STK 38 kinase acts as XPO 1 gatekeeper regulating the nuclear export of autophagy proteins and other cargoes
2019; Springer Nature; Volume: 20; Issue: 11 Linguagem: Inglês
10.15252/embr.201948150
ISSN1469-3178
AutoresAlexandre Martin, Maarten Jacquemyn, Joanna Lipecka, Cérina Chhuon, Vasily N. Aushev, Brigitte Meunier, Manish Kumar Singh, Nicolas Carpi, Matthieu Piel, Patrice Codogno, Alexander Hergovich, Maria Carla Parrini, Gérard Zalcman, Ida Chiara Guerrera, Dirk Daelemans, Jacques Camonis,
Tópico(s)Endoplasmic Reticulum Stress and Disease
ResumoArticle23 September 2019free access Transparent process STK38 kinase acts as XPO1 gatekeeper regulating the nuclear export of autophagy proteins and other cargoes Alexandre PJ Martin orcid.org/0000-0003-3875-6754 ART Group, Inserm U830, Paris, France Institut Curie, Centre de Recherche, Paris Sciences et Lettres Research University, Paris, France Search for more papers by this author Maarten Jacquemyn Laboratory of Virology and Chemotherapy, KU Leuven Department of Microbiology, Immunology and Transplantation, Rega Institute for Medical Research, KU Leuven, Leuven, Belgium Search for more papers by this author Joanna Lipecka Inserm U894, Center of Psychiatry and Neuroscience, Paris, France Université Paris Descartes, Sorbonne Paris Cité, Paris, France Search for more papers by this author Cerina Chhuon Université Paris Descartes, Sorbonne Paris Cité, Paris, France Proteomics Platform 3P5-Necker, Université Paris Descartes - Structure Fédérative de Recherche Necker, INSERM US24/CNRS UMS3633, Paris, France Search for more papers by this author Vasily N Aushev Icahn School of Medicine at Mount Sinai, New York, NY, USA Search for more papers by this author Brigitte Meunier ART Group, Inserm U830, Paris, France Institut Curie, Centre de Recherche, Paris Sciences et Lettres Research University, Paris, France Search for more papers by this author Manish K Singh ART Group, Inserm U830, Paris, France Institut Curie, Centre de Recherche, Paris Sciences et Lettres Research University, Paris, France Search for more papers by this author Nicolas Carpi Institut Curie, Centre de Recherche, Paris Sciences et Lettres Research University, Paris, France CNRS, UMR 144, Paris, France Search for more papers by this author Matthieu Piel Institut Curie, Centre de Recherche, Paris Sciences et Lettres Research University, Paris, France CNRS, UMR 144, Paris, France Search for more papers by this author Patrice Codogno Université Paris Descartes, Sorbonne Paris Cité, Paris, France Inserm U1151/CNRS UMR 8253, Institut Necker Enfants-Malades, Paris, France Search for more papers by this author Alexander Hergovich Cancer Institute, University College London, London, UK Search for more papers by this author Maria Carla Parrini ART Group, Inserm U830, Paris, France Institut Curie, Centre de Recherche, Paris Sciences et Lettres Research University, Paris, France Search for more papers by this author Gerard Zalcman ART Group, Inserm U830, Paris, France Institut Curie, Centre de Recherche, Paris Sciences et Lettres Research University, Paris, France Sorbonne Paris Cité, Université Paris Diderot, Paris, France Search for more papers by this author Ida Chiara Guerrera Université Paris Descartes, Sorbonne Paris Cité, Paris, France Proteomics Platform 3P5-Necker, Université Paris Descartes - Structure Fédérative de Recherche Necker, INSERM US24/CNRS UMS3633, Paris, France Search for more papers by this author Dirk Daelemans Laboratory of Virology and Chemotherapy, KU Leuven Department of Microbiology, Immunology and Transplantation, Rega Institute for Medical Research, KU Leuven, Leuven, Belgium Search for more papers by this author Jacques H Camonis Corresponding Author [email protected] orcid.org/0000-0003-2047-4150 ART Group, Inserm U830, Paris, France Institut Curie, Centre de Recherche, Paris Sciences et Lettres Research University, Paris, France Search for more papers by this author Alexandre PJ Martin orcid.org/0000-0003-3875-6754 ART Group, Inserm U830, Paris, France Institut Curie, Centre de Recherche, Paris Sciences et Lettres Research University, Paris, France Search for more papers by this author Maarten Jacquemyn Laboratory of Virology and Chemotherapy, KU Leuven Department of Microbiology, Immunology and Transplantation, Rega Institute for Medical Research, KU Leuven, Leuven, Belgium Search for more papers by this author Joanna Lipecka Inserm U894, Center of Psychiatry and Neuroscience, Paris, France Université Paris Descartes, Sorbonne Paris Cité, Paris, France Search for more papers by this author Cerina Chhuon Université Paris Descartes, Sorbonne Paris Cité, Paris, France Proteomics Platform 3P5-Necker, Université Paris Descartes - Structure Fédérative de Recherche Necker, INSERM US24/CNRS UMS3633, Paris, France Search for more papers by this author Vasily N Aushev Icahn School of Medicine at Mount Sinai, New York, NY, USA Search for more papers by this author Brigitte Meunier ART Group, Inserm U830, Paris, France Institut Curie, Centre de Recherche, Paris Sciences et Lettres Research University, Paris, France Search for more papers by this author Manish K Singh ART Group, Inserm U830, Paris, France Institut Curie, Centre de Recherche, Paris Sciences et Lettres Research University, Paris, France Search for more papers by this author Nicolas Carpi Institut Curie, Centre de Recherche, Paris Sciences et Lettres Research University, Paris, France CNRS, UMR 144, Paris, France Search for more papers by this author Matthieu Piel Institut Curie, Centre de Recherche, Paris Sciences et Lettres Research University, Paris, France CNRS, UMR 144, Paris, France Search for more papers by this author Patrice Codogno Université Paris Descartes, Sorbonne Paris Cité, Paris, France Inserm U1151/CNRS UMR 8253, Institut Necker Enfants-Malades, Paris, France Search for more papers by this author Alexander Hergovich Cancer Institute, University College London, London, UK Search for more papers by this author Maria Carla Parrini ART Group, Inserm U830, Paris, France Institut Curie, Centre de Recherche, Paris Sciences et Lettres Research University, Paris, France Search for more papers by this author Gerard Zalcman ART Group, Inserm U830, Paris, France Institut Curie, Centre de Recherche, Paris Sciences et Lettres Research University, Paris, France Sorbonne Paris Cité, Université Paris Diderot, Paris, France Search for more papers by this author Ida Chiara Guerrera Université Paris Descartes, Sorbonne Paris Cité, Paris, France Proteomics Platform 3P5-Necker, Université Paris Descartes - Structure Fédérative de Recherche Necker, INSERM US24/CNRS UMS3633, Paris, France Search for more papers by this author Dirk Daelemans Laboratory of Virology and Chemotherapy, KU Leuven Department of Microbiology, Immunology and Transplantation, Rega Institute for Medical Research, KU Leuven, Leuven, Belgium Search for more papers by this author Jacques H Camonis Corresponding Author [email protected] orcid.org/0000-0003-2047-4150 ART Group, Inserm U830, Paris, France Institut Curie, Centre de Recherche, Paris Sciences et Lettres Research University, Paris, France Search for more papers by this author Author Information Alexandre PJ Martin1,2, Maarten Jacquemyn3, Joanna Lipecka4,5, Cerina Chhuon5,6, Vasily N Aushev7, Brigitte Meunier1,2, Manish K Singh1,2, Nicolas Carpi2,8, Matthieu Piel2,8, Patrice Codogno5,9, Alexander Hergovich10, Maria Carla Parrini1,2, Gerard Zalcman1,2,11, Ida Chiara Guerrera5,6, Dirk Daelemans3 and Jacques H Camonis *,1,2 1ART Group, Inserm U830, Paris, France 2Institut Curie, Centre de Recherche, Paris Sciences et Lettres Research University, Paris, France 3Laboratory of Virology and Chemotherapy, KU Leuven Department of Microbiology, Immunology and Transplantation, Rega Institute for Medical Research, KU Leuven, Leuven, Belgium 4Inserm U894, Center of Psychiatry and Neuroscience, Paris, France 5Université Paris Descartes, Sorbonne Paris Cité, Paris, France 6Proteomics Platform 3P5-Necker, Université Paris Descartes - Structure Fédérative de Recherche Necker, INSERM US24/CNRS UMS3633, Paris, France 7Icahn School of Medicine at Mount Sinai, New York, NY, USA 8CNRS, UMR 144, Paris, France 9Inserm U1151/CNRS UMR 8253, Institut Necker Enfants-Malades, Paris, France 10Cancer Institute, University College London, London, UK 11Sorbonne Paris Cité, Université Paris Diderot, Paris, France *Corresponding author. Tel: +33 1 56 24 66 54; Fax: +33 1 56 24 66 50; E-mail: [email protected] EMBO Rep (2019)20:e48150https://doi.org/10.15252/embr.201948150 