Triggering MSR1 promotes JNK‐mediated inflammation in IL‐4‐activated macrophages
2019; Springer Nature; Volume: 38; Issue: 11 Linguagem: Inglês
10.15252/embj.2018100299
ISSN1460-2075
AutoresManman Guo, Anetta Härtlová, Marek Gierliński, Alan R. Prescott, Josep Castellví, Javier Hernández‐Losa, Sine Kragh Petersen, Ulf Alexander Wenzel, Brian D. Dill, Christoph H. Emmerich, Santiago Ramón y Cajal, David G. Russell, Matthias Trost,
Tópico(s)Immunotherapy and Immune Responses
ResumoArticle26 April 2019Open Access Transparent process Triggering MSR1 promotes JNK-mediated inflammation in IL-4-activated macrophages Manman Guo Manman Guo orcid.org/0000-0002-7928-8902 MRC Protein Phosphorylation and Ubiquitylation Unit, University of Dundee, Dundee, UK Search for more papers by this author Anetta Härtlova Corresponding Author Anetta Härtlova [email protected] orcid.org/0000-0002-8152-4361 MRC Protein Phosphorylation and Ubiquitylation Unit, University of Dundee, Dundee, UK Institute for Cell and Molecular Biosciences, Newcastle University, Newcastle upon Tyne, UK Wallenberg Centre for Molecular and Translational Medicine, University of Gothenburg, Gothenburg, Sweden Department of Microbiology and Immunology, Institute for Biomedicine, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden Search for more papers by this author Marek Gierliński Marek Gierliński Data Analysis Group, School of Life Sciences, University of Dundee, Dundee, UK Search for more papers by this author Alan Prescott Alan Prescott Division of Cell Signalling and Immunology, School of Life Sciences, University of Dundee, Dundee, UK Search for more papers by this author Josep Castellvi Josep Castellvi Department of Pathology, Hospital Universitario Vall d'Hebron, Barcelona, Spain Search for more papers by this author Javier Hernandez Losa Javier Hernandez Losa Department of Pathology, Hospital Universitario Vall d'Hebron, Barcelona, Spain Spanish Biomedical Research Network Centre in Oncology (CIBERONC), Barcelona, Spain Search for more papers by this author Sine K Petersen Sine K Petersen Wallenberg Centre for Molecular and Translational Medicine, University of Gothenburg, Gothenburg, Sweden Department of Microbiology and Immunology, Institute for Biomedicine, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden Search for more papers by this author Ulf A Wenzel Ulf A Wenzel Wallenberg Centre for Molecular and Translational Medicine, University of Gothenburg, Gothenburg, Sweden Department of Microbiology and Immunology, Institute for Biomedicine, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden Search for more papers by this author Brian D Dill Brian D Dill MRC Protein Phosphorylation and Ubiquitylation Unit, University of Dundee, Dundee, UK Search for more papers by this author Christoph H Emmerich Christoph H Emmerich MRC Protein Phosphorylation and Ubiquitylation Unit, University of Dundee, Dundee, UK Search for more papers by this author Santiago Ramon Y Cajal Santiago Ramon Y Cajal Department of Pathology, Hospital Universitario Vall d'Hebron, Barcelona, Spain Spanish Biomedical Research Network Centre in Oncology (CIBERONC), Barcelona, Spain Search for more papers by this author David G Russell David G Russell Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, NY, USA Search for more papers by this author Matthias Trost Corresponding Author Matthias Trost [email protected] orcid.org/0000-0002-5732-700X MRC Protein Phosphorylation and Ubiquitylation Unit, University of Dundee, Dundee, UK Institute for Cell and Molecular Biosciences, Newcastle University, Newcastle upon Tyne, UK Search for more papers by this author Manman Guo Manman Guo orcid.org/0000-0002-7928-8902 MRC Protein Phosphorylation and Ubiquitylation Unit, University of Dundee, Dundee, UK Search for more papers by this author Anetta Härtlova Corresponding Author Anetta Härtlova [email protected] orcid.org/0000-0002-8152-4361 MRC Protein Phosphorylation and Ubiquitylation Unit, University of Dundee, Dundee, UK Institute for Cell and Molecular Biosciences, Newcastle University, Newcastle upon Tyne, UK Wallenberg Centre for Molecular and Translational Medicine, University of Gothenburg, Gothenburg, Sweden Department of Microbiology