylinositol phosphate 5-kinase i5 (PIPKIγi5) regulates sorting of endosomal epidermal growth factor receptor (EGFR). PIPKIγi5-deficiency results in reduced transition of the EGFR from endosomes to lysosomes and consequently prolonged signalling (Sun et al, 2013). Paladin is a membrane-associated protein encoded by Pald1 or x99384/mKIAA1274 in mouse and PALD1 or KIAA1274 in human. Its expression is primarily restricted to endothelial cells during development (Wallgard et al, 2008; Suzuki et al, 2010; Wallgard et al, 2012). Although Paladin contains a phosphatase domain, it reportedly lacks protein phosphatase activity and was thus suggested to be a catalytically inactive pseudophosphatase (Huang et al, 2009; Roffers-Agarwal et al, 2012; Kharitidi et al, 2014; Reiterer et al, 2014). However, Paladin has been implicated in various cell signalling pathways. A broad phenotypic screen in Pald1 null mice covering all organ systems revealed a specific lung phenotype, i.e., an emphysema-like lung histology and increased turnover of lung endothelial cells (Egana et al, 2017). In addition, studies on chick embryos support a role for Paladin in neural crest migration (Roffers-Agarwal et al, 2012). Cell culture studies suggest that Paladin negatively regulates expression and phosphorylation of the insulin receptor, as well as the phosphorylation of the downstream serine/threonine kinase AKT (Huang et al, 2009). Furthermore, Paladin is a negative regulator of Toll-like receptor 9 (TLR9) signalling (Li et al, 2011). Collectively, these observations suggest that Paladin is an important player in cell signalling. Nevertheless, the mechanism whereby Paladin achieves those effects on diverse signalling pathways has remained unknown. Here, we provide evidence that Paladin is a PI(4,5)P2 phosphatase that lacks phospho-tyrosine/serine/threonine phosphatase activity. Paladin localized to endosomal vesicles where it interacted with VEGFR2, thereby positioned as a potential regulator of endosomal trafficking. In line with this, loss of Pald1 expression led to faster VEGFR2 internalization to EEA1+ endosomes and increased pERK1/2 levels in vitro and in vivo after VEGF-A stimulation. Phenotypically, Pald1 deficiency promoted enhanced pathological retinal angiogenesis. Pald1 deficiency also resulted in retinal vascular hypersprouting, which was normalized by inhibition of MEK. Results and Discussion Paladin is an endosomal PI(4,5)P2 phosphatase interacting with VEGFR2 Despite the lack of published experimental evidence, Paladin had been postulated to be a catalytically inactive pseudophosphatase (Huang et al, 2009; Kharitidi et al, 2014; Reiterer et al, 2014). However, more recently, Alonso and Pulido suggested that Paladin is a Cys-based phosphatase, which forms its own subclass (IV). Whereas all the neighbouring phosphatase subclasses dephosphorylate PI, they proposed, based on structural similarity, that Paladin might possess inositol phosphatase activity (Alonso & Pulido, 2015). The Paladin amino acid sequence contains four repeats of the minimal protein tyrosine phosphatases (PTP) consensus sequence CX5R (Fig EV1A) (Wallgard et al, 2012). Two of these repeats share high similarity with the extended conserved signature motif of the PTP active site, but importantly Paladin lacks the conserved histidine residue preceding the CX5R motif (Fig EV1B) (Andersen et al, 2001). However, an increasing number of PTPs are shown to have phosphoinositides as substrates (Pulido et al, 2013) and several new candidate PI phosphatases have been proposed, including Paladin (Alonso & Pulido, 2015). We therefore used a colorimetric screen based on the release of free phosphate to evaluate such phosphatase activity of Paladin. We expressed and immunoprecipitated V5-tagged Paladin and the phosphatase and tensin homolog (PTEN) in HEK293 cells. Wild-type PTEN and dephosphorylation of PI(3,4,5)P3 was used as positive control and the C124S phosphatase-dead PTEN variant as negative control. Similarly, we used a Paladin variant with a cysteine to serine (C/S) substitution of all four cysteines in the CX5R motifs as a negative control (Fig EV1A). Indeed, wild-type Paladin showed specific phosphatase activity towards PI(4,5)P2 and tended to also dephosphorylate PI(3,4,5)P3 but not