Akt‐mediated phosphorylation of MICU 1 regulates mitochondrial Ca 2+ levels and tumor growth
2018; Springer Nature; Volume: 38; Issue: 2 Linguagem: Inglês
10.15252/embj.201899435
ISSN1460-2075
AutoresSaverio Marchi, Mariangela Corricelli, Alessio Branchini, Veronica Angela Maria Vitto, Sonia Missiroli, Giampaolo Morciano, Mariasole Perrone, Mattia Ferrarese, Carlotta Giorgi, Mirko Pinotti, Lorenzo Galluzzi, Guido Kroemer, Paolo Pinton,
Tópico(s)Protein Tyrosine Phosphatases
ResumoArticle30 November 2018free access Source DataTransparent process Akt-mediated phosphorylation of MICU1 regulates mitochondrial Ca2+ levels and tumor growth Saverio Marchi Corresponding Author [email protected] orcid.org/0000-0003-2708-1843 Laboratory for Technologies of Advanced Therapies (LTTA), Department of Morphology, Surgery and Experimental Medicine, Section of Pathology, Oncology and Experimental Biology, University of Ferrara, Ferrara, Italy Search for more papers by this author Mariangela Corricelli Laboratory for Technologies of Advanced Therapies (LTTA), Department of Morphology, Surgery and Experimental Medicine, Section of Pathology, Oncology and Experimental Biology, University of Ferrara, Ferrara, Italy Search for more papers by this author Alessio Branchini orcid.org/0000-0002-6113-2694 Department of Life Sciences and Biotechnology, University of Ferrara, Ferrara, Italy Search for more papers by this author Veronica Angela Maria Vitto Laboratory for Technologies of Advanced Therapies (LTTA), Department of Morphology, Surgery and Experimental Medicine, Section of Pathology, Oncology and Experimental Biology, University of Ferrara, Ferrara, Italy Search for more papers by this author Sonia Missiroli Laboratory for Technologies of Advanced Therapies (LTTA), Department of Morphology, Surgery and Experimental Medicine, Section of Pathology, Oncology and Experimental Biology, University of Ferrara, Ferrara, Italy Search for more papers by this author Giampaolo Morciano Laboratory for Technologies of Advanced Therapies (LTTA), Department of Morphology, Surgery and Experimental Medicine, Section of Pathology, Oncology and Experimental Biology, University of Ferrara, Ferrara, Italy Maria Cecilia Hospital, GVM Care & Research, Cotignola, Italy Search for more papers by this author Mariasole Perrone Laboratory for Technologies of Advanced Therapies (LTTA), Department of Morphology, Surgery and Experimental Medicine, Section of Pathology, Oncology and Experimental Biology, University of Ferrara, Ferrara, Italy Search for more papers by this author Mattia Ferrarese Department of Life Sciences and Biotechnology, University of Ferrara, Ferrara, Italy Search for more papers by this author Carlotta Giorgi Laboratory for Technologies of Advanced Therapies (LTTA), Department of Morphology, Surgery and Experimental Medicine, Section of Pathology, Oncology and Experimental Biology, University of Ferrara, Ferrara, Italy Search for more papers by this author Mirko Pinotti Department of Life Sciences and Biotechnology, University of Ferrara, Ferrara, Italy Search for more papers by this author Lorenzo Galluzzi orcid.org/0000-0003-2257-8500 Department of Radiation Oncology, Weill Cornell Medical College, New York, NY, USA Université Paris Descartes, Sorbonne Paris Cité, Paris, France Search for more papers by this author Guido Kroemer orcid.org/0000-0002-9334-4405 Université Paris Descartes, Sorbonne Paris Cité, Paris, France Equipe 11 Labellisée Ligue Nationale Contre le Cancer, Centre de Recherche des Cordeliers, Paris, France Institut National de la Santé et de la Recherche Médicale, U1138, Paris, France Université Pierre et Marie Curie, Paris, France Metabolomics and Cell Biology Platforms, Gustave Roussy Cancer Campus, Villejuif, France Center of Clinical Investigations in Biotherapies of Cancer (CICBT), Villejuif, France Pôle de Biologie, Hôpital