and Immunology, Institute for Biomedicine, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden Search for more papers by this author Marek Gierliński Marek Gierliński Data Analysis Group, School of Life Sciences, University of Dundee, Dundee, UK Search for more papers by this author Alan Prescott Alan Prescott Division of Cell Signalling and Immunology, School of Life Sciences, University of Dundee, Dundee, UK Search for more papers by this author Josep Castellvi Josep Castellvi Department of Pathology, Hospital Universitario Vall d'Hebron, Barcelona, Spain Search for more papers by this author Javier Hernandez Losa Javier Hernandez Losa Department of Pathology, Hospital Universitario Vall d'Hebron, Barcelona, Spain Spanish Biomedical Research Network Centre in Oncology (CIBERONC), Barcelona, Spain Search for more papers by this author Sine K Petersen Sine K Petersen Wallenberg Centre for Molecular and Translational Medicine, University of Gothenburg, Gothenburg, Sweden Department of Microbiology and Immunology, Institute for Biomedicine, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden Search for more papers by this author Ulf A Wenzel Ulf A Wenzel Wallenberg Centre for Molecular and Translational Medicine, University of Gothenburg, Gothenburg, Sweden Department of Microbiology and Immunology, Institute for Biomedicine, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden Search for more papers by this author Brian D Dill Brian D Dill MRC Protein Phosphorylation and Ubiquitylation Unit, University of Dundee, Dundee, UK Search for more papers by this author Christoph H Emmerich Christoph H Emmerich MRC Protein Phosphorylation and Ubiquitylation Unit, University of Dundee, Dundee, UK Search for more papers by this author Santiago Ramon Y Cajal Santiago Ramon Y Cajal Department of Pathology, Hospital Universitario Vall d'Hebron, Barcelona, Spain Spanish Biomedical Research Network Centre in Oncology (CIBERONC), Barcelona, Spain Search for more papers by this author David G Russell David G Russell Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, NY, USA Search for more papers by this author Matthias Trost Corresponding Author Matthias Trost [email protected] orcid.org/0000-0002-5732-700X MRC Protein Phosphorylation and Ubiquitylation Unit, University of Dundee, Dundee, UK Institute for Cell and Molecular Biosciences, Newcastle University, Newcastle upon Tyne, UK Search for more papers by this author Author Information Manman Guo1,‡,‡, Anetta Härtlova *,1,2,3,4,‡, Marek Gierliński5, Alan Prescott6, Josep Castellvi7, Javier Hernandez Losa7,8, Sine K Petersen3,4, Ulf A Wenzel3,4, Brian D Dill1, Christoph H Emmerich1, Santiago Ramon Y Cajal7,8, David G Russell9 and Matthias Trost *,1,2 1MRC Protein Phosphorylation and Ubiquitylation Unit, University of Dundee, Dundee, UK 2Institute for Cell and Molecular Biosciences, Newcastle University, Newcastle upon Tyne, UK 3Wallenberg Centre for Molecular and Translational Medicine, University of Gothenburg, Gothenburg, Sweden 4Department of Microbiology and Immunology, Institute for Biomedicine, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden 5Data Analysis Group, School of Life Sciences, University of Dundee, Dundee, UK 6Division of Cell Signalling and Immunology, School of Life Sciences, University of Dundee, Dundee, UK 7Department of Pathology, Hospital Universitario Vall d'Hebron, Barcelona, Spain 8Spanish Biomedical Research Network Centre in Oncology (CIBERONC), Barcelona, Spain 9Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, NY, USA ‡These authors contributed equally to this work ‡Present address: Botnar Research Centre, Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, University of Oxford, Oxford, UK *Corresponding author. Tel: +46 31 786 6241; E-mail: [email protected] *Corresponding author. Tel: +44 191 2087009; E-mail: [email protected] The EMBO Journal (2019)38:e100299https://doi.org/10.15252/embj.2018100299 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 Alternatively activated M2 macrophages play an important role in maintenance of tissue homeostasis by scavenging dead cells, cell debris and lipoprotein aggregates via phagocytosis. Using proteomics, we investigated how alternative