PI monophosphates or inositol phosphates (Figs 1A and EV1C). Further, using a radioactively labelled phosphopeptide substrate and the protein tyrosine phosphatase, T cell (TC)-PTP, as a positive control we confirmed the data by Huang and co-workers that Paladin lacks phospho-tyrosine activity (Fig EV1D; Huang et al, 2009). Similarly, no phosphatase activity against a protein kinase C (PKC)-phosphorylated phosphoserine/phosphothreonine peptide was apparent (Fig EV1E). These observations support the conclusion that Paladin is a phosphoinositide phosphatase. Click here to expand this figure. Figure EV1. Paladin is lipid phosphatase interacting with VEGFR2 Schematic of Paladin protein depicting the four putative phosphatase domains (white boxes, CX5R; X: any amino acid; mouse: amino acids 121–127, 160–166, 315–321, and 664–670; human: amino acids 118–124, 157–163, 312–318, and 661–667). Phosphatase domains predicted by Interpro (black box) and SCOP SUPERFAMILY algorithm (grey). A full-length phosphatase-dead variant was generated by substituting the four cysteine residues for serine (C/S). Amino acid sequence alignment of the third (amino acids: mouse, 312-–322; human, 309–319) and fourth (amino acids: mouse, 661–671; human, 658–668) putative phosphatase domains of Paladin with the consensus sequence of catalytic domain motif 9 of cysteine-based protein tyrosine phosphatases revealed serine instead of histidine residue in front of the cysteine in the phosphatase domains. By contrast, the phosphatase domain of vascular endothelial PTP (VE-PTP, Ptprb) contains the complete PTP motif. Screening of the phosphatase activity towards phosphoinositides and inositol phosphates (IP4 = Ins(1,3,4,5)P4 and IP6 = Ins(1,2,3,4,5,6)P6 of immunoprecipitated wild-type Paladin and phosphatase-dead (C/S) mutant variant using an in vitro colorimetric molybdate dye assay. Commercial SHIP2 enzyme reaction buffer was used (Echelon, USA). Mean ± SEM, n = 3 technical replicates. In vitro radioactive phosphatase assay using Paladin, wild-type, or C/S variant, immunoprecipitated from HEK293, and as a substrate, Src-optimized peptide phosphorylated on tyrosines. Immunoprecipitates from cells transfected with empty vector or endogenous TC-PTP served as negative and positive controls, respectively. Data were normalized to 32P input. Mean ± SEM. n = 7 for wild-type Paladin, negative control, and substrate, n = 3 for C/S Paladin variant and TC-PTP (biological replicates). Immunoprecipitated full-length wild-type Paladin or its phosphatase-dead C/S variant expressed in HEK293 cells were analysed in an in vitro radioactive phosphatase assay using phosphorylated PKC-optimal peptide containing phosphoserine and phosphothreonine residues as a substrate. Immunoprecipitates of cells transfected with an empty vector served as a negative control. Data were normalized to 32P input. Mean ± SEM. n = 3 biological replicates. Confocal representative image of HDMEC stained for Paladin (green) and Giantin (Golgi apparatus marker) (red). Cells were treated with VEGF-A (50 ng/ml) for 10 min. Scale bar: 5 μm. Controls for proximity ligation assay (PLA) to determine complex formation between Paladin and VEGFR2 as presented in Fig 1E. The indicated component was omitted in the reaction. HDMEC were counterstained for VE cadherin (red) and nuclei (DAPI, blue). Scale bar: 10 µm. Formation of VEGFR2/Paladin complex in vivo. Immunoprecipitation (IP) of VEGFR2 from lysate of wild-type adult mouse lung retrieved 2 min after tail vein-injection of VEGF-A (0.25 µg/g body weight) and/or peroxyvanadate (PV) (50 µmol/g body weight) or PBS, and immunoblotting for Paladin and VEGFR2. IP with isotype control IgG as negative control (IgG con). Each lane represents lysate from one mouse lung. Total lysate samples indicate levels of total and phosphorylated VEGFR2 and Erk and Paladin after above treatment. Beta-catenin serves as loading control. Formation of VEGFR2/Paladin complex in vitro. Immunoprecipitation (IP) of VEGFR2 from untransfected HDMEC stimulated with 50 ng/ml VEGF-A alone or in combination with phosphatase inhibitor 100 µM peroxyvanadate (PV) for indicated time points, and immunoblotting for Paladin and VEGFR2. IP with isotype control IgG as negative control (IgG con). Total lysate