TPL‐2 kinase induces phagosome acidification to promote macrophage killing of bacteria
2021; Springer Nature; Volume: 40; Issue: 10 Linguagem: Inglês
10.15252/embj.2020106188
ISSN1460-2075
AutoresFelix Breyer, Anetta Härtlová, Teresa L. M. Thurston, Helen R. Flynn, Probir Chakravarty, Julia Janzen, Julien Peltier, Tiaan Heunis, Ambrosius P. Snijders, Matthias Trost, Steven C. Ley,
Tópico(s)Immune Response and Inflammation
ResumoArticle21 April 2021Open Access Transparent process TPL-2 kinase induces phagosome acidification to promote macrophage killing of bacteria Felix Breyer Felix Breyer orcid.org/0000-0002-9957-3209 The Francis Crick Institute, London, UK Search for more papers by this author Anetta Härtlova Anetta Härtlova orcid.org/0000-0002-8152-4361 Biosciences Institute, Newcastle University, Newcastle-upon-Tyne, UK Wallenberg Centre for Molecular and Translational Medicine, University of Gothenburg, Gothenburg, Sweden Search for more papers by this author Teresa Thurston Teresa Thurston orcid.org/0000-0001-6139-3723 Department of Infectious Diseases, MRC Centre for Molecular Bacteriology & Infection, Imperial College London, London, UK Search for more papers by this author Helen R Flynn Helen R Flynn orcid.org/0000-0001-7002-9130 The Francis Crick Institute, London, UK Search for more papers by this author Probir Chakravarty Probir Chakravarty orcid.org/0000-0003-1146-8824 The Francis Crick Institute, London, UK Search for more papers by this author Julia Janzen Julia Janzen The Francis Crick Institute, London, UK Search for more papers by this author Julien Peltier Julien Peltier Biosciences Institute, Newcastle University, Newcastle-upon-Tyne, UK Search for more papers by this author Tiaan Heunis Tiaan Heunis Biosciences Institute, Newcastle University, Newcastle-upon-Tyne, UK Search for more papers by this author Ambrosius P Snijders Ambrosius P Snijders The Francis Crick Institute, London, UK Search for more papers by this author Matthias Trost Matthias Trost orcid.org/0000-0002-5732-700X Biosciences Institute, Newcastle University, Newcastle-upon-Tyne, UK Search for more papers by this author Steven C Ley Corresponding Author Steven C Ley [email protected] orcid.org/0000-0001-5911-9223 The Francis Crick Institute, London, UK Department of Immunology & Inflammation, Centre for Molecular Immunology & Inflammation, Imperial College London, London, UK Search for more papers by this author Felix Breyer Felix Breyer orcid.org/0000-0002-9957-3209 The Francis Crick Institute, London, UK Search for more papers by this author Anetta Härtlova Anetta Härtlova orcid.org/0000-0002-8152-4361 Biosciences Institute, Newcastle University, Newcastle-upon-Tyne, UK Wallenberg Centre for Molecular and Translational Medicine, University of Gothenburg, Gothenburg, Sweden Search for more papers by this author Teresa Thurston Teresa Thurston orcid.org/0000-0001-6139-3723 Department of Infectious Diseases, MRC Centre for Molecular Bacteriology & Infection, Imperial College London, London, UK Search for more papers by this author Helen R Flynn Helen R Flynn orcid.org/0000-0001-7002-9130 The Francis Crick Institute, London, UK Search for more papers by this author Probir Chakravarty Probir Chakravarty orcid.org/0000-0003-1146-8824 The Francis Crick Institute, London, UK Search for more papers by this author Julia Janzen Julia Janzen The Francis Crick Institute, London, UK Search for more papers by this author Julien Peltier Julien Peltier Biosciences Institute, Newcastle University, Newcastle-upon-Tyne, UK Search for more papers by this author Tiaan Heunis Tiaan Heunis Biosciences Institute, Newcastle University, Newcastle-upon-Tyne, UK Search for more papers by this author Ambrosius P Snijders Ambrosius P Snijders The Francis Crick Institute, London, UK Search for more papers by this author Matthias Trost Matthias Trost orcid.org/0000-0002-5732-700X Biosciences Institute, Newcastle University, Newcastle-upon-Tyne, UK Search for more papers by this author Steven C Ley Corresponding Author Steven C Ley [email protected] orcid.org/0000-0001-5911-9223 The Francis Crick Institute, London, UK Department of Immunology & Inflammation, Centre for Molecular Immunology & Inflammation, Imperial College London, London, UK