activation, driven by IL-4, modulated the phagosomal proteome to control macrophage function. Our data indicate that alternative activation enhances homeostatic functions such as proteolysis, lipolysis and nutrient transport. Intriguingly, we identified the enhanced recruitment of the TAK1/MKK7/JNK signalling complex to phagosomes of IL-4-activated macrophages. The recruitment of this signalling complex was mediated through K63 polyubiquitylation of the macrophage scavenger receptor 1 (MSR1). Triggering of MSR1 in IL-4-activated macrophages leads to enhanced JNK activation, thereby promoting a phenotypic switch from an anti-inflammatory to a pro-inflammatory state, which was abolished upon MSR1 deletion or JNK inhibition. Moreover, MSR1 K63 polyubiquitylation correlated with the activation of JNK signalling in ovarian cancer tissue from human patients, suggesting that it may be relevant for macrophage phenotypic shift in vivo. Altogether, we identified that MSR1 signals through JNK via K63 polyubiquitylation and provides evidence for the receptor's involvement in macrophage polarization. Synopsis Macrophage scavenger receptor MSR1 is a key phagocytic receptor for the uptake of lipids and cell debris in macrophages. In IL-4 activated macrophages MSR1 becomes ubiquitylated, recruits the Tak1/MKK7 kinase complex and signals directly through JNK, which induces pro-inflammatory cytokine production. Proteomics analysis of phagosomes indicate that alternative activation by IL-4 enhances phagosomal homeostatic functions in macrophages. Triggering of MSR1 in IL-4 activated macrophages leads to its ubiquitylation, which recruits the TAK1/MKK7/JNK kinase complex. This leads to pro-inflammatory signalling, inducing a phenotypic switch of the macrophages. MSR1 ubiquitylation and enhanced JNK signalling is present in human tumour associated macrophages. Introduction Phagocytosis is a highly conserved process essential for host defence and tissue remodelling. It involves the recognition of particles by a variety of cell surface receptors, followed by cargo processing and delivery to lysosomes via phagosome–lysosome fusion, process known as phagosome maturation. This leads to gradual acidification of the phagosomal lumen and acquisition of digestive enzymes required for the degradation of phagosomal cargo. Therefore, phagocytosis is not only responsible for elimination of bacterial pathogens, but also responsible for the clearance of apoptotic cells, cell debris and senescence cells and orchestrates the subsequent immune response (Rothlin et al, 2007; Murray & Wynn, 2011; Lemke, 2013, 2017). Central to this process is phagosome function. If uncontrolled, the inappropriate clearance of apoptotic bodies can give rise to autoimmune diseases, atherosclerosis and cancer, while failure to ingest or kill pathogens can result in deadly infections (Johnson & Newby, 2009; Nagata et al, 2010; Colegio et al, 2014; Arandjelovic & Ravichandran, 2015). Therefore, it is of great importance to understand which signalling pathways regulate phagocytosis and phagosomal maturation. It has recently been acknowledged that the phagosome serves as a signalling platform and interacts with innate immune signalling (Stuart et al, 2007; Martinez et al, 2011, 2015; Kagan, 2012; Heckmann et al, 2017). However, whether phagosome-associated cell signalling is independent of its role in cargo degradation has not been well understood. Supporting this notion, recent proteomic studies demonstrated that phagosomes are dynamic organelles that change their composition and function in response to infection or inflammation (Trost et al, 2009; Boulais et al, 2010; Dill et al, 2015; Guo et al, 2015; Naujoks et al, 2016; Hartlova et al, 2018). While the regulation of phagosomal maturation in so-called M1 inflammatory macrophages has been extensively studied, the mechanisms facilitating phagosomal maturation in macrophages involved in tissue repair remain poorly understood (Balce et al, 2011). Th2-derived cytokines, such as interleukin-4 (IL-4) and interleukin-13 (IL-13), induce a strong anti-inflammatory macrophage phenotype, also called alternative-activated macrophages (M2). M2 macrophages and tissue-resident macrophages, which