samples indicate level of total and phosphorylated VEGFR2 and Paladin after above treatment. Beta2-microglobulin serves as loading control. Source data are available online for this figure. Download figure Download PowerPoint Figure 1. Paladin is a lipid phosphatase Lipid phosphatase activity of Paladin, wild-type and phosphatase-dead C/S mutant, toward PI(4,5)P2 and PI(3,4,5)P3 substrates. Positive control; wild-type phosphatase and tensin homolog (PTEN); negative control; lipid phosphatase-dead C124S PTEN. Mean ± SEM, Paired t-test, n = 3 biological replicates. Representative confocal image of HDMEC stained for Paladin (green), VE Cadherin (VEC, red), and nuclei (DAPI, blue), scale bar: 10 μm. White boxes in the upper image are showed at higher magnification below, scale bar: 3 µm. HDMEC stained for VEGFR2 (green), Paladin (red), and nuclei (DAPI, blue) and stimulated with 50 ng/ml VEGF-A for 0, 2, or 10 min. Scale bar: 10 µm. Quantification of VEGFR2-Paladin co-staining before and after VEGF-A stimulation as shown in (C). Mean ± SEM, one-way ANOVA. n = 3 biological replicates. HDMEC analysed using Proximity Ligand Assay (PLA) for Paladin and VEGFR2. Green dots indicate complex formation, VE cadherin (red), and nuclei (DAPI, blue). Cells stimulated with 50 ng/ml VEGF-A for 0, 2, or 10 min. Scale bar: 10 µm. Quantification of (E), the number of PLA Paladin-VEGFR2 complexes per cell at 0, 2, and 10 min after VEGF-A stimulation (top). Quantification of (E), average distance for the PLA complexes to the nearest VE cadherin positive junction at 0, 2, and 10 min after VEGF-A stimulation (bottom). Mean ± SEM, one-way ANOVA. n = 3 biological replicates. HDMEC stained for Paladin (green) and EEA1 (red), colocalization in yellow, and nuclei (DAPI, blue) after VEGF-A stimulation for 0, 2, or 10 min, a representative image from a single confocal plane is shown, scale bar: 10 µm. White boxes in the upper image are showed at higher magnification below, scale bar 3 µm. See Appendix Fig S1 for siRNA PALD1 knockdown controls. Quantification of (G), EEA1/Paladin double-positive particles per field of view at 0, 2, and 10 min after VEGF-A stimulation. Mean ± SEM, one-way ANOVA. n = 3 biological replicates. Data information: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Download figure Download PowerPoint Paladin is preferentially expressed in endothelial cells during development (Wallgard et al, 2012). Accordingly, we used immunostaining to evaluate the subcellular localization of Paladin in primary human dermal microvascular endothelial cells (HDMEC). The analysis revealed a vesicular staining pattern of Paladin with enrichment in the perinuclear region overlapping with Golgi staining, but not with the plasma membrane identified by immunostaining of the junctional protein vascular endothelial (VE) cadherin (Figs 1B and EV1F). However, we observed Paladin co-localization with VEGFR2-positive vesicles which was increased after VEGF-A stimulation (Fig 1C and D). Further, Proximity Ligation Assay (PLA) was used to assess a possible interaction between Paladin and VEGFR2 over time after VEGF-A stimulation. A low level of proximity between Paladin and VEGFR2 existed in the basal state that rapidly increased after 2 min of VEGF-A stimulation and was still maintained, but at a lower level, at 10 min (Figs 1E and F, and EV1G). The VEGF-A-induced complexes appeared close to the cell border, compared with basal complexes (Fig 1E and F). VEGFR2 immunoprecipitation confirmed complex formation with Paladin in vitro (in primary endothelial cells) and in vivo (in mouse), but the interaction required blocking of dephosphorylation by peroxyvanadate treatment (Fig EV1H and I). Given the early induction of complex formation between Paladin and VEGFR2 after VEGF-A treatment, we explored the relationship between Paladin and the early endosome antigen 1 (EEA1). Paladin decorated microdomains of EEA1+ vesicles and the number of vesicles positive for both EEA1 and Paladin increased after VEGF-A stimulation (Fig 1G and H, and Appendix Fig S1). Taken together, Paladin catalyses PI(4,5)P2 dephosphorylation and is present in endosomal vesicles while quickly depleting towards the cell periphery. Paladin appears in close proximity to VEGFR2 in response to VEGF-A