Search for more papers by this author Author Information Felix Breyer1, Anetta Härtlova2,3, Teresa Thurston4, Helen R Flynn1, Probir Chakravarty1, Julia Janzen1, Julien Peltier2, Tiaan Heunis2, Ambrosius P Snijders1, Matthias Trost2 and Steven C Ley *,1,5 1The Francis Crick Institute, London, UK 2Biosciences Institute, Newcastle University, Newcastle-upon-Tyne, UK 3Wallenberg Centre for Molecular and Translational Medicine, University of Gothenburg, Gothenburg, Sweden 4Department of Infectious Diseases, MRC Centre for Molecular Bacteriology & Infection, Imperial College London, London, UK 5Department of Immunology & Inflammation, Centre for Molecular Immunology & Inflammation, Imperial College London, London, UK *Corresponding author. Tel: +44 203 796 2207; E-mail: [email protected] The EMBO Journal (2021)40:e106188https://doi.org/10.15252/embj.2020106188 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 Tumour progression locus 2 (TPL-2) kinase mediates Toll-like receptor (TLR) activation of ERK1/2 and p38α MAP kinases in myeloid cells to modulate expression of key cytokines in innate immunity. This study identified a novel MAP kinase-independent regulatory function for TPL-2 in phagosome maturation, an essential process for killing of phagocytosed microbes. TPL-2 catalytic activity was demonstrated to induce phagosome acidification and proteolysis in primary mouse and human macrophages following uptake of latex beads. Quantitative proteomics revealed that blocking TPL-2 catalytic activity significantly altered the protein composition of phagosomes, particularly reducing the abundance of V-ATPase proton pump subunits. Furthermore, TPL-2 stimulated the phosphorylation of DMXL1, a regulator of V-ATPases, to induce V-ATPase assembly and phagosome acidification. Consistent with these results, TPL-2 catalytic activity was required for phagosome acidification and the efficient killing of Staphylococcus aureus and Citrobacter rodentium following phagocytic uptake by macrophages. TPL-2 therefore controls innate immune responses of macrophages to bacteria via V-ATPase induction of phagosome maturation. Synopsis TPL-2 kinase plays an important role in innate immune responses to bacteria, mediating Toll-like receptor (TLR) activation of ERK1/2 and p38α MAP kinases in myeloid cells. Here, TPL-2 is shown to also directly promote macrophage bacterial killing by MAP kinase-independent induction of phagosome maturation. TPL-2 catalytic activity is required for phagosome acidification in macrophages following uptake of latex beads. TPL-2 induces phagosome acidification independently of MAP kinase activation. TPL-2 induces phagosome acidification by promoting phosphorylation of DMXL1, a V-ATPase-associated protein. TPL-2 induces phagosome acidification and bacterial killing following internalization of Staphylococcus aureus or Citrobacter rodentium by macrophages. Introduction A key component of the innate immune response involves the killing of phagocytosed microbes, such as bacteria, by macrophages (Pauwels et al, 2017). Phagocytosis internalises bacteria into membrane-bound vacuoles in the cytoplasm called phagosomes (Flannagan et al, 2012). The nascent phagosome is innocuous, and to kill internalised bacteria, the phagosome must mature via an ordered series of membrane fusion and fission events with endosomes and ultimately with lysosomes to form the phagolysosome, a potent microbicidal organelle (Flannagan et al, 2009). During the maturation process, phagosomes become increasingly acidic, highly oxidative and enriched with proteases and hydrolases that can degrade the internalised bacteria (Flannagan et al, 2009). Phagosome acidification is mediated by V-ATPases, ATP-dependent proton pumps, which are recruited to the phagosome during maturation (Lukacs et al, 1990; Sun-Wada et al, 2009). Acidification inhibits bacterial growth (Downey et al, 1999; Ip et al, 2010), activates cathepsin proteases that degrade internalised bacteria (Yates et al, 2005) and promotes NOX2 production of reactive oxygen species (ROS) that damage bacterial proteins, lipids and nucleic acids (Savina et al, 2006). Bacterial infection also engages macrophage Toll-like receptors (TLRs) on the plasma and endosomal membranes, triggering