often resemble an M2-like state, clear cell debris and dead cells through phagocytosis. They are therefore essential for maintenance and tissue homeostasis. M2 alternatively activated macrophages (AAMs) inhibit inflammatory responses and promote angiogenesis and tissue repair by synthetizing mediators required for collagen deposition, which is important for wound healing (Gordon & Martinez, 2010). It has been shown that IL-4 enhanced phagosomal protein degradation (Balce et al, 2011). Whether IL-4 regulates other phagosomal functions, and through which molecular mechanisms, remains unclear. Here, we investigated the phagosome proteome of IL-4-activated macrophages. In line with the known role in homeostasis, the phagosome of AAMs has increased abilities to degrade incoming apoptotic cells and transport the resulting nutrients. Furthermore, we demonstrate that the TAK1/MKK7/JNK signalling complex showed an enhanced association with the phagosome upon IL-4 macrophage activation. The assembly of the signalling complex is mediated through K63 polyubiquitylation. By combining K63-polyubiquitylation enrichment and mass spectrometry approaches, we identified macrophage scavenger receptor 1 (MSR1) as the upstream receptor that promotes the recruitment of the TAK1/MKK7/JNK signalling complex to the phagosome. Triggering MSR1 induces JNK activation in M2 macrophages. This MSR1/JNK signalling pathway activation leads to a M2/M1 macrophage phenotypic switch that is abolished in macrophages lacking MSR1. We demonstrate that MSR1 is K63-ubiquitylated and signals through JNK in human patient ovarian cancer, thus suggesting a potential role in human cancer. Results Alternative activation regulates phagosomal proteolysis and lipolysis To determine the impact of IL-4 on phagocytosis and phagosomal functions, we examined the rate of phagocytosis and phagosomal functions in IL-4 AAMs (M2) and resting macrophages (M0). We found that both IL-4- and IL-13-activated M2 macrophages have enhanced uptake of apoptotic cells, while uptake of necrotic cells was comparable to M0 resting macrophages (Fig 1A). To determine whether the enhanced uptake was because of the negative charge of apoptotic cells, we compared the uptake of fluorescently labelled carboxylated negatively charged and positively charged amino silica microspheres in M2 and M0 macrophages. The analysis revealed an increased uptake of negatively charged microspheres in M2 macrophages, which are taken up through scavenger receptors (Tanaka et al, 1996; Platt et al, 1999; Stephen et al, 2010), while the engulfment of positively charged microspheres was similar to M0 macrophages (Fig 1B). This indicates that, due to their similar uptake behaviour, carboxylated microspheres may serve as a surrogate for apoptotic cells (Kiss et al, 2006). Next, we analysed the functional parameters of the phagosomal lumen. In these assays, we use fluorescent probes that allow the measurement of proteolysis (a readout for maturation), acidification and lipolysis in real time (Yates et al, 2005; Podinovskaia et al, 2013). Consistent with the previous reports, we observed enhanced proteolytic activity in phagosomes of M2 macrophages (Balce et al, 2011). Furthermore, we found that IL-4 increased phagosomal lipid degradation and facilitated phagosomal acidification (Fig 1C–E) indicating that IL-4 activation promotes phagosome maturation and the ability of macrophages to degrade lipid-rich particles through phagosomes. Altogether, these data indicate that that alternative activation increases the ability to take up and degrade apoptotic cells and other lipid-rich particles by increasing the degradative potential of phagolysosomes from M2 (IL4) MΦs. Figure 1. Alternative activation affects phagosomal proteolysis and lipolysis as well as phagocytosis of negatively charged particle A. Phagocytosis assay of apoptotic or necrotic GFP-expressing RAW264.7 cells in M2 (IL-4 or IL13) and untreated M0 BMDMs. B. Phagocytosis of fluorescent negatively charged carboxylated and positively charged amino microspheres (B) in primary M2 (IL-4) and M0 macrophages. Cytochalasin D (Cyto) (6 μM) was used as an inhibitor of phagocytosis, 1 h before phagocytosis. C–E. Real-time fluorescence