stimulation. Paladin regulates VEGFR2 internalization and early endosomal trafficking To test whether Paladin affects VEGFR2 trafficking and signalling, the effect of siRNA-mediated knockdown of PALD1 in HDMEC was analysed. PALD1 siRNA treatment resulted in a marked, 35–51% increase of the total basal VEGFR2 pool (Figs 2A and C, and EV2A). However, the receptor was degraded similarly over time after VEGF-A stimulation when comparing PALD1 siRNA and control-treated cells (Figs 2A and EV2B). To study the effect of the presence and absence of Paladin on the trafficking of surface VEGFR2, endothelial cells, in which PALD1 expression had been silenced or not, were treated with VEGF-A for different time periods. Cell surface biotinylation after VEGF-A stimulation was used to pull down VEGFR2 by streptavidin beads, separating the cell surface-localized VEGFR2 pool from the internal pool. When normalized to total VEGFR2 levels, the amount of VEGFR2 at the cell surface in control and PALD1 siRNA-treated cells was not significantly different after VEGF-A treatment (Figs 2A and B, and EV2C). In a parallel analysis, we evaluated the size of the internalized VEGFR2 pool over time, by cell surface biotinylation prior to VEGF-A stimulation and subsequent stripping of remaining cell surface biotin, allowing the pull down of only protected, internalized proteins. After 15-min treatment with VEGF-A and normalization to total VEGFR2, the internalized VEGFR2 pool in PALD1-silenced endothelial cells was almost twice that of the control culture (Figs 2C and D, and EV2D). This suggests that Paladin controls the rate of VEGFR2 internalization at the early time points after VEGF-A stimulation. Figure 2. Paladin regulates VEGFR2 internalization and endosomal trafficking Cell surface VEGFR2 levels detected by cell surface biotinylation, using thiol-cleavable sulfo-NHS-SS-biotin, of HDMEC transfected with PALD1 siRNA (#1 and #2) or non-targeting control ("c") siRNA, followed by VEGF-A stimulation (50 ng/ml) for indicated time periods. Total lysates (input) and streptavidin (SA) pull down, immunoblotted for VEGFR2, Paladin, and actin. 'No biotin ctrl', cells not treated with sulfo-NHS-SS-biotin. Quantification of data in (A). VEGFR2 surface levels (data pooled for the indicated time points) normalized to total VEGFR2 levels and compared between control and siRNA-treated HDMEC. n = 4 for each time point, biological replicates, Mean ± SEM. Internalized pool of VEGFR2 after VEGF-A treatment (50 ng/ml) of non-transfected HDMEC ("NT") or HDMEC transfected with PALD1 siRNA (#1 and #2) or non-targeting control siRNA ("c"). Cell surface biotinylation was performed prior to VEGF-A stimulation and at indicated time points, remaining cell surface biotin was stripped and the internalized pool of VEGFR2 was collected by SA pull down. Immunoblotting of the total lysate (input) and SA pull down fraction for VEGFR2, Paladin, and actin. Quantification of data in (C). Data were normalized to total VEGFR2 levels in the lysate after subtraction of signals in biotinylated and stripped samples. Mean ± SEM, unpaired t-test for indicated time points, normalized to control siRNA sample. n = 3 for each time point, biological replicates. Analysis of EEA1 and VEGFR2 vesicles following PALD1 knockdown. Representative images of VEGFR2 (green)/EEA1 (red) double-positive (yellow) vesicles in negative control siRNA, and PALD1 KD#2 siRNA-silenced HDMEC at 0, 2, and 10 min of VEGF-A stimulation (50 ng/ml). DAPI in blue, scale bar: 10 µm. Inset shows only EEA1 channel, scale bar: 10 µm. Quantification of (E), number of EEA1 positive (top) or VEGFR2-EEA1 double-positive vesicles (bottom) per field of view. Mean ± SEM, two-way ANOVA, n = 3 biological replicates. HDMEC stained for PI(4,5)P2 (cyan), VE cadherin (red), and Paladin (yellow in inset) following treatment using negative control or PALD1 (KD#2) siRNA. VEGF-A stimulation for 0, 2, or 10 min (50 ng/ml). DAPI in blue. Scale bar: 10 µm. Quantification of (G), Total PI(4,5)P2 signal (top), or intracellular PI(4,5)P2 not overlapping with VE cadherin (bottom). Mean ± SEM, two-way ANOVA, n = 3 biological replicates. Data information: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Source d
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