intracellular signalling pathways that control activation of NF-κB transcription factors and each of the major mitogen-activated protein (MAP) kinases (extracellular signal-regulated kinases 1 and 2 [ERK1/2], Jun amino-terminal kinases 1 and 2 [JNK1/2] and p38α) (Arthur & Ley, 2013). Together, these stimulate the expression of multiple genes, the products of which can directly target the invading bacterium (e.g. antimicrobial peptides and NOS2) or induce recruitment of additional immune cells (e.g. cytokines and chemokines) (Smale, 2010; Kawasaki & Kawai, 2014). This inflammatory response is essential for killing of bacteria by macrophages and also the subsequent induction of the adaptive immune response (Nau et al, 2002). TLR activation of ERK1/2 and p38α in macrophages is mediated by TPL-2 (tumour progression locus 2, also known as MAP3K8), a MAP 3-kinase that directly phosphorylates and activates the MAP 2-kinases MKK1, 2, 3 and 6 (Gantke et al, 2012; Pattison et al, 2016). TPL-2 is critical for inflammatory immune responses to invading bacteria, fungi and viruses. TPL-2 forms a stoichiometric complex with NF-κB1 p105 and A20 binding inhibitor of NF-κB (ABIN)-2 in unstimulated macrophages (Lang et al, 2004). NF-κB1 p105 functions as an inhibitor of TPL-2 and TLR activation of ERK1/2, and p38α requires the liberation of TPL-2 from p105-mediated inhibition (Beinke et al, 2003, 2004). This results from p105 proteolysis by the proteasome triggered by IκB kinase (IKK)-induced p105 phosphorylation (Belich et al, 1999). ABIN-2 is also released from p105 after TLR stimulation but is not required for TPL-2 activation, and its function in innate immune responses remains unclear. TPL-2 expression is required for efficient immune responses to Citrobacter rodentium, decreasing the bacterial burden and dissemination to the liver and spleen (Acuff et al, 2017). TPL-2 deficiency reduces neutrophil recruitment to the colon at peak infection. In vitro experiments have shown that TPL-2 expression in neutrophils is required for LPS activation of ERK1/2 and induction of tumour necrosis factor (TNF) secretion, similar to that in macrophages (Acuff et al, 2017). TPL-2 is also required for efficient killing of phagocytosed C. rodentium, although phagocytic uptake of bacteria is TPL-2-independent (Acuff et al, 2017). However, the mechanism by which TPL-2 regulates bacterial killing has not been established. A recent study by one of our groups using quantitative mass spectrometry demonstrated that TPL-2 catalytic activity potentially regulates intracellular trafficking. Tpl2D270A kinase-dead mutation reduced the phosphorylation of several proteins linked to endocytosis, vesicle trafficking and GTPase signalling in LPS-stimulated macrophages (Pattison et al, 2016). Together, these results raised the interesting possibility that TPL-2 promotes the killing of internalised bacteria by controlling phagosome maturation in myeloid cells. In the present study, the potential regulation of phagosome maturation by TPL-2 in macrophages was investigated. TPL-2 catalytic activity was found to induce macrophage phagosome proteolytic activity and phagosome acidification, both independently of its ability to activate MAP kinase signalling. These effects were mediated by direct regulation of V-ATPase assembly and function via controlling the phosphorylation of the V-ATPase-associated regulatory protein DMXL1. Consistent with these findings, TPL-2 catalytic activity was necessary for efficient macrophage killing of phagocytosed Staphylococcus aureus and Citrobacter rodentium. These results indicate that TPL-2 has two key functions in the innate immune response of macrophages to bacterial infection. TPL-2 mediates TLR activation of MAP kinase signalling to control inflammation by modulation of gene expression and directly promotes bacterial killing by MAP kinase-independent induction of phagosome acidification. Results TPL-2 catalytic activity promotes macrophage phagosome maturation To investigate the role of TPL-2 signalling in phagosome maturation, bone marrow-derived macrophages (BMDMs) were generated from Tpl2D270A/D270A knock-in mice, which express catalytically inactive TPL-2 (Sriskantharajah et al, 2014). Consistent