Stress‐induced vesicular assemblies of dual leucine zipper kinase are signaling hubs involved in kinase activation and neurodegeneration
2022; Springer Nature; Volume: 41; Issue: 14 Linguagem: Inglês
10.15252/embj.2021110155
ISSN1460-2075
AutoresElena Tortosa, Arundhati Sengupta Ghosh, Qingling Li, Weng Ruh Wong, Trent Hinkle, Wendy Sandoval, Christopher M. Rose, Casper C. Hoogenraad,
Tópico(s)Ion Channels and Receptors
ResumoArticle25 May 2022Open Access Source DataTransparent process Stress-induced vesicular assemblies of dual leucine zipper kinase are signaling hubs involved in kinase activation and neurodegeneration Elena Tortosa Elena Tortosa orcid.org/0000-0003-3910-2314 Department of Neuroscience, Genentech, Inc., South San Francisco, CA, USA Contribution: Conceptualization, Data curation, Software, Formal analysis, Supervision, Validation, Investigation, Visualization, Methodology, Writing - original draft, Project administration, Writing - review & editing Search for more papers by this author Arundhati Sengupta Ghosh Arundhati Sengupta Ghosh Department of Neuroscience, Genentech, Inc., South San Francisco, CA, USA Contribution: Investigation, Methodology Search for more papers by this author Qingling Li Qingling Li Department of Microchemistry, Proteomics and Lipidomics, Genentech, Inc., South San Francisco, CA, USA Contribution: Resources, Methodology Search for more papers by this author Weng Ruh Wong Weng Ruh Wong Department of Microchemistry, Proteomics and Lipidomics, Genentech, Inc., South San Francisco, CA, USA Contribution: Resources, Methodology Search for more papers by this author Trent Hinkle Trent Hinkle Department of Microchemistry, Proteomics and Lipidomics, Genentech, Inc., South San Francisco, CA, USA Contribution: Resources, Formal analysis, Methodology Search for more papers by this author Wendy Sandoval Wendy Sandoval Department of Microchemistry, Proteomics and Lipidomics, Genentech, Inc., South San Francisco, CA, USA Contribution: Resources, Supervision, Methodology Search for more papers by this author Christopher M Rose Christopher M Rose orcid.org/0000-0002-7502-3368 Department of Microchemistry, Proteomics and Lipidomics, Genentech, Inc., South San Francisco, CA, USA Contribution: Resources, Supervision, Methodology Search for more papers by this author Casper C Hoogenraad Corresponding Author Casper C Hoogenraad [email protected] orcid.org/0000-0002-2666-0758 Department of Neuroscience, Genentech, Inc., South San Francisco, CA, USA Contribution: Conceptualization, Supervision, Writing - original draft, Project administration, Writing - review & editing Search for more papers by this author Elena Tortosa Elena Tortosa orcid.org/0000-0003-3910-2314 Department of Neuroscience, Genentech, Inc., South San Francisco, CA, USA Contribution: Conceptualization, Data curation, Software, Formal analysis, Supervision, Validation, Investigation, Visualization, Methodology, Writing - original draft, Project administration, Writing - review & editing Search for more papers by this author Arundhati Sengupta Ghosh Arundhati Sengupta Ghosh Department of Neuroscience, Genentech, Inc., South San Francisco, CA, USA Contribution: Investigation, Methodology Search for more papers by this author Qingling Li Qingling Li Department of Microchemistry, Proteomics and Lipidomics, Genentech, Inc., South San Francisco, CA, USA Contribution: Resources, Methodology Search for more papers by this author Weng Ruh Wong Weng Ruh Wong Department of Microchemistry, Proteomics and Lipidomics, Genentech, Inc., South San Francisco, CA, USA Contribution: Resources, Methodology Search for more papers by this author Trent Hinkle Trent Hinkle Department of Microchemistry, Proteomics and Lipidomics, Genentech, Inc., South San Francisco, CA, USA Contribution: Resources, Formal analysis, Methodology Search for more papers by this author Wendy Sandoval Wendy Sandoval Department of Microchemistry, Proteomics and Lipidomics, Genentech, Inc., South San Francisco, CA, USA Contribution: Resources, Supervision, Methodology Search for more papers by this author Christopher M Rose Christopher M Rose orcid.org/0000-0002-7502-3368 Department of Microchemistry, Proteomics and Lipidomics, Genentech, Inc., South San