assays for intraphagosomal proteolysis (C), acidification (D) and lipolysis (E) show substantially increased proteolysis, acidification and lipolysis in the phagosomes of M2 (IL-4) macrophages. The kinetics of proteolysis, acidification and lipolysis of phagocytosed beads were plotted as a ratio of substrate fluorescence to calibration fluorescence. Beads were added to macrophages at 0 min. (E) is a representative of three independent experiments. Leupeptin (100 nM) and bafilomycin (100 nM) treatments serve as negative controls in (C) and (D), respectively. Data information: The statistical significance of data is denoted on graphs by asterisks where *P < 0.05, ***P < 0.001 or ns = not significant. (A, B) Data are shown as means of relative fluorescence units (RFU) ± standard error of the mean (SEM), Student's t-test used. (C, D) Shaded area represents SEM. (A–D) Three replicates were used. Download figure Download PowerPoint Quantitative proteomic analysis of phagosomes from IL-4 alternatively activated macrophages To obtain further molecular details about the changes on phagosomes of M2 macrophages, we isolated highly pure phagosomes from M2 and M0 macrophages by floatation on a sucrose gradient using carboxylated microspheres and analysed their proteomes by quantitative LC-MS/MS (Fig 2A; Appendix Figs S1A–C and S2A; Desjardins et al, 1994; Peltier et al, 2017; Trost et al, 2009). Comparative analysis led to the identification of 20,614 distinct peptides corresponding to 1,948 unique proteins across three independent replicates at a false-discovery rate (FDR) of < 1%, of which 1,766 proteins were quantified in at least two of the three biological replicates. IL-4 activation induced strong changes to the phagosome proteome with 121 proteins significantly up-regulated and 62 proteins significantly down-regulated (twofold change, P < 0.05; Fig 2B; Dataset EV1), some of which we validated by Western blot analysis (Appendix Fig S2B). Consistent with the above observations, a subset of proteins involved in lipid metabolism (Lpl lipoprotein lipase, ABHD12 lipase and phospholipase D1), acidification (v-ATPase complex) and lysosomal enzymes including cathepsins L1 and D were highly enriched on the phagosome of M2 macrophages (Fig 2C; Dataset EV1). Moreover, GO term (Fig 2D) and protein network analysis (Appendix Fig S2C) further showed that IL-4 alternative activation also increased phagosome abundance of scavenger receptors such as MARCO, CD36, Colec12 and MSR1 required for clearance of dead cells while Toll-like receptors (TLRs) involved in inflammatory response were reduced (Fig 2D, Appendix Fig S2C). Furthermore, M2 phagosomes showed higher phosphatidylinositol-binding proteins, suggesting changes to the phagosome membrane lipid content (Fig 2D). Consistent with a previous report, superoxide anion generation including the NADPH oxidase complex proteins NCF1 (p47-phox), Cyba (p22-phox), Cybb (gp91-phox) and superoxide dismutase SOD1 was strongly down-regulated in phagosomes from M2 macrophages (Fig 2D, Appendix Fig S2C; Balce et al, 2011). Interestingly, M2 macrophages also enriched a large number of specific carbohydrate-binding proteins such as lectins, while carbohydrate hydrolases were significantly reduced. This indicates a conservation of phagocytosed glycans, potentially for antigen presentation via MHC class II molecules (Avci et al, 2013). Taken together, these results indicate that the phagosome of M2 macrophages has mainly a homeostatic role with its increased ability to hydrolyse proteins and lipids of incoming cargo. Figure 2. Experimental workflow and the phagosome proteome of M2 (IL-4) macrophages Workflow of the phagosome proteomic experiment. Volcano plot of the phagosome proteome data. 1,766 proteins were quantified of which 121 proteins were significantly up-regulated and 62 proteins were down-regulated in M2 (IL-4) macrophages. Selected proteins are indicated. Heatmap of proteomic data shows high reproducibility between biological replicates. Selected proteins are highlighted. Selected Gene Ontology (GO) terms of biological processes significantly up-regulated and down-regulated on phagosomes of M2 (IL-4) macrophages. Selected proteins of these GO-terms are