with earlier experiments using Tpl2−/− neutrophils (Acuff et al, 2017), phagocytic uptake of fluorescently labelled beads by Tpl2D270A/D270A BMDMs was similar to wild-type (WT) controls (Fig 1A). To investigate whether TPL-2 regulated phagosome maturation, bulk intra-phagosomal proteolysis was monitored using latex beads coupled to the fluorescent substrate DQ green BSA (Yates & Russell, 2008; Russell et al, 2009). Alexa Fluor 594 labelling of beads was used to normalise phagocytic uptake. Real-time measurements showed that phagosome proteolysis was significantly reduced in Tpl2D270A/D270A BMDMs compared to WT controls (Fig 1B). Figure 1. Tpl2[D270A] mutation impairs phagosome maturation Phagocytic uptake of fluorescently labelled latex beads by WT and Tpl2[D270A] (Tpl2D270/D270A) BMDMs. Intracellular fluorescence was monitored following uptake of AF488 latex beads by BMDMs (n = 4 wells). Intra-phagosomal proteolysis in WT and Tpl2[D270A] BMDMs was assayed following uptake of DQ Green BSA / AF594 latex beads. As positive assay control, BMDMs were separately pre-treated with 100 μg/ml leupeptin for 1 h to inhibit serine-cysteine proteases (n = 4 wells). Cathepsin activity assay in WT and Tpl2[D270A] BMDMs 0.5 h after uptake of latex beads. BMDMs were stained with the Magic Red cathepsin L substrate (red). Average fluorescence intensity of the cathepsin probe per cell was quantified (n = 40–51 cells). Intra-phagosomal acidification in WT and Tpl2[D270A] BMDMs was monitored following uptake of BCECF-coupled latex beads. BMDMs were pre-treated with 1 μM bafilomycin A1 for 15 min to inhibit V-ATPases (n = 4 wells). pH assay in WT and Tpl2[D270A] BMDMs upon 0.5 h after incubation with latex beads. BMDMs were stained with the LysoTracker Red DND-99 dye (red). Average fluorescence intensity of the LysoTracker Red probe per cell was quantified (n = 95–126 cells). Intra-phagosomal oxidative burst in WT and Tpl2[D270A] BMDMs was assayed following uptake of OxyBURST Green BSA / AF594 latex beads. BMDMs were pre-treated with 1 μM bafilomycin A1 for 15 min to inhibit ROS production (n = 4 wells). Reactive oxygen species assay in WT and Tpl2[D270A] BMDMs upon 0.5 h after incubation with latex beads. BMDMs were stained with the ROS Deep Red dye. Average fluorescence intensity of the ROS Deep Red probe per cell was quantified (n = 80–110 cells). Data information: One representative experiment out of three shown. Error bars and shaded areas represent SEM. ****P < 0.0001. Panels (B, D, F) Paired Mann–Whitney t-test; All differences relative to WT are ****. Panels (C, E, G) Student's unpaired t-test. Download figure Download PowerPoint Next, a confocal microscopy assay was used to monitor cathepsin protease activation following phagocytic uptake of latex beads by Tpl2D270A/D270A BMDMs compared to WT control cells, using a fluorescently labelled cathepsin L target peptide. Thirty mins after bead uptake, phagosome cathepsin activity was significantly reduced by Tpl2D270A mutation (Fig 1C). These results showed that TPL-2 catalytic activity is required to increase protease activity inside phagosomes of primary macrophages. During maturation, the lumens of phagosomes become increasingly acidic due to the action of V-ATPases that are recruited by fusion of phagosomes initially with endocytic vesicles and ultimately lysosomes (Kinchen & Ravichandran, 2008). Intra-phagosomal acidification was assayed using latex beads coupled to the pH indicator BCECF (Russell et al, 1995). Real-time measurements showed that phagosomal acidification was significantly decreased in Tpl2D270A/D270A BMDMs compared to WT BMDMs (Fig 1D). Tpl2D270A mutation reduced phagosome acidification to a similar degree to the pre-treatment of WT BMDMs with the V-ATPase inhibitor bafilomycin A1 (Fig 1D). Consistent with these results, analysis of phagosomal pH by staining with LysoTracker Red, a dye that labels acidic compartments, and confocal microscopy demonstrated that Tpl2D270A mutation reduced LysoTracker Red staining following latex bead uptake (Fig 1E). The production of reactive oxygen species (ROS) in phagosomes by NAPDH oxidase is essential for killing internalised bacteria and is dependent on an intra-phagosomal proton gradient by V-ATPases (Mantegazza et al, 2008). Consistent with its inhibitory effects on phagosome acidification, Tpl2D270A mutation significantly reduced ROS generation in BMDMs after uptake of latex beads coupled to the fluorescence reporter substrate OxyBURST Green BSA (Fig 1F) (Vanderven et al, 2009). Tpl2D270A mutation also significantly reduced ROS generation detected microscopically using ROS Deep Red dye following latex bead uptake (Fig 1G). Together, these results show that TPL-2 catalytic activity promotes the maturation of phagosomes in macrophages. TPL-2 induces phagosome maturation independently of MAP kinase activation TPL-2's established functions in innate immunity are mediated by activation of MAP kinase pathways (Gantke et al, 2011). However, uptake of latex beads by BMDMs did not induce ERK1/2 and p38α activation, as detected by phospho-antibody immunoblotting, suggesting that TPL-2 modulated phagosome maturation independently of its ability to activate MAP kinases. Genetic and pharmacological experiments were used to investigate whether MAP kinase activation was required for TPL-2 catalytic activity to promote phagosome maturation. IKK triggers proteolysis of NF-κB1 p105 by phosphorylating Ser930 and Ser935 in the p105 PEST region (Lang et al, 2003). Nfkb1SSAA mutation, which changes both serines to alanine, blocks IKK-induced p105 proteolysis, thereby preventing release of TPL-2 and ABIN-2 from p105 and blocking TPL-2 activation of ERK1/2 and p38α MAP 2 kinases (Yang et al, 2012; Pattison et al, 2016). Phagosome proteolysis and acidification, however, were not affected by Nfkb1SSAA mutation (Fig 2A and B). Importantly, however, both processes were reduced by Nfkb1SSAA Tpl2D270A compound mutation confirming their dependence on TPL-2 catalytic activity (Fig 2A and B). Inhibition of ERK1/2 phosphorylation in Nfkb1SSAA and Nfkb1SSAA Tpl2D270A BMDMs following LPS stimulation was confirmed by immunoblotting (Fig 2C). In line with these genetic data, simultaneous pharmacological inhibition of ERK1/2 (PD0325901) and p38α (VX-745) activity did not alter phagosome proteolysis or acidification (Fig 2D and E). In contrast, TPL-2 inhibition with the small molecule inhibitor C34 (Wu et al, 2009) significantly reduced both processes to a similar degree as Tpl2D270A mutation (Fig 2D and E). Substantial reductions of MAP kinase activation by pharmacological inhibitors were confirmed by immunoblotting of LPS-stimulated BMDM lysates (Fig 2F). Figure 2. Tpl2[D270A] mutation promotes phagosome maturation independently of MAP kinase activation A–F. Experiments were performed using murine BMDMs. (A) Intra-phagosomal proteolysis in WT, Nfkb1[SSAA] (Nfkb1SSAA/SSAA), and Nfkb1[SSAA]/Tpl2[D270A] BMDMs were monitored as in Fig 1B (n = 4 wells). (B) Intra-phagosomal acidification in WT, Nfkb1[SSAA], and Nfkb1[SSAA]/Tpl2[D270A] BMDMs was monitored as in Fig 1C (n = 4 wells). (C) Cell extracts from LPS-stimulated Nfkb1[SSAA] and Nfkb1[SSAA]/Tpl2[D270A] BMDMs were immunoblotted for the indicated antigens. (D) Intra-phagosomal proteolysis in WT and inhibitor-treated BMDMs (n = 4 wells) was monitored as in Fig 1B. BMDMs were pre-treated with 0.1 μM PD0325901 (10 min) to inhibit MEK1, pre-treated with 1 μM VX-745 (1 h) to inhibit p38α, and pre-treated with 10 μM C34 (1 h) to inhibit TPL-2. (E). Intra-phagosomal acidification in WT and inhibitor-treated BMDMs (see (D) for conditions) was assayed as in Fig 1C (n = 4 wells). (F) Cell extracts from LPS-stimulated, inhibitor-treated WT, and Tpl2[D270A] BMDMs were immunoblotted for the indicated antigens. 1 μM bafilomycin A1 was added to inhibit V-ATPases. G–K. Experiments were performed using human primary monocyte-derived macrophages. (G) Intra-phagosomal proteolysis in human macrophages was monitored following uptake of DQ Green BSA / AF594 latex beads. TPL-2 catalytic kinase activity was blocked by pre-treatment with 10 μM C34 for 1 h (n = 4 wells). (H) Intra-phagosomal acidification in human macrophages was monitored following uptake of BCECF-coupled latex beads (n = 4 wells). (I) Intra-phagosomal proteolysis in human macrophages was assayed as in Fig 2G. MAP kinase activity was inhibited by combinatorial pre-treatment with 0.1 μM PD0325901 (MEK1 inhibition) for 10 min and 1 μM