Francisco, CA, USA Contribution: Resources, Supervision, Methodology Search for more papers by this author Casper C Hoogenraad Corresponding Author Casper C Hoogenraad [email protected] orcid.org/0000-0002-2666-0758 Department of Neuroscience, Genentech, Inc., South San Francisco, CA, USA Contribution: Conceptualization, Supervision, Writing - original draft, Project administration, Writing - review & editing Search for more papers by this author Author Information Elena Tortosa1, Arundhati Sengupta Ghosh1, Qingling Li2, Weng Ruh Wong2, Trent Hinkle2, Wendy Sandoval2, Christopher M Rose2 and Casper C Hoogenraad *,1 1Department of Neuroscience, Genentech, Inc., South San Francisco, CA, USA 2Department of Microchemistry, Proteomics and Lipidomics, Genentech, Inc., South San Francisco, CA, USA *Corresponding author (Lead contact). Tel: +1 650 467 2877; E-mail: [email protected] The EMBO Journal (2022)41:e110155https://doi.org/10.15252/embj.2021110155 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 Mitogen-activated protein kinases (MAPKs) drive key signaling cascades during neuronal survival and degeneration. The localization of kinases to specific subcellular compartments is a critical mechanism to locally control signaling activity and specificity upon stimulation. However, how MAPK signaling components tightly control their localization remains largely unknown. Here, we systematically analyzed the phosphorylation and membrane localization of all MAPKs expressed in dorsal root ganglia (DRG) neurons, under control and stress conditions. We found that MAP3K12/dual leucine zipper kinase (DLK) becomes phosphorylated and palmitoylated, and it is recruited to sphingomyelin-rich vesicles upon stress. Stress-induced DLK vesicle recruitment is essential for kinase activation; blocking DLK-membrane interaction inhibits downstream signaling, while DLK recruitment to ectopic subcellular structures is sufficient to induce kinase activation. We show that the localization of DLK to newly formed vesicles is essential for local signaling. Inhibition of membrane internalization blocks DLK activation and protects against neurodegeneration in DRG neurons. These data establish vesicular assemblies as dynamically regulated platforms for DLK signaling during neuronal stress responses. Synopsis Mitogen-activated protein kinases (MAPKs) cascades are key signaling pathways mediating neuronal survival and degeneration. Although MAPK localization is critical to control kinase (in)activation, the mechanisms that regulate their localization are not fully understood. Neuronal stress increases DLK phosphorylation, palmitoylation and recruitment to vesicles. DLK-vesicle recruitment is essential for kinase activation and signaling upon stress. Sphingomyelin controls DLK- vesicle localization and kinase activity. DLK uses the endocytic pathway as a platform for signaling, and inhibition of endocytosis blocks DLK activation and protects against neurodegeneration. Introduction Neuronal death is a tightly regulated process, necessary for the proper development of the nervous system and a key pathological event in many neurodegenerative diseases. Elimination of excessive neurons during the developing nervous system occurs as a failure to establish trophic relationships with a target cell. Neuronal loss becomes highly restricted as the nervous system matures; however, it emerges in response to stroke and traumatic brain injury and contributes to neurodegenerative diseases such as amyotrophic lateral sclerosis and Alzheimer's disease (Mattson, 2000; Yamaguchi & Miura, 2015). Neuronal death is an active and well-orchestrated process with numerous signaling pathways implicated. Among others, mitogen-activated protein kinase (MAPK) signaling pathways have been shown to play key roles in balancing cell survival and death during both development and pathological conditions in numerous cell types including neurons (Kim & Choi, 2010, 2015). Despite the critical importance of MAPK cascades, it remains largely unknown how MAPKs signaling is spatially and temporally controlled in complex neuronal cells under the various stress conditions. Different MAPK cascades have been reported to differentially contribute to neuronal survival and