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 STK38 (also known as NDR1) is a Hippo pathway serine/threonine protein kinase with multifarious functions in normal and cancer cells. Using a context-dependent proximity-labeling assay, we identify more than 250 partners of STK38 and find that STK38 modulates its partnership depending on the cellular context by increasing its association with cytoplasmic proteins upon nutrient starvation-induced autophagy and with nuclear ones during ECM detachment. We show that STK38 shuttles between the nucleus and the cytoplasm and that its nuclear exit depends on both XPO1 (aka exportin-1, CRM1) and STK38 kinase activity. We further uncover that STK38 modulates XPO1 export activity by phosphorylating XPO1 on serine 1055, thus regulating its own nuclear exit. We expand our model to other cellular contexts by discovering that XPO1 phosphorylation by STK38 regulates also the nuclear exit of Beclin1 and YAP1, key regulator of autophagy and transcriptional effector, respectively. Collectively, our results reveal STK38 as an activator of XPO1, behaving as a gatekeeper of nuclear export. These observations establish a novel mechanism of XPO1-dependent cargo export regulation by phosphorylation of XPO1's C-terminal auto-inhibitory domain. Synopsis Cytoplasmic accumulation of STK38 kinase is essential for starvation-induced autophagy. STK38 phosphorylates and activates the nuclear export factor XPO1, thereby supporting the shuttling of the autophagy regulator Beclin1 and other XPO1 cargoes to the cytoplasm. XPO1 is a substrate for the STK38 kinase. XPO1 is activated by this phosphorylation event on Ser1055. STK38 behaves as a regulator of XPO1 activity, which is inactive in absence of phosphorylation of its Ser1055. Introduction The serine/threonine kinase 38 STK38, also known as NDR1, is important in multifarious and unrelated biological functions, playing roles in cell cycle progression 1, 2, apoptosis 3, and centrosome duplication 4, 5. As a member of the Hippo core signaling, STK38 can directly phosphorylate the Hippo effector YAP1 on S127, resulting in YAP1 inactivation by cytoplasmic sequestration 6. In addition, STK38 has pro-cancer cell survival functions in stress response and adaptation. STK38 responds to osmotic shock and to the expression of RASSF1A, a Hippo pathway regulator 7. Moreover, STK38 kinase activity is required for autophagy induction in response to starvation by regulating Beclin1 8. Finally, STK38 was also reported to be involved in resistance to anoïkis of cancer cells 9. This non-comprehensive inventory illustrates the variety and multiplicity of cellular functions driven by and/or dependent on STK38. How a single kinase can perform these crucial, but clearly distinct, functions? Would it have function-specific substrates or a yet-unknown common regulator being permissive for these functions? To address this question, we performed a proximity-dependent biotinylation to map STK38 partnership in different cellular contexts. This revealed that STK38 interacts mainly with cytoplasmic proteins upon starvation-induced autophagy, and with nuclear proteins after ECM detachment, suggesting that the subcellular localization of STK38 plays a regulatory role in response to these diverse stimuli. In addition, we confirmed a nuclear/cytoplasmic shuttling of STK38 being under the dependency of its own kinase activity and on XPO1 (exportin-1, aka CRM1) nuclear export. Moreover, we discovered that STK38 phosphorylates the serine 1055 in the auto-inhibitory domain of XPO1, thereby triggering the nuclear export of STK38 itself as well as other XPO1 cargoes such as Beclin1 and YAP1. These results suggest that STK38 regulates the subcellular localization and thereby the function of central cellular components by modulating their nuclear export via phosphorylation of XPO1 on its auto-inhibitory domain. Results STK38 interacts with different sets of partners depending on the cellular context To identify the proteins that interact with STK38 upon nutrient starvation-induced autophagy, or when cells resist to anoïkis in suspension, we undertook a context-dependent proteomic approach 10 by establishing cell lines stably expressing APEX2 N-terminally fused to STK38 (Fig EV1). HeLa cells were used for nutrient starvation-induced autophagy condition and HEK-HT-HRasG12V cells (hereafter referred to as HekRasV12) 11, 12 for anoïkis resistance condition, to match with previous studies of STK38's roles in autophagy 8 and survival upon ECM detachment 9, respectively. A quantitative SILAC proteomic methodology was applied, comparing quantitatively, on the one hand, complete medium incubation versus nutrient starvation-induced autophagy (see Fig EV1 for this example) and, on the other hand, attached growth versus suspension growth. Validation of autophagy process (Appendix Fig S1A and B) and biotinylation efficiency in both contexts (Appendix Fig S1C and D) was performed before mass spectrometry (MS) identification. Each replicate displayed a good correlation between experiments (Appendix Fig S1E), indicating a high reproducibility between biological triplicate samples. Click here to expand this figure. Figure EV1. STK38 partner identification strategy (related to Fig 1)Figure indicating the strategy used to identify STK38 partner association dynamic depending on the context (example is shown for autophagy condition here). Stable cell lines expressing the fusion construct APEX2-STK38 were generated and then subdivided for amino acid replacement (SILAC). Context was then induced (4 h of EBSS incubation for autophagy induction and suspension growth) as well as a control condition (nutrient-rich medium incubation and attached growth, respectively). Proximity labeling of STK38 partners was performed as described 56: Briefly, cells were incubated with phenol–biotin for a minimum of 30 min followed by H2O2 incubation for 1 min precisely. Finally, biotinylated proteins (=STK38 partners) were purified from whole-cell lysates using streptavidin-coated magnetic beads and subjected to mass spectrometry identification. Download figure Download PowerPoint Ninety-seven binding partners of STK38 were identified in the context of autophagy, and 221 partners were identified in the context of suspension growth (Fig 1A) (see Dataset EV1 for the complete list), including several known interactors of STK38 such as MAP4K4 7, HIST2H2AC 13, EWSR1 14, NPM1 15, YWHAZ 16, and MAGOH 17. Among these 97 interactors, 32 displayed an interaction with STK38 that was significantly increased upon nutrient starvation-induced autophagy as compared to rich medium, while interaction with only one protein was decreased. Upon ECM detachment, 44 proteins displayed an increased interaction with STK38, while for 72 proteins, the interaction with STK38 was decreased. Interestingly, 50 partners were common to both studied conditions, but displayed a differential association status with STK38, depending on the context (Fig 1A). These 50 common partners were classified by unsupervised hierarchical clustering, based on their association status with STK38, resulting in two main clusters as highlighted in orange and purple in Fig 1B. The upper cluster (orange) is composed of 31 proteins mainly increasing their interaction with STK38 upon nutrient starvation-induced autophagy while decreasing their interaction with STK38 upon ECM detachment. The lower cluster (purple) consists of 19 proteins that increase their interaction with STK38 in suspension but do not see their interaction with STK38 modified upon nutrient starvation-induced autophagy. Gene Ontology analysis of these two groups of interactors revealed a striking difference (Fig 1C): The most enriched terms upon nutrient starvation-induced autophagy characterize these proteins as localized in the cytoplasm, while the cluster enriched upon ECM detachment contains mainly nuclear proteins. These results suggest that upon autophagy induction, STK38 associates with cytoplasmic partners while, upon ECM detachment, STK38 appears to preferentially interact with nuclear proteins. Figure 1. STK38 associates with cytoplasmic interactors upon nutrient starvation and with nuclear interactors upon suspension growth Venn diagram of STK38 partners identified in both starvation and suspension conditions by proximity biotinylation assay coupled to mass spectrometry identification (see Appendix Table S1 for the complete list of STK38 context-dependent interactors and Fig EV1 for the context-dependent protein labeling strategy). Heatmap