VX-745 (p38 inhibition) for 1 h (n = 4 wells). (J) Intra-phagosomal acidification in human macrophages was monitored as in Fig 2H (n = 4 wells). (K) Cell extracts from LPS-stimulated, inhibitor-treated primary human macrophages were immunoblotted for p-ERK1/2, ERK1/2, p-p38, p38, and HSP90. Data information: One representative experiment out of three shown. Error bars and shaded areas represent SEM. ****P < 0.0001. Panels (A, B, D, E, G, H, I, J) Paired Mann–Whitney t-test; all differences relative to WT are ****. Download figure Download PowerPoint TPL-2 catalytic activity therefore stimulated phagosome maturation in macrophages independently of MAP kinase activation (via unknown substrates). The inhibitory effects of C34 on phagosome proteolysis and acidification in WT BMDMs also demonstrated that the effects of TPL-2 catalytic activity on phagosome maturation were mediated acutely following latex bead uptake. To investigate the role of TPL-2 in phagosome maturation in human primary macrophages, macrophages were generated from monocytes isolated from peripheral blood by culturing in GM-CSF and phagosome maturation was assayed as for BMDMs. C34 inhibition of TPL-2 catalytic activity reduced intra-phagosomal proteolysis (Fig 2G) and acidification (Fig 2H) in monocyte-derived human macrophages compared to cells pre-treated with vehicle control. Pharmacological inhibition of MEK1/2 (PD0325901) (Ciuffreda et al, 2009) and p38α (VX-745) (Duffy et al, 2011) did not alter either phagosome proteolysis (Fig 2I) or acidification (Fig 2J), although these inhibitors potently inhibited phosphorylation of ERK1/2 and p38α following LPS stimulation (Fig 2K). These experiments showed that MAP kinase-independent stimulation of phagosome maturation by TPL-2 catalytic activity was conserved between mouse and human primary macrophages. TPL-2 catalytic activity regulates the protein composition of phagosomes To further study the molecular mechanisms by which TPL-2 kinase activity promotes phagosome maturation, the composition of phagosomes isolated from Tpl2D270A/D270A and WT BMDMs following uptake of latex beads was analysed by mass spectrometry. Tpl2D270A mutation significantly altered the composition of the phagosome proteome (Fig 3A and B, Dataset EV1). Phagosome abundance of numerous Rab GTPases was decreased by Tpl2D270A mutation (Fig 3A). These included RAB5 and RAB7, which have key roles in regulating the maturation of phagosomes (Vieira et al, 2003; Huynh et al, 2007). The abundance of LAMP1, which is essential for late phagosome/lysosome fusion, was also decreased in Tpl2D270A/D270A phagosomes compared with WT cells (Fig 3A and D). The levels of seven V-ATPase subunits, including V-ATPase subunits D, D1 and E1, were also significantly reduced (Fig 3C). This finding was particularly interesting given the inhibitory effect of Tpl2D270A mutation on phagosome acidification. Phagosome abundance of four members of the LAMTOR protein complex, a key regulator of lysosomal trafficking (Colaço & Jäättelä, 2017), was also reduced in Tpl2D270A/D270A BMDMs compared to controls. Immunoblotting confirmed that Tpl2D270A mutation reduced levels of RAB5 GTPase and LAMP-1 associated with purified phagosomes (Fig 3D). The effect of TPL-2 catalytic activity on the abundance of phagosomal proteins was selective since abundance of numerous proteins on phagosomes, including vimentin, SNARE, vacuolar protein sorting (VPS) and ESCRT proteins, was similar between WT and Tpl2D270A/D270A BMDMs (Figs 3D and EV1). Figure 3. Tpl2[D270A] mutation alters the protein composition of phagosomes WT and Tpl2[D270A] BMDMs were incubated with latex beads for 0.5 h. Latex bead phagosomes were purified from Tpl2[D270A] and WT BMDMs and analysed by mass spectrometry. Biological triplicates were analysed for each genotype. Heatmap of selected proteins that were significantly downregulated in BMDMs from Tpl2[D270A] mice relative to WT (P < 0.05). Selected hits were grouped into clusters according to their molecular functions. Gene set enrichment analysis (GSEA) of significantly downregulated biological processes in phagosomal fractions. Changes of Tpl2[D270A] phagosomes relative to WT. Dot colour; enrichment score. Dot size; statistical significance. Protein