highlighted. Error bars represent standard deviations from three biological replicates. Download figure Download PowerPoint TAK1/MKK7/JNK is recruited to the phagosome of M2 macrophages via K63 polyubiquitylation Interestingly, anti-inflammatory IL-4 activation also led to an increased phagosome abundance of the pro-inflammatory MAP kinase signalling complex around TAK1 (Map3k7, 2.1-fold) and MKK7 (Map2k7, 3.1-fold; Dataset EV1; Appendix Fig S2C) indicating cross-regulation between anti- and pro-inflammatory pathways. Given the increased abundance of these pro-inflammatory kinases was surprising on phagosomes of IL-4-stimulated macrophages, we next investigated how this complex was translocated to the phagosome. Immunoblot analyses of total cell lysates and phagosomal fractions revealed significant enrichment of TAK1 and MKK7 on phagosomes of M2 macrophages compared to resting M0 macrophages (Fig 3A and B). Activated TAK1 can phosphorylate two MAPK kinases, MKK4 and MKK7, which both can activate JNK. While MKK4 can activate p38 and JNK MAPK signalling pathways, MKK7 selectively activates JNK (Tournier et al, 2001). Noteworthy, our mass spectrometry data revealed that only MKK7 was enriched on phagosomes upon IL-4 alternative activation. Consistent with our LC-MS/MS data, MKK4 was not detected on phagosomes of M2 macrophages by immunoblot analysis indicating that MKK7 alone was important in this phagosome signalling pathway. Further immunoblot analysis also confirmed enrichment of JNK of M2 macrophage phagosomes (Appendix Fig S4C). Figure 3. TAK1/MKK7/JNK complex is recruited to phagosomes of M2 (IL-4) macrophages in a K63-polyubiquitylation-dependent manner Immunoblot (IB) analysis showing the recruitment of TAK1, MKK7, JNK and TAB1/TAB2 to the phagosome in M2 (IL-4) macrophages, while MKK4 is not recruited. Rab7a, a phagosomal marker, was used as a loading control for phagosomes, and vimentin was used as a loading control for total cell lysates. The white line in the JNK blot shows other bands were cut out. Full blot can be seen in Appendix Figure S4. Quantitation of three independent IB experiments by ImageJ of non-saturated blots for TAK1, MKK7, TAB1 and TAB2 expression on phagosomes of resting M0 macrophages and M2 (IL-4) macrophages. Error bars represent SEM. *P < 0.01, **P < 0.001 (Student's t-test). Three replicates were used. IB showing enrichment of K63-polyubiquitylated proteins on the phagosome of M2 (IL-4) macrophages compared to M0 macrophages. Treatment with the UBC13 inhibitor NSC697923 reduces recruitment of TAB1, TAB2, TAK1 and MKK7 to the phagosome of M2 (IL-4) macrophages, indicating a K63-polyubiquitylation-dependent translocation for these proteins. Data information: (C) and (D) are representatives of three and two independent experiments, respectively. Download figure Download PowerPoint Previous data have shown that upon pro-inflammatory interleukin-1 receptor or Toll-like receptor (TLR) activation, the TAK1/MKK7/JNK complex binds to the TAB1/TAB2 protein complex, which in turn is recruited to K63-polyubiquitin chains (Xia et al, 2009; Emmerich et al, 2013). We next tested whether TAB1/TAB2 is recruited to the phagosome of M2 macrophages. Indeed, immunoblot analysis demonstrated enrichment of TAB1/TAB2 on phagosomes of M2 macrophages compared to M0. To further validate the recruitment of the protein complex to phagosomes, we performed confocal fluorescence microscopy and showed vesicular distribution in the cytoplasm with enhanced recruitment of TAB1, TAB2 and MKK7 to the M2 macrophage phagosome compared to M0 macrophages (Appendix Fig S3A and B). As it is well-established that TAK1 binds via TAB1/2/3 to free and protein-anchored K63-polyubiquitin chains in inflammatory innate immune responses (Xia et al, 2009; Emmerich et al, 2013), we tested whether phagosomes from M2 macrophages contain K63-polyubiquitylated proteins independent of inflammatory stimuli. Immunoblot analysis of phagosome extracts probed with anti-K63-polyubiquitin antibodies revealed that phagosomes contain a large amount of K63-polyubiquitylated proteins compared to the total cell lysate, which was even more increased by alternative activation (Fig 3C). To determine whether recruitment of TAB1, TAB2, TAK1 and MKK7 to the M2 phagosome was indeed K63-polyubiquitylation-dependent, we treated cells with NSC697923, a pharmacological inhibitor of the K63-specific E2-conjugating enzyme UBC13-UEV1A (Pulvino et al, 2012) and probed isolated phagosomes for K63 polyubiquitylation. As shown in Fig 3D, recruitment of the protein complex was virtually abolished under these conditions, which we also confirmed for MKK7 by immunofluorescence microscopy (Appendix Fig S4A and B). These data indicate that IL-4 activation of macrophages promotes K63 polyubiquitylation, which recruits the TAK1/MKK7/JNK complex to the phagosome. Macrophage scavenger receptor 1 is K63-polyubiquitylated and interacts with TAK1/MKK7/JNK on the phagosome of M2 macrophages In order to identify the K63-polyubiquitylated proteins, which bind the TAK1/MKK7/JNK complex on the phagosome of M2 macrophages, we enriched polyubiquitylated phagosomal proteins from M2 macrophages using tandem ubiquitin-binding entities (TUBEs) of a repeat of the Npl4 Zinc Finger (NZF) domain of TAB2 tagged with Halo (termed here Halo-TAB2). These constructs have been shown to bind specifically to K63-polyubiquitin chains (Fig 4A; Hjerpe et al, 2009; Emmerich et al, 2013; Heap et al, 2017). Quantitative mass spectrometric analysis of these pull-downs identified 538 phagosomal proteins that were reproducibly captured by Halo-TAB2 compared to mutant, ubiquitin-non-binding Halo (T674A/F675A) TAB2 control beads (based on a twofold, P < 0.05 cut-off; Dataset EV2). Moreover, we identified 62 novel direct ubiquitylation (−GlyGly) sites on 33 different phagosomal proteins. Quantitation of the data revealed that the Gly-Gly peptide derived from K63-linked polyubiquitin was by far the most abundant, proving that we achieved good enrichment. However, we also identified peptides for K11-, K48-, linear/M1-, K29-, K6-, K27- and K33 (in order of decreasing abundances)-linked polyubiquitin, suggesting that there are either mixed chains or K63-polyubiquitylated proteins might also be modified with other polyubiquitin chains. Figure 4. Phagosomal MSR1 is K63-polyubiquitylated and interacts with Tab1/Tab2/Tak1/MKK7 Workflow for TUBE pull-down of K63-polyubiquitylated proteins from phagosomal extracts. Selected ubiquitylated phagosomal proteins identified by TUBE-MS approach. Sequences alignment of N-terminal region of murine and human MSR1 shows high-sequence identity and conserved ubiquitylated lysine. The arrow points out the ubiquitylated lysine residue. Tab2-TUBE pull-downs from phagosome extracts of M2 (IL-4) macrophages treated with the K63-specific deubiquitylase (DUB) AMSH-LP or the unspecific DUB USP2. MSR1 immunoprecipitation from M2 (IL-4) macrophage phagosomes shows that TAB1, TAB2, TAK1 and MKK7 bind to polyubiquitylated MSR1. TAK1 shows a specific pattern of post-translational modifications indicative of its activation. Data information: (D) and (E) are representative of two independent experiments. Download figure Download PowerPoint Other identified ubiquitylated proteins included many known phagolysosomal proteins such as the large neutral amino acid transporter SLC43A2/LAT4 (K283, K293, K402, K557), the cholesterol transporter ABCG1 (K55), Fc- and B-cell receptor adaptor LAT2 (K39, K84), LYN kinase (K20) and the TLR chaperone UNC93B1 (K197, K582; Fig 4B; Appendix Table S1). Interestingly, one of the most abundant Gly-Gly-modified peptides was a peptide containing lysine 27 (K27) of macrophage scavenger receptor 1/scavenger receptor A (MSR1/SR-A; CD204). This site is highly conserved between human and mouse (Fig 4C). MSR1 is a multifunctional phagocytic receptor, highly expressed in macrophages, involved in uptake of apoptotic cells and modified lipoproteins (Kelley et al, 2014). In addition to its scavenging function, MSR1 has been implicated in the innate immune response to bacteria (Platt & Gordon, 2001). Our MS and immunoblot data showed an increase in MSR1 on phagosomes from M2 macrophages compared to M0 macrophages (Dataset EV1; Appendix Fig S5A and C), while both total cell and cell surface expression levels of MSR1 were unchanged between the two conditions (Append
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