representation of common STK38 interactors identified in both starvation and suspension conditions according to their dynamic of association with STK38 (with a minimal fold increase of 1.38 ± 0.06). Unsupervised hierarchical clustering was generated based on their association fold using Pearson correlation. Representation of the 10 most cellular component enriched terms of the two clusters (assessed by using the Gene Ontology (GO) database; http://www.geneontology.org/). Download figure Download PowerPoint STK38 interacts with XPO1 The above observations imply that STK38 shuttles between the nucleus and the cytoplasm. Exportin-1 (XPO1), also known as the chromosomal region maintenance protein 1 (CRM1), the main nuclear export factor (karyopherin) that transports a wide diversity of proteins from the nucleus to the cytoplasm 18-21, was identified in our screen as a novel STK38 interactor (Dataset EV1). To investigate whether STK38 interacts with XPO1, we performed a pull-down experiment. myc-tagged STK38 was transiently co-expressed with Flag-XPO1, or Flag-Sirt3 as a control. Upon pull-down using Flag antibody, STK38 co-immunoprecipitated with XPO1 but not with Flag-Sirt3 (Fig 2A), suggesting that STK38 physically interacts with XPO1. In addition, inhibiting protein phosphatase type 2A (PP2A) with okadaic acid (OA), which increases STK38 phosphorylation state and activity 22, did not modify the binding of STK38 and XPO1. Figure 2. XPO1 activity is required for STK38 cytoplasmic accumulation and autophagy upon nutrient starvation A. STK38 interacts with XPO1. HekRasV12 cells were transiently transfected with myc-STK38(wt) together with either Flag-XPO1(wt) plasmid or Flag-control (ctrl = Sirt3) plasmid or without DNA. Twenty-four hours later, cells were incubated with okadaic acid (OA) (final concentration = 1 μM) for 1 h or with DMSO. Flag fusions were pulled down, and co-immunoprecipitated proteins were analyzed by Western blotting (WB). The upper panel displays whole-cell lysates (WCL), and the lower panel represents immunoprecipitated proteins. B, C. STK38 accumulates in the cytoplasm upon nutrient starvation in a XPO1-dependent manner. (B) HeLa cells were transfected with myc-STK38(wt) plasmid. The next day, cells were incubated with DMEM or EBSS in the presence of XPO1 inhibitor KPT-185 or KPT-330 as indicated (final concentration = 1 μM) or DMSO for 4 h. Cells were then fixed and stained for myc-tag. Representative images are shown, and scale bars are 40 μm. (C) Quantification of myc-STK38(wt) nuclear/cytoplasmic staining (n > 30 cells from 3 independent experiments, mean ± SEM; *P < 0.05, **P < 0.01, ***P < 0.001, Mann–Whitney test). D, E. XPO1 activity is required for nutrient starvation-induced autophagy. (D) Immunoblotting with the indicated antibodies of whole-cell lysates of cells in (B) and (C) and its graphical representation (n = 3 independent experiments, mean ± SEM; ***P < 0.001, ns, not significant, Student's t-test). As expected, p62 degradation in starvation conditions was inhibited in the presence of XPO1 inhibitors. (E) HeLa cells stably expressing the GFP-LC3-RFP-LC3ΔG reporter autophagic probe 26 were incubated with DMEM or EBSS in the presence of XPO1 inhibitor KPT-185 or KPT-330 (final concentration = 1 μM) or DMSO for 4 h. The GFP-LC3 (degraded upon autophagy) and RFP-LC3ΔG (non-degraded upon autophagy) signals were recorded by FACS analysis and are shown as a ratio recapitulating overall LC3 level (n = 3 independent experiments, mean ± SEM; *P < 0.05, **P < 0.01, ns, not significant, Mann–Whitney test). Here again, incubation with XPO1 inhibitors significantly impaired the autophagy process (see Appendix Fig S2C and D for stable cell line validation). Download figure Download PowerPoint STK38 accumulates in the cytoplasm upon nutrient starvation-induced autophagy in a XPO1-dependent manner Our proximity-labeling experiment suggested that STK38 interacts mainly with cytoplasmic proteins upon nutrient starvation, suggesting that in this condition, STK38 is localized to the cytoplasm. Due to the lack of specific and sensitive antibodies to follow endogenous STK38 subcellular localization using immunofluorescence (IF), HeLa cells were transfected with a plasmid expressing myc-tagged STK38. Immunostaining of STK38 in nutrient-starved cells (EBSS medium) demonstrated that STK38 localizes mainly in the cytoplasm, while in nutrient-rich medium (complete DMEM) STK38 is mainly localized in the nucleus (Fig 2B and C). The highly selective XPO1 inhibitors KPT-185 and KPT-330 23, 24 inhibited STK38's exit form the nucleus. XPO1 inhibition by KPT-185 and KPT-330 was validated in parallel by monitoring the nucleo-/cytoplasmic localization of a well-known cargo of XPO1: IκBα (Appendix Fig S2A and B). Together, these results indicate that STK38 shuttles from the nucleus to the cytoplasm upon nutrient starvation in a XPO1-dependent manner. To study whether the XPO1-dependent transport of STK38 contributes to STK38's function in autophagy 8, 25, we monitored the levels of p62/SQSTM1, a well-known autophagy substrate. As expected, p62 levels decreased upon starvation (Fig 2D). However, the inhibition of XPO1 with the subsequent inhibition of STK38 cytoplasmic localization reduced p62 degradation (Fig 2D), indicating a defect in autophagy flux. As a complementary approach, we generated a stable cell line expressing the autophagic flux probe GFP-LC3-RFP-LC3Δ 26. HeLa GFP-LC3-RFP-LC3Δ cells were validated by silencing autophagy regulators such as ATG5 and Beclin1 followed by measuring the GFP-LC3/RFP-LC3Δ ratio by FACS. Similar to the silencing of ATG5 or Beclin1, knockdown of STK38 significantly impaired the reduction of the GFP-LC3/RFP-LC3Δ ratio observed in the control condition upon EBSS treatment, indicating a defect in autophagy in these conditions (Appendix Fig S2C and D). This method was used to examine XPO1's contribution to nutrient starvation-induced autophagy (Fig 2E). Significantly, XPO1 inhibition by both KPT-185 and KPT-330 significantly reduced autophagy, as measured by the GFP-LC3/RFP-LC3Δ ratios. Taken together, these results indicate that STK38 is exported to the cytoplasm by XPO1 upon starvation-induced autophagy and that XPO1 has to be functional for the resulting starvation-induced autophagy process, but are in contrast to a previous report where blockage of XPO1 led to the nuclear accumulation of TFEB and to the induction of autophagy 27. This apparent discrepancy arises in two different settings: with XPO1 being inhibited for 6 h for the transcription-dependent autophagy induction 27, while in our case, 4 h of inhibition led to a blockage of autophagy. On the other hand, the TFEB-dependent impact concerned basal autophagy as opposed to our observation related to starvation-induced autophagy. STK38 kinase activity is necessary and sufficient to induce its own cytoplasmic relocalization and autophagy To investigate whether STK38's kinase activity is required for its nuclear/cytoplasmic shuttling, HeLa cells were silenced for endogenous STK38 followed by transient expression of wild-type STK38 (wt), kinase-dead (K118R) 3, or constitutively active (PIF) STK38 28. Transfected cells were subsequently submitted to nutrient starvation and stained for STK38 subcellular localization (Fig 3A and B). As expected, STK38(wt) accumulated in the cytoplasm of nutrient-starved cells. However, kinase-dead STK38(K118R) remained nuclear upon starvation while a constitutively active version of STK38(PIF) accumulated in the cytoplasm, irrespective of culture conditions (Fig 3A and B). These results indicate that STK38 kinase activity is required and sufficient to induce its export to the cytoplasm, independently of physiological conditions. Figure 3. STK38 kinase activity is necessary and sufficient to induce its cytoplasmic relocalization and autophagy A, B. STK38 kinase activity is required and sufficient for its cytoplasmic accumulation upon nutrient starvation. (A) HeLa cells silenced for endogenous STK38 were transfected with myc-STK38(wt) expressing plasmid, myc-STK38(K118R) (STK38 kinase-dead version) plasmid, or HA-STK38(PIF) (STK38 constitutively active version) plasmid. Twenty-four hours later, cells were incubated with DMEM or EBSS for 4 h, fixed, and stained for myc-tag or HA-tag. Representative images are shown, and scale bars are 40 μm. (B) Graphical representation of tag-STK38 variant nuclear staining/cytoplasmic staining (n > 30 cells from three independent experiments, mean ± SEM; **P < 0.01, ***P < 0.001, ns, not significant, Mann–Whitney test). C, D. STK38 kinase activity is required and sufficient for nutrient starvation-induced autophagy. (C) HeLa cells stably expressing the GFP-LC3-RFP-LC3ΔG autophagic probe 26 were transiently transfected with siRNA targeting the 3′UTR region of endogenous STK38 (or with non-targeting siRNA (siNT)). The next day, cells were transiently transfected with the indicated STK38 mutants expressing plasmids (identical to (A–B)). Twenty-four hours after plasmid transfection, cells were incubated with DMEM or EBSS for 4 h. The GFP-LC3 (degraded upon autophagy) and RFP-LC3ΔG (non-degraded upon autophagy) signals were recorded by FACS analysis and are shown as a ratio recapitulating overall LC3 level (n = 4 independent experiments, mean ± SEM; *P < 0.05, ***P < 0.001, ns, not significant, Mann–Whitney test). As published 8, depleting STK38 prevents autophagy to take place. This effect was partially reversed by expressing the wt ORF as well as constitutively active STK38, whereas kinase-dead version failed to reproduce endogenous STK38 effect. Expression of constitutively active STK38 is sufficient to induce a substantial change in the autophagic flux upon nutrient-rich conditions (see Appendix Fig S3A for STK38 replacement). (D) HeLa cells were transiently transfected with siRNA targeting the 3′UTR region of endogenous STK38 (or with non-targeting siRNA (siNT)) and then transiently transfected with the indicated STK38 mutants expressing plasmids 48 h after. The next day, the cells were incubated with DMEM or EBSS for 4 h and overall p62 level was quantified for autophagy estimation (see Appendix Fig S3B for blots) (n = 3 independent experiments, mean ± SEM; ***P < 0.001, ns, not significant, Student's t-test). Download figure Download PowerPoint To further investigate whether STK38 kinase activity is also involved in nutrient starvation-induced autophagy, we knocked down endogenous STK38 in HeLa GFP-LC3-RFP-LC3ΔG cells, then complemented these cells with RNAi-resistant STK38(wt), K118R, or PIF variants (Appendix Fig S3A), and measured the autophagic flux (Fig 3C). As expected 8, STK38 depletion significantly impaired autophagy upon nutrient starvation, which was restored by reintroducing wild-type STK38 (Fig 3C). In stark contrast, expression of kinase-dead version of STK38(K118R) failed to restore nutrient starvation-induced autophagy, while constitutively active STK38(PIF) supported autophagy. Interestingly, expression of constitutively active STK38(PIF) is sufficient to induce a significant decrease in the GFP-LC3/RFP-LC3ΔG in nutrient-rich conditions, indicating an increase in autophagy (Fig 3C). The same effects were observed while using a complementary approach for autophagy measurement: by monitoring the p62 levels (Fig 3D). In conclusion, these experiments support that the kinase activity of STK38 is important for both its subcellular localization and autophagy and that STK38 is instructive and permissive for autophagy (a situation similar to the one observed in flies 8). STK38 phosphorylates XPO1 on serine 1055 The presence of a bona fide STK38 HxRxxS/T phosphorylation motif 29 in XPO1 protein sequence offered a patent implication of a potential kinase–substrate relationship between STK38 and XPO1. This motif is centered on serine 1055, reported as being phosphorylated in human cells by an undetermined kinase without any functional relevance 30-33, and S1055 phosphorylation was required for XPO1 interaction with a 14-3-3 34. We generated a phospho-specific anti-S1055-P antibody that was validated using wt or phospho-acceptor (S1055A) mutant of XPO1 (Fig EV2A). The anti-XPO1-S1055-P antibody exhibited a strong specificity for XPO1(wt) upon OA incubation, a potent inhibitor of protein phosphatase type 2A (PP2A) that boosts most phosphorylation in cells, including STK38 22, leading to XPO1 serine phosphorylation. Concurrently, as expected, the XPO1(S1055A) mutant was not detected with this antibody in both whole-cell lysates and after pulling down Flag-XPO1 variants (Fig EV2A). Click here to expand this figure. Figure EV2. Validation of pS1055_XPO1 antibody (related to Fig 4) HeLa cells were transiently transfected with the indicated Flag-XPO1 mutants
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