intensities of V-ATPase subunits from phagosomes purified from WT and Tpl2[D270A] BMDMs, (n = 3 biological replicates). Protein intensities of RAB5 and LAMP-1 from phagosome proteome analysis of WT and Tpl2[D270A] BMDMs (n = 3 biological replicates) (left). Immunoblot of isolated phagosomes from WT and Tpl2[D270A] BMDMs probed for RAB5, LAMP-1, and vimentin. Phagosomal fractions of two biological replicates were pooled. One representative experiment out of two shown (right) (n = 2). Data information: Data were analysed by Student's t-test. Error bars represent SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Tpl2[D270A] mutation does not alter abundance of several key phagosomal proteins on isolated phagosomes Protein intensities of selected proteins from phagosomes purified from WT and Tpl2[D270A] BMDMs (n = 3 biological replicates). Error bars represent SEM. Download figure Download PowerPoint Consistent with these changes in phagosome composition, gene set enrichment analyses to identify significantly downregulated biological processes revealed that Tpl2D270A mutation decreased the abundance of proteins involved in vesicle-mediated transport, receptor-mediated transport, ion transport, vesicle proteolysis and regulation of pH (Fig 3B). These processes are all important in the generation of mature phagolysosomes (Flannagan et al, 2009; Pauwels et al, 2017). Together, these experiments indicated that blocking TPL-2 catalytic activity substantially changed the composition of phagosomes in BMDMs, reducing the abundance of several proteins with key roles in regulating different stages of phagosome maturation. Importantly, this analysis revealed that TPL-2 catalytic activity promoted the recruitment of V-ATPase subunits to phagosomes during their maturation suggesting a possible mechanism for TPL-2 control of phagosome acidification. TPL-2 induces phosphorylation of DMXL1, a component of the V-ATPase complex To further investigate the mechanism by which TPL-2 kinase activity regulated phagosome maturation, the phosphoproteome in Tpl2D270A/D270A and WT BMDMs was characterised 30 min after phagocytic uptake of latex beads. TMT (tandem mass tag) labelling and mass spectrometry were used to quantify differences in phosphopeptide abundance (Dataset EV2). The phospho-site most highly downregulated by Tpl2D270A mutation was Ser141 on TPL-2 itself (Fig 4A), a known autophosphorylation site (Stafford et al, 2006; Xu et al, 2018). Two of the next most downregulated phospho-sites were Ser1903 and Ser1904 of DMXL1, which were both reduced over threefold. Phosphorylation of DMXL1 on Ser434, Ser915, Thr1373 and Ser1900 was not altered by Tpl2D270A mutation indicating that TPL-2 catalytic activity did not change DMXL1 protein abundance (Fig 4A). Figure 4. TPL-2 induces phosphorylation of DMXL1, a V-ATPase regulatory protein A. TPL-2-dependent phosphoproteome following phagocytosis of latex beads (0.5 h) was determined by TMT mass spectrometry. Volcano plot representing the significance (-log10 P-values after Welch's t-test) versus phosphorylation fold change (Welch difference ratios) between WT and Tpl2[D270A] BMDMs. Five biological replicates were analysed per genotype (n = 5). Three of the most highly and significantly downregulated phospho-sites in Tpl2[D270A] BMDMs relative to WT, as well as unaltered DMXL1 phospho-sites, are shown. B. Total cell lysates from WT and Tpl2[D270A] BMDMs 0.5 h after incubation with latex beads were immunoblotted for phospho-DMXL1 (S1903) and HSP90 (loading control). C–F. Dmxl1 was knocked down in WT iBMDMs or Tpl2[D270A] iBMDMs by RNA interference using a SMARTpool ON-TARGETplus siRNA for 48 h. ON-TARGETplus non-targeting pool functioned as siRNA control. (C) qRT–PCR analysis of RNA extracted from iBMDMs was used to check the efficiency of Dmxl1 knockdown (D, E). Dmxl1 mRNA levels were normalised to Hprt mRNA levels and fold changes calculated (ΔCt values) (n = 4). (D) Intra-phagosomal acidification was assayed following uptake of BCECF-coupled latex beads by WT iBMDMs. As a control, BMDMs were pre-treated with 1 μM bafilomycin A1 for 15 min to directly block V-ATPase function (n = 4 wells). (E) Intra-phagos
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