Européen Georges Pompidou, AP-HP, Paris, France Department of Women's and Children's Health, Karolinska University Hospital, Stockholm, Sweden Search for more papers by this author Paolo Pinton Corresponding Author [email protected] orcid.org/0000-0001-7108-6508 Laboratory for Technologies of Advanced Therapies (LTTA), Department of Morphology, Surgery and Experimental Medicine, Section of Pathology, Oncology and Experimental Biology, University of Ferrara, Ferrara, Italy Maria Cecilia Hospital, GVM Care & Research, Cotignola, Italy Search for more papers by this author Saverio Marchi Corresponding Author [email protected] orcid.org/0000-0003-2708-1843 Laboratory for Technologies of Advanced Therapies (LTTA), Department of Morphology, Surgery and Experimental Medicine, Section of Pathology, Oncology and Experimental Biology, University of Ferrara, Ferrara, Italy Search for more papers by this author Mariangela Corricelli Laboratory for Technologies of Advanced Therapies (LTTA), Department of Morphology, Surgery and Experimental Medicine, Section of Pathology, Oncology and Experimental Biology, University of Ferrara, Ferrara, Italy Search for more papers by this author Alessio Branchini orcid.org/0000-0002-6113-2694 Department of Life Sciences and Biotechnology, University of Ferrara, Ferrara, Italy Search for more papers by this author Veronica Angela Maria Vitto Laboratory for Technologies of Advanced Therapies (LTTA), Department of Morphology, Surgery and Experimental Medicine, Section of Pathology, Oncology and Experimental Biology, University of Ferrara, Ferrara, Italy Search for more papers by this author Sonia Missiroli Laboratory for Technologies of Advanced Therapies (LTTA), Department of Morphology, Surgery and Experimental Medicine, Section of Pathology, Oncology and Experimental Biology, University of Ferrara, Ferrara, Italy Search for more papers by this author Giampaolo Morciano Laboratory for Technologies of Advanced Therapies (LTTA), Department of Morphology, Surgery and Experimental Medicine, Section of Pathology, Oncology and Experimental Biology, University of Ferrara, Ferrara, Italy Maria Cecilia Hospital, GVM Care & Research, Cotignola, Italy Search for more papers by this author Mariasole Perrone Laboratory for Technologies of Advanced Therapies (LTTA), Department of Morphology, Surgery and Experimental Medicine, Section of Pathology, Oncology and Experimental Biology, University of Ferrara, Ferrara, Italy Search for more papers by this author Mattia Ferrarese Department of Life Sciences and Biotechnology, University of Ferrara, Ferrara, Italy Search for more papers by this author Carlotta Giorgi Laboratory for Technologies of Advanced Therapies (LTTA), Department of Morphology, Surgery and Experimental Medicine, Section of Pathology, Oncology and Experimental Biology, University of Ferrara, Ferrara, Italy Search for more papers by this author Mirko Pinotti Department of Life Sciences and Biotechnology, University of Ferrara, Ferrara, Italy Search for more papers by this author Lorenzo Galluzzi orcid.org/0000-0003-2257-8500 Department of Radiation Oncology, Weill Cornell Medical College, New York, NY, USA Université Paris Descartes, Sorbonne Paris Cité, Paris, France Search for more papers by this author Guido Kroemer orcid.org/0000-0002-9334-4405 Université Paris Descartes, Sorbonne Paris Cité, Paris, France Equipe 11 Labellisée Ligue Nationale Contre le Cancer, Centre de Recherche des Cordeliers, Paris, France Institut National de la Santé et de la Recherche Médicale, U1138, Paris, France Université Pierre et Marie Curie, Paris, France Metabolomics and Cell Biology Platforms, Gustave Roussy Cancer Campus, Villejuif, France Center of Clinical Investigations in Biotherapies of Cancer (CICBT), Villejuif, France Pôle de Biologie, Hôpital Européen Georges Pompidou, AP-HP, Paris, France Department of Women's