degeneration. For example, it is widely accepted that MAPKs such as c-Jun N-terminal kinase (JNK) and p38 MAPK (p38) promote cell death, whereas extracellular signal-regulated kinase 1/2 (ERK1/2) contribute to neuron survival (Xia et al, 1995; Hetman & Gosdz, 2004; Yarza et al, 2016; Asih et al, 2020). MAP3Ks have been shown to function as critical nodes in neuronal signaling pathways. For instance, MAP3K12/ dual leucine zipper kinase (DLK) mediates neuronal death induced by nerve growth factor (NGF) deprivation in sympathetic neurons, and DLK, together with leucine zipper-bearing kinase (LZK), MAPK/ERK kinase kinase 4 (MEKK4), and mixed lineage kinase 2 (MLK2), are key mediators of cell death in response to traumatic injury (Ghosh et al, 2011; Yang et al, 2015). Interestingly, pharmacological and genetic inhibition of MAP3Ks such as DLK and LZK are sufficient to attenuate neuronal death in different models of acute neuronal injury and neurodegenerative diseases (Chen et al, 2008; Miller et al, 2009; Ghosh et al, 2011; Pozniak et al, 2013; Watkins et al, 2013; Fernandes et al, 2014; Patel et al, 2015; Le Pichon et al, 2017; Welsbie et al, 2013; Welsbie et al, 2017; Welsbie et al, 2019; Wlaschin et al, 2018; Hu et al, 2019; Ma et al, 2021). Considering the broad range of stimuli that activate MAPK signaling cascades and the large number of processes that these kinases regulate, MAPK specificity needs to be tightly controlled. A precise and dynamic subcellular localization of the MAPK cascade components is critical to tightly control kinase (in)activation and the access to downstream effectors (Witzel et al, 2012; Wainstein & Seger, 2016; Zhang et al, 2021). The mechanisms that control kinase localization are not fully understood but may include interactions with specific adaptor proteins or lipids. For example, in resting conditions, specific anchor proteins retain components of the Raf-MEK-ERK cascade such as ERK1/2 in the cytoplasm in different cell lines (Fukuda et al, 1997; Brunet et al, 1999; Formstecher et al, 2001; Chen et al, 2005; Mebratu et al, 2008). Upon stimulation, the components of the cascade rapidly change their subcellular location: Rafs are known to move to the plasma membranes and other intracellular membranes, whereas active MEK1/2 and ERK1/2 translocate from the cytosol to the nucleus in numerous cell types including neurons (Eblen, 2018). In addition, lipid modifications such as palmitoylation have been shown to target MAPKs such as DLK and JNK3 to membranes, controlling not only localization and activity but also protein stability in neurons (Yang et al, 2012; Holland et al, 2016; Niu et al, 2022). Understanding the complex subcellular localization of MAPK cascades components in neurons and their dynamic changes upon stress will provide a better understanding of the regulation of signaling pathways involved in neuronal survival and degeneration. Here, we systematically analyze changes in phosphorylation and localization of MAPKs expressed in dorsal root ganglia (DRG) neurons, in control and stress conditions. MAPKs such as c-Raf, MEKK2, and ERK1/2 decrease their phosphorylation levels in response to NGF deprivation. Interestingly, DLK is the only MAPK that responds by increasing its phosphorylation and membrane localization. NGF deprivation-induced stress also promotes DLK palmitoylation which changes its localization from the cytoplasm to axonal transport vesicles. Preventing DLK vesicle assembly inhibits DLK-mediated signaling pathways, and conversely, ectopic recruitment of DLK to other subcellular structures is sufficient to induce its activation. We show that DLK vesicle recruitment is independent of DLK dimerization or kinase activation, but relies on an active endocytic pathway and sphingomyelin levels in the neuron. These findings provide new insights into the mechanism of DLK activation in neurons and suggest that stress-induced DLK vesicle assemblies act as local signaling platforms that drive kinase activation and neurodegeneration. Results Neuronal stress increases DLK recruitment to membranes MAPK cascades respond to various stimuli and control both survival and apoptosis signaling. The subcellular localization of MAPK components has been shown to be key in controlling their activity and specificity. We decided to systematically characterize the different MAPK family members and their membrane localization during neuronal stress using an in vitro model that mimics the competition for trophic factors experienced by sensory neurons in vivo. Cultured DRG neurons in vitro require the presence of NGF to survive, and NGF depletion initiate an active and tightly controlled process of degeneration (Deshmukh & Johnson, 1997; Freeman et al, 2004). Interestingly, the molecular machinery involved in this developmental neurodegeneration process is activated in adult axonal injury and various neurodegenerative diseases (Asghari Adib et al, 2018). We first analyzed total protein expression and phosphorylation of the different MAPK cascade components by mass spectrometry. Many different MAPKs were expressed in DRG primary cultures, including MAPK, MAP2K, and MAP3K members (Fig 1A). Although none of the MAPK family member changed their expression at total protein levels (Dataset EV1), some of them, such as DLK, ERK1/2, MEKK2, and c-Raf, altered their phosphorylation levels after 1 and 4 h of NGF deprivation (Fig 1B, Appendix Fig S1A). In addition to the changes in DLK phosphorylation, we also found increased phosphorylation levels of the downstream effector c-Jun after NGF deprivation, but could not detect sustained changes in phosphorylation levels of intermediate kinases such as JNK1/2/3 or MKK4/7 during the 4-h time window in this particular mass spectrometry experiment (Dataset EV1). We next tested the membrane association of the different MAPKs present in DRG neurons and determined whether their localization was altered upon NGF deprivation. By performing membrane fractionation experiments of DRG neurons after NGF withdrawal, we found that kinases such as DLK, MKK5, or ERK1/2 partially or fully coincided with membranes. However, only DLK changed its membrane association upon NGF withdrawal (Fig 1C and D, Appendix Fig S1B). Membrane anchoring of cytosolic protein can be mediated by palmitoylation (Iwanaga et al, 2009). We confirmed that several MAPK can be palmitoylated in DRG neurons using click-it chemistry (Fig 1E, Appendix Fig S1C) and observed that, in agreement with the stress-induced membrane recruitment, DLK increased its palmitoylation levels upon NGF deprivation while total protein levels remained unchanged (Fig 1F, Appendix Fig S1D). These findings are in line with previous studies in which DLK was described to respond to cellular stress (Tedeschi & Bradke, 2013; Asghari Adib et al, 2018). As DLK is the only MAPK in DRG neurons that responded by increasing its protein phosphorylation and membrane association, we decided to further characterize the role of DLK-membrane associations and its impact on DLK activity and neurodegeneration. Figure 1. Neuronal stress increases DLK recruitment to membranes Schematic diagram of MAPK signaling cascades in mammalian cells. Only representative signaling molecules are shown. MAPK cascade components detected in 3 days in vitro (DIV) cultured embryonic DRG neurons by TMT-based mass spectrometry analysis are colored. In grey, MAPKs not detected (n = 2 independent experiments). Volcano plot of differently-regulated phosphosites of MAPK cascade components from 3DIV cultured embryonic DRG neurons upon 4 h of NGF deprivation, with protein-level cut-offs set at log2 fold change > 1.0 and –log10 adjusted P-value > 1.3 (P < 0.05), marked by dashed lines. Downregulated sites represented as blue points, and up-regulated sites as orange points (n = 2 independent experiments). Representative Western blots of the different MAPKs expressed in DRG neurons (DIV3) cultured in the presence or absence of NGF for 3 h and subjected to a membrane/cytosolic fractionation. α-tubulin and STMN-1 are used as controls of cytosolic proteins and Na+/K+-ATPase and STMN-2 as controls of membrane proteins. Quantification of the ratio of protein localized in the membrane fraction after 3 h of NGF deprivation (n = 6–14 biological replicates; series of unpaired t-tests followed by a Holm-Sidak correction). Flow chart showing the approach used to detect palmitoylation levels in DLK from cultured embryonic DRG neurons. Palmitoylated proteins were labeled with click-it palmitic acid, conjugated with biotin and subjected to immunoprecipitation using streptavidin beads. Representative Western blots of total and palmitoylated DLK (upper panel) and quantification of DLK palmitoylation levels (lower panel) from cultured DRG neurons subjected to 3 h of NGF deprivation (n = 5 independent experiments; Mann–Whitney U test). Data information: Graphs represent mean ± SEM in (D) and (F). *P < 0.05 and **P < 0.01. Diagrams in A and E were created with BioRender.com. See also Appendix Fig S1. Source data are available online for this figure. Source Data for Figure 1 [embj2021110155-sup-0004-SDataFig1.zip] Download figure Download PowerPoint DLK vesicle recruitment induces kinase activation and signaling upon stress We next analyzed the subcellular localization of DLK under control and stress induced by NGF deprivation. Using live imaging experiments in DRG neurons, we found that GFP-tagged DLK was present in vesicle-like structures that were transported along the axon, in both anterograde and retrograde directions (Fig 2A and B). Similar vesicle localization was observed for endogenous DLK (Fig 2C, Appendix Fig S2A). DLK localization was prevented by the pharmacological inhibitor of protein palmitoylation, 2-bromopalmitate (2-BP) or by point mutation of DLK's palmitoylation site, Cys-127 (Appendix Fig S2B and C) (Holland et al, 2016). We observed an increase in the number of DLK-positive vesicles after NGF withdrawal for both endogenous and overexpressed GFP-tagged DLK in DRG neurons (Fig 2D–G), consistent with the increase in membrane association in the fractionation experiments. Figure 2. DLK vesicle recruitment induces kinase activation and signaling upon stress A. Representative stills from time-lapse recordings of DIV6 DRG neurons overexpressing DLK-GFP. Arrowheads point to individual moving DLK-GFP positive structures. B. Kymographs made from the complete time-lapse recording shown in A. The left shows the original kymograph, and the right shows an illustration of the manually traced structure displacements for better visualization. C. Representative images of DIV3 DRG neurons nucleofected with a non-targeting siRNA (NT siRNA) or a siRNA against DLK (DLK siRNA), and stained for endogenous DLK and β3-tubulin (TUBB3). On the right, quantification of the DLK mean intensity in neurons expressing control NT or DLK siRNAs (n = 25 neurons/condition; Unpaired t-test). D–F. Representative kymographs (D), quantification of GFP-positive particle number (E) and percentage of anterograde, retrograde, reverse or non-moving GFP-positive particles (F) from DLK-GFP time-lapse recordings in DIV6 DRG neurons, in control situation (+NGF) or after 3 h of NGF deprivation (−NGF) (n = 45–46 neurons/ condition; Mann–Whitney U test in E and Chi-square test in F). G. Representative images of DIV3 cultured embryonic DRG neurons maintained in the presence (+NGF) or absence (−NGF) of NGF for 3 h and stained for endogenous DLK. On the right, quantification of the number of DLK- positive puncta in control situation or after 3 h of NGF deprivation (n = 18–19 neurons/condition; Mann–Whitney U test). H. Representative kymograph and stills from a time lapse recording of 6DIV neurons overexpressing a GFP-tagged N-terminal region of DLK (NtermDLK-GFP). Arrowheads point to individual moving NtermDLK-GFP positive structures. I, J. Representative Western blots of p-cJun and GAPDH (I), and quantification of relative c-Jun phosphorylation levels (J) from DIV6 cultured embryonic DRG neurons overexpressing GFP or NtermDLK-GFP, and subjected to NGF withdrawal for 3 h (n = 7–8 biological replicates; series of unpaired t-tests /Mann–Whitney U tests followed by a Holm–Sidak correction). K. Diagram of different DLK constructs fused with the transmembrane domain of VAPB (DLK-ER), the C-terminal CAAX motif of Ras proteins (DLK-CAAX) or the membrane targeting sequence of the ActA protein of Listeria monocytogenes (DLK-MTS), and their expected localization. L. Representative images of HeLa cells expressing the different HA-tagged wild-type (WT) and palmitoyl-mutant (CS) DLK constructs (DLK-WT-ER, DLK-CS-ER, DLK-WT-CAAX, DLK-CS-CAAX, DLK-WT-MTS, DLK-CS-MTS), and stained with markers of the endoplasmic reticulum (KDEL), plasma membrane (CellMask) and mitochondria (cytochrome C). M, N. Representative Western blots of DLK, p-cJun and GAPDH (M) and quantification of relative c-Jun phosphorylation levels (N) from HeLa cells transfected with HA alone (HA) and the different HA-tagged wild-type (WT) and palmitoyl-mutant (CS) DLK constructs (DLK-WT-ER, DLK-CS-ER, DLK-WT-CAAX, DLK-CS-CAAX, DLK-WT-MTS, DLK-CS-MTS) (n = 7–8 biological replicates; series of unpaired t-tests /Mann–Whitney U tests with DLK-WT or DLK-CS followed by a Holm–Sidak correction). Data information: All graphs represent mean ± SEM. *P < 0.05 **P < 0.01 and ***P < 0.001. Scale bar represents 10 µm in (B) and (C), 5 µm in (A), (D), (G), (H) and (L), and 2 µm in zooms in (C). See also Figure Appendix Fig S2. Source data are available online for this figure. Source Data for Figure 2 [embj2021110155-sup-0005-SDataFig2.zip] Download figure Download PowerPoint To further explore the role of vesicle recruitment during DLK signaling, we next tested whether displacing DLK from vesicles could block DLK activity. We overexpressed an N-terminal portion of DLK (1–162 aa) including the palmitoylation region but without the kinase domain, and analyzed the levels of p-c-Jun as a readout of endogenous DLK activation. We observed that the N-terminal domain is recruited to vesicles and reduces the activation of DLK in response to NGF withdrawal (Fig 2H–J). To further determine the importance of DLK recruitment for the kinase activity, we measured the activity of DLK upon recruitment to ectopic membrane of structures such as the endoplasmic reticulum, the plasma membrane, or mitochondria using the transmembrane domain of VAPB (DLK-WT-ER), the C-terminal CAAX motif of Ras proteins (DLK-WT-CAAX) or the membrane targeting sequence of the ActA protein of Listeria monocytogenes (DLK-WT-MTS), respectively (Fig 2K). We observed that upon intracellular membrane recruitment to structures such as the endoplasmic reticulum or mitochondria DLK increases the phosphorylation of c-Jun, indicating that recruiting DLK to ectopic subcellular structures is sufficient to induce kinase activation. To exclude the possibility of an additional recruitment of DLK to Golgi membranes, we tested the activity of the C127S palmitoyl mutant DLK (DLK-CS) ectopically recruited to similar structures. As observed before, palmitoyl mutant DLK is activated when recruited to structures such as endoplasmic reticulum or mitochondria (Fig 2L–N, Appendix Fig S2D and E). Together, these data suggest that DLK vesicle localization is critical for the kinase activation and downstream signaling in response to stress. DLK-vesicle localization is independent of its activation state To further elucidate whether DLK vesicle recruitment requires dimerization and kinase activity, we generated a series of point mutations in the kinase and leucine zipper domains of DLK, DLK-KD-GFP, and DLK-LZ-GFP, respectively (Fig 3A, Appendix Fig S2F). It has been shown that DLK overexpression is sufficient for DLK activation and phosphorylation of c-Jun in cell lines (Mata et al, 1996). We expressed the mutant constructs in HeLa cells and found that, with the exception of the GFP- tagged wild-type DLK (DLK-WT-GFP), all mutated constructs were inactive and did not increase the phosphorylation levels of c-Jun (Fig 3B and C). We next analyzed the localization of the different mutants and found that DLK-WT-GFP was present at intracellular vesicles. The vesicular association was dependent on palmitoylation because the palmitoyl mutant (DLK-CS-GFP) shifted DLK to a cytoplasmic localization. Interestingly, both DLK-KD-GFP and DLK-LZ-GFP also showed a punctate localization (Fig 3D). Consistently, subcellular fractionations showed that all constructs tested were equally present in both membrane and cytosolic compartments, with the exception of DLK-CS-GFP, that was highly enriched in the cytosolic fraction (Fig 3E and F). We next performed similar experiments in DRG neurons and found that, consistently with results obtained in HeLa cells, all DLK mutants were recruited to vesicles, with the exception of DLK-CS (Fig 3G–I). In agreement with these data, treatments with DLK inhibitor (GNE-3511) did not block DLK vesicle localization. Interestingly, DLK inhibitor increased the number of DLK-positive vesicles in DRG neurons (Fig 3J–L). These results demonstrate that DLK vesicle localization is dependent on protein palmitoylation but is independent of protein dimerization or kinase activity. Figure 3. DLK- vesicle localization is independent of its activation state A. Schematic diagram of DLK indicating point mutations generated for the different GFP-tagged DLK constructs. B, C. Representative Western blots of GFP, p-cJun and GAPDH (B) and quantification of relative c-Jun phosphorylation levels (C) from HeLa cells non transfected (NT) or transfected with GFP alone (GFP), GFP-tagged DLK wild-type (DLK-WT-GFP), kinase dead mutant (DLK-KD-GFP), leucine zipper domain mutant (DLK-LZ-GFP) and a palmitoyl-site mutant (DLK-CS-GFP) (n = 4–7 biological replicates; series of unpaired t-tests /Mann–Whitney U tests with DLK-WT-GFP followed by a Holm–Sidak correction). D. Representative images and zooms of HeLa cells expressing GFP alone or the different GFP-tagged DLK mutants. E, F. Representative Western blots of GFP, α-tubulin and Na+/K+-ATPase from cytosolic and membrane fractions (E) and quantification of the percentage of DLK localized in the membrane and cytosolic fraction (F) from HeLa cells expressing GFP alone or the different GFP-tagged DLK mutants (n = 4–5 biological replicates; series of unpaired t-tests /Mann–Whitney U tests with DLK-WT-GFP followed by a Holm–Sidak correction). G–I. Representative stills (G), representative kymographs (H) and quantification of the number of GFP-positive particles (I) from time-lapse recordings of the different GFP-tagged DLK mutants in DIV6 cultured embryonic DRG neurons (n = 31–65 neurons/condition; series of Mann-Whitney U tests with DLK-WT-GFP followed by a Holm-Sidak correction). J–L. Representative stills (J), kymographs (K) and quantification of the number of DLK-GFP positive particles (L) from time-lapse recordings of DIV6 DRG neurons overexpressing GFP-tagged DLK and treated with control (DMSO) or DLK inhibitor GNE-3511 (DLKi) for 3 h (n = 22–46 neurons/condition; Unpaired t-test). Data information: Arrowheads point to individual DLK-GFP positive structures in G and J. All graphs represent mean ± SEM. *P < 0.05 and ***P < 0.001. Scale bar represents 100 µm in (D), 10 µm in (G) and (J), 5 µm in (H), (K) and zooms in (D). See also Appendix Fig S2. Source data are available online for this figure. Source Data for Figure 3 [embj2021110155-sup-0006-SDataFig3.zip] Download figure Download PowerPoint Sphingomyelin regulates DLK-vesicle localization and kinase activity We next determined the identity of the DLK-positive vesicles by staining for various vesicular structures, including markers for the secretory pathway, such as post-Golgi secretory vesicles (Rab6) and synaptic precursor vesicles (Rab3), and markers for vesicles from the endocytic pathway, including late endosomes (Rab7) and recycling endosomes (Rab11) (Fig 4A). Object-based colocalization analysis revealed that DLK was present on various vesicles in the secretory and endosomal pathway, and was not associated with a specific vesicular subcompartment (Fig 4B, Appendix Fig S3A–C). Based on these results, we investigated whether DLK-positive vesicles have a unique lipid signature. We expressed control HA, DLK-WT-HA, and DLK-CS-HA in HeLa cells, performed anti-HA pull downs, and ran an unbiased lipidomic analysis on the samples. We found that vesicles containing overexpressed HA-tagged DLK were enriched in lipids such as phosphatidylethanolamine, phosphatidylcholines, and sphingomyelin (Fig 4C–E). From these lipids only sphingomyelin levels and species were significantly different between DLK-WT-HA and DLK-CS-HA (Fig 4E and F). Consistently, inhibition of sphingomyelin synthase with compounds such as D609 blocked both DLK-vesicle localization and the increase in c-Jun phosphorylation induced by NGF withdrawal, while did not affect vesicular structures, axon integrity and neuron viability (Fig 4G–L, Appendix Fig S3D–G). Together, the data suggest that DLK vesicle localization and further kinase activation requires the presence of sphingomyelin in membrane vesicles. Figure 4. Sphingomy
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