and Children's Health, Karolinska University Hospital, Stockholm, Sweden Search for more papers by this author Paolo Pinton Corresponding Author [email protected] orcid.org/0000-0001-7108-6508 Laboratory for Technologies of Advanced Therapies (LTTA), Department of Morphology, Surgery and Experimental Medicine, Section of Pathology, Oncology and Experimental Biology, University of Ferrara, Ferrara, Italy Maria Cecilia Hospital, GVM Care & Research, Cotignola, Italy Search for more papers by this author Author Information Saverio Marchi *,1, Mariangela Corricelli1, Alessio Branchini2, Veronica Angela Maria Vitto1, Sonia Missiroli1, Giampaolo Morciano1,3, Mariasole Perrone1, Mattia Ferrarese2, Carlotta Giorgi1, Mirko Pinotti2, Lorenzo Galluzzi4,5, Guido Kroemer5,6,7,8,9,10,11,12 and Paolo Pinton *,1,3 1Laboratory for Technologies of Advanced Therapies (LTTA), Department of Morphology, Surgery and Experimental Medicine, Section of Pathology, Oncology and Experimental Biology, University of Ferrara, Ferrara, Italy 2Department of Life Sciences and Biotechnology, University of Ferrara, Ferrara, Italy 3Maria Cecilia Hospital, GVM Care & Research, Cotignola, Italy 4Department of Radiation Oncology, Weill Cornell Medical College, New York, NY, USA 5Université Paris Descartes, Sorbonne Paris Cité, Paris, France 6Equipe 11 Labellisée Ligue Nationale Contre le Cancer, Centre de Recherche des Cordeliers, Paris, France 7Institut National de la Santé et de la Recherche Médicale, U1138, Paris, France 8Université Pierre et Marie Curie, Paris, France 9Metabolomics and Cell Biology Platforms, Gustave Roussy Cancer Campus, Villejuif, France 10Center of Clinical Investigations in Biotherapies of Cancer (CICBT), Villejuif, France 11Pôle de Biologie, Hôpital Européen Georges Pompidou, AP-HP, Paris, France 12Department of Women's and Children's Health, Karolinska University Hospital, Stockholm, Sweden *Corresponding author. Tel: +39 0532 455858; E-mail: s[email protected] *Corresponding author. Tel: +39 0532 455802; E-mail: [email protected] EMBO J (2019)38:e99435https://doi.org/10.15252/embj.201899435 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 Although mitochondria play a multifunctional role in cancer progression and Ca2+ signaling is remodeled in a wide variety of tumors, the underlying mechanisms that link mitochondrial Ca2+ homeostasis with malignant tumor formation and growth remain elusive. Here, we show that phosphorylation at the N-terminal region of the mitochondrial calcium uniporter (MCU) regulatory subunit MICU1 leads to a notable increase in the basal mitochondrial Ca2+ levels. A pool of active Akt in the mitochondria is responsible for MICU1 phosphorylation, and mitochondrion-targeted Akt strongly regulates the mitochondrial Ca2+ content. The Akt-mediated phosphorylation impairs MICU1 processing and stability, culminating in reactive oxygen species (ROS) production and tumor progression. Thus, our data reveal the crucial role of the Akt-MICU1 axis in cancer and underscore the strategic importance of the association between aberrant mitochondrial Ca2+ levels and tumor development. Synopsis The role of mitochondrial calcium uniporter (MCU) and mitochondrial calcium homeostasis in cancer progression is poorly understood. Active Akt in mitochondria phosphorylates MICU1 to functionally inhibit the MCU complex, thereby increasing basal mitochondrial calcium levels and promoting tumour progression. Akt phosphorylates a serine residue in the N-terminal region of MICU1. MICU1 phosphorylation increases mitochondrial [Ca2+] at resting conditions. Akt-mediated phosphorylation affects MICU1 maturation and stability. The Akt-MICU1 axis plays a critical role in cancer growth by regulating mitochondrial Ca2+ levels and ROS production. Introduction Mitochondrial Ca2+ accumulation controls cellular energetics and cell metabolism by favoring ATP production, and mitochondrial Ca2+ also operates as a key regulator of cell fate (Giorgi et al, 2018). Multiple pathological contexts, including tumor formation and development, are strictly linked to mitochondrial deregulation, and a hallmark feature of cancer cells is the re-programming of their mitochondrial metabolism (Hanahan & Weinberg, 2011; Boroughs & DeBerardinis, 2015). The connection between mitochondrial dysfunction and cancer is not only limited to the metabolic transformation of cancer cells but also triggers tumor-promoting epigenetic changes (Gottlieb & Tomlinson, 2005; Gaude & Frezza, 2014). Therefore, it is not surprising that several oncogenes and tumor suppressors exert their activities by regulating mitochondrial function (Galluzzi et al, 2012; Frezza, 2014) and that many of them act on key Ca2+-transport molecules to provoke deep mitochondrial Ca2+ homeostasis remodeling and promote certain malignant phenotypes (Prevarskaya et al, 2011; Danese et al, 2017). The mitochondrial calcium uniporter (MCU), which is the channel that is responsible for Ca2+ accumulation inside the mitochondrial matrix, has been recently characterized at the molecular level (Baughman et al, 2011; De Stefani et al, 2011). Moreover, a series of proteins that contribute to the formation of the so-called MCU complex have also been identified (Giorgi et al, 2018). Such proteins include the dominant-negative form MCUb (Raffaello et al, 2013), the essential regulator EMRE (Sancak et al, 2013), and others that regulate MCU channel activity, such as MICU1 (Perocchi et al, 2010) and its paralog MICU2 (Plovanich et al, 2013). Among the different components of the mitochondrial Ca2+ uptake machinery, MICU1 functions have been the subject of extensive studies that revealed that MICU1 acts as a gatekeeper for the MCU complex, setting the threshold for mitochondrial Ca2+ uptake (Mallilankaraman et al, 2012; Csordas et al, 2013). Although genetic MICU1 ablations in both cells and tissues and loss-of-function mutations in the MICU1 gene have been associated with different pathological scenarios (Logan et al, 2014; Antony et al, 2016), little is known about the role of MICU1-related mitochondrial Ca2+ homeostasis in tumor progression. Recent evidence indicates that both prostate and colon cancer cells overexpress cancer-related microRNAs that target the channel pore-forming subunit MCU, thus conferring resistance to apoptotic stimuli (Marchi et al, 2013). Moreover, MCU expression correlates with breast cancer progression, and the deletion of MCU reduces tumor growth and metastasis formation (Tosatto et al, 2016). No correlation has been observed between the expression of MCU regulators and tumor size, raising the possibility that instead of quantitative changes, qualitative alterations determined by post-translational modifications could account for the altered MCU function in oncogenesis (Tosatto et al, 2016). Here, we show that phosphorylation at the N-terminal domain of the MICU1 protein robustly alters the basal mitochondrial Ca2+ content under resting conditions. The target amino acid residue is contained in an Akt consensus phosphorylation motif, and MICU1 is readily phosphorylated upon Akt activation inside the mitochondrial compartment. Akt-mediated phosphorylation affects MICU1 proteolytic maturation and stability, thereby explaining the altered mitochondrial Ca2+ homeostasis. Importantly, the expression of a nonphosphorylatable MICU1 mutant significantly reduces the in vivo growth rate of tumors, even in the presence of activated Akt, suggesting a key role for the mitochondrial Akt-MICU1 axis in cancer progression. Results N-terminal MICU1 phosphorylation increases the basal mitochondrial Ca2+ levels We investigated the potentially phosphorylated residues in the MICU1 sequence. Using the Scansite 3 software program (http://scansite3.mit.edu), we searched for motifs within the wild-type (WT) MICU1 protein (NM_144822) that are likely to be phosphorylated by specific protein kinases. The following three candidates were identified: Ser124, Ser195, and Thr256 (Fig 1A). Among them, Ser124 displayed the highest value of surface accessibility, as well as a high phosphorylation prediction score (Fig 1A). Ser124 is localized in the N-terminal region of MICU1, which has been proposed to extend into the intermembrane space (Csordas et al, 2013). As a result, we generated two MICU1 mutants, a nonphosphorylatable S124A and a phosphomimetic S124D MICU1 mutant. When transfected into cells, both mutated MICU1 proteins localized correctly to mitochondria (Fig 1B). It is now widely accepted that MICU1 functions as a gatekeeper of the MCU channel, meaning that loss of MICU1 facilitates Ca2+ accumulation inside the mitochondrial matrix even at low cytosolic Ca2+ levels (Mallilankaraman et al, 2012). Thus, we transfected the mitochondrial matrix-targeted Ca2+ biosensor GCaMP6m into cells expressing the MICU1 WT or SD and SA mutants. Due to its characteristics as a ratiometric sensor, GCaMP6m is reputed to quantitatively measure even small differences in the resting mitochondrial Ca2+ concentration ([Ca2+]m) (Hill et al, 2014). We expressed WT or mutant forms of MICU1 in cells from which endogenous MICU1 was constitutively depleted using a short hairpin RNA (shRNA) (Fig EV1A). As expected, the resting mitochondrial Ca2+ levels were largely increased when MICU1 was stably downregulated, and the expression of both the WT MICU1 and SA mutant reduced the baseline [Ca2+]m levels in ShMICU1 cells. In contrast, the MICU1 SD variant failed to restore [Ca2+]m in ShMICU1 cells (Fig 1C and D). To verify the role of Ser124 phosphorylation in the regulation of MICU1 functionality, we analyzed the mitochondrial Ca2+ uptake following treatment with the sarcoplasmic/endoplasmic reticulum Ca2+ ATPase (SERCA) inhibitor 2,5-di-tert-butylhydroquinone (TBHQ), which induces slow and weak ER Ca2+ depletion (Waldeck-Weiermair et al, 2015). The co-transfection of the MICU1 WT, SD mutant, or SA mutant with an aequorin-based mitochondrial Ca2+ probe revealed that only mock and MICU1 SD-expressing cells were able to accumulate [Ca2+]m under these conditions, whereas the MICU1 WT and SA mutant failed to do so (Fig 1E and F). We obtained similar results when TBHQ was replaced with another SERCA antagonist, cyclopiazonic acid (CPA) (Fig 1G and H). The higher Ca2+ affinity of the MICU1 SD mutant-expressing mitochondria was also investigated in permeabilized cells. After permeabilization in an EGTA-containing Ca2+-free buffer (IB/EGTA) that mimics the physiological ion milieu, Ca2+ uptake was generated by switching the perfusion buffer to IB, containing EGTA-buffered Ca2+. Using different [Ca2+]s (500, 800 nM, and 1.5 μM), the rates of mitochondrial Ca2+ uptake were substantially increased in both MICU1-depleted and SD-expressing cells (Fig EV1B). Interestingly, MICU1 SA mutant slightly lowered the Ca2+ threshold for channel activation, showing reduced Ca2+ uptake rate compared to MICU1 WT (Fig EV1B). Overall, these data suggest that upon phosphorylation of serine 124, MICU1 loses its inhibitory function on the MCU complex. Figure 1. MICU1 phosphorylation at the Ser124 position increases the mitochondrial basal Ca2+ levels A. Potentially phosphorylated residues in the MICU1 (NM_144822) sequence were detected using the Scansite 3 software (http://scansite3.mit.edu). The different values refer to the surface accessibility scores (Scansite) or the phosphorylation scores, which were obtained with both NetPhos 3.1 (http://www.cbs.dtu.dk/services/NetPhos/) and NetPhorest (http://www.netphorest.info) software. B. HeLa cells overexpressing the HA-tagged MICU1 WT, MICU1 S124D, or MICU1 S124A mutants were stained for HA or HSP60 (mitochondrial marker). Merged images are indicated (merge). Scale bar 10 μm. C. Representative images of the 2mt-GCaMP6m 474/410 ratio of ShMICU1 HeLa stable cells expressing an empty vector (ctrl) or the MICU1 WT, MICU1 SD, and MICU1 SA. Scale bar 10 μm. D. Resting mitochondrial calcium levels, evaluated through ratiometric imaging of the mitochondrial-targeted GCaMP6m, in ShRNA control (plko) or ShRNA MICU1 HeLa stable clone cells transfected with the indicated constructs (n = 5 independent experiments; 55–67 cells). E, F. Representative kinetics (E) and analysis (F) of aequorin-based [Ca2+]m measurements in ShRNA MICU1 HeLa stable clone cells transfected with the indicated constructs and challenged with 20 μM 2,5-di-tert-butylhydroquinone (TBHQ) in the absence of extracellular Ca2+ (n = 3 independent experiments). G, H. Representative kinetics (G) and analysis (H) of aequorin-based [Ca2+]m measurements in intact ShRNA MICU1 HeLa stable clone cells transfected with the indicated constructs and challenged with 10 μM cyclopiazonic acid (CPA) in the presence of 100 μM EGTA (n = 3 independent experiments). I. Western blot analysis for the presence of MICU1 in clones arising from single cells generated by CRISPR/Cas9-mediated genome editing. The results for 12 of the 36 clones that were examined are shown. J. Resting mitochondrial calcium levels, evaluated through ratiometric imaging of the mitochondrial-targeted GCaMP6m, in MICU1 KO cells generated using the CRISPR/Cas9 technique and transfected with the indicated constructs (n = 3 independent experiments; 30–56 cells). K, L. Representative kinetics (K) and analysis (L) of aequorin-based [Ca2+]m measurements in intact MICU1 KO cells generated using the CRISPR/Cas9 technique, transfected with the indicated constructs, and challenged with 20 μM TBHQ in the absence of extracellular Ca2+ (n = 3 independent experiments). M, N. Representative kinetics (M) and analysis (N) of aequorin-based [Ca2+]m measurements in intact MICU1 KO cells generated using the CRISPR/Cas9 technique, transfected with the indicated constructs, and challenged with 10 μM CPA in the presence of 100 μM EGTA (n = 3 independent experiments). Data information: (D, F, H, J, L, N) Means ± SEM. ***P < 0.001; ****P < 0.0001 (one-way ANOVA). Source data are available online for this figure. Source Data for Figure 1 [embj201899435-sup-0003-SDataFig1.pdf] Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Akt-mediated MICU1 phosphorylation increases mitochondrial basal Ca2+ levels MICU1 mRNA levels in plko.1 and ShRNA MICU1 HeLa stable clone cells (n = 3 independent experiments). Analysis of aequorin-based [Ca2+]m measurements in permeabilized ShRNA MICU1-HeLa cells transfected with the indicated constructs and challenged with 500, 800 nM or 1.5 μM buffered [Ca2+] (n = 3 independent experiments). HEK293 cells were transfected with GFP-tagged wild-type (WT) MICU1 and then treated with 1 μM rapamycin (Rapa.) alone for 4 h or in combination with the Akt inhibitor triciribine (10 μM). The cell lysates were immunoprecipitated with PAS (phospho-Akt substrate) antibody and then analyzed by Western blotting. IgH: immunoglobulin heavy chain. HEK293 cells were transfected with either GFP-tagged wild-type (WT) MICU1 or MICU1 S124A-GFP mutant and treated as in (C). MICU1-GFP immuno complexes were precipitated with a GFP antibody and analyzed with PAS (phospho-Akt substrate) and phosphoserine (p-Ser) antibodies by Western blotting. Resting mitochondrial calcium levels, evaluated through ratiometric imaging of the mitochondrial-targeted GCaMP6m, in ShRNA MICU1 HeLa stable clone cells transfected with the indicated constructs, treated with vehicle or 1 μM rapamycin for 4 h (n = 3 independent experiments; 27–30 cells). Western blot analysis of Akt levels in HeLa cells silenced with scramble (ctrl) or Akt siRNAs. Resting mitochondrial calcium levels, evaluated through ratiometric imaging of the mitochondrial-targeted GCaMP6m, in HeLa cells silenced with the indicated constructs, treated with vehicle or 1 μM rapamycin for 4 h (n = 3 independent experiments; 40–63 cells). FURA-2 AM ratiometric measurements in HeLa cells upon stimulation with vehicle (DMSO) or 1 μM rapamycin (n = 3 independent experiments; 46–75 cells). Calibrated FURA-2 AM cytosolic [Ca2+] in HeLa cells treated with vehicle or 1 μM rapamycin for 4 h (n = 3 independent experiments; 50 cells). Data information: (A, I) Means ± SEM. N.S. not significant; **P < 0.01 (Student's t-test); (B) Means ± SEM. N.S. not significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 (one-way ANOVA); (E, G) Means ± SEM. ****P < 0.0001 (two-way ANOVA). Source data are available online for this figure. Download figure Download PowerPoint To further demonstrate this concept, we analyzed the basal mitochondrial Ca2+ levels in intact HeLa cells. Overexpression of the MICU1 WT or MICU1 SA mutant caused no significant changes in the basal mitochondrial Ca2+ levels compared with those found in untransfected control cells. However, the basal mitochondrial Ca2+ content in the MICU1 SD-overexpressing cells was significantly increased compared to that in the MICU1 WT- and MICU1 SA-expressing cells (Appendix Fig S1A). These data are independent from changes in the mitochondrial membrane potential (Appendix Fig S1B) and were confirmed using both TBHQ and CPA inhibitors (Appendix Fig S1C–F). Because the protein levels of MICU1 and MCU and their stoichiometry are crucial aspects in regulating [Ca2+]m (Paillard et al, 2017), we reconstituted the MICU1 WT and mutants in a MICU1-null background, generated using a CRISPR/CAS9-mediated approach (Fig 1I). Using the mito-GCaMP6m indicator, we observed higher Ca2+ levels only in MICU1-null and MICU1 SD-expressing cells (Fig 1J). Similar findings were obtained with an aequorin-based approach upon stimulation with either TBHQ or CPA (Fig 1K–N). Therefore, the results achieved in three different cell lines and using three different techniques to measure [Ca2+]m, namely (i) in untreated intact cells, (ii) upon treatment of intact cells with SERCA antagonists, and (iii) upon Ca2+ addition to permeabilized cells, indicate that phosphorylation of MICU1 at position 124 limits its negative effect on MCU and thus induces Ca2+ accumulation inside the mitochondrial matrix. Based on these results, we became interested in identifying the kinase responsible for MICU1 phosphorylation. MICU1 phosphorylation is mediated by mitochondrial Akt The MICU1 sequence surrounding Ser124 matches the Akt consensus phosphorylation motif, R-X-R-X-X-S/T (Manning & Cantley, 2007), which is highly conserved among different species (Fig 2A). We observed that upon treatment with rapamycin, which is a known activator of Akt, a significant number of cells displayed an obvious mitochondrial localization of activated (Ser473 phosphorylated) Akt (Fig 2B and C and Appendix Fig S2A). Subcellular fractionation followed by immunoblot analyses suggested that the treatment of HEK293T cells with rapamycin resulted in an increased Akt activation/phosphorylation to mitochondria (Appendix Fig S2B). This effect was observed not only in the crude mitochondrial fractions (Appendix Fig S2B), but also highly purified mitochondria without any detectable plasma membrane contamination (Fig 2D). Consistent with these results, pure mitochondria extracted from mouse livers after in vivo exposure to rapamycin also contained higher levels of Akt with phosphorylated Ser473 (Fig 2E). Having established the existence of a rapamycin-induced pool of active Akt in mitochondria, we sought to determine its submitochondrial localization. Proteinase K (PK) digestion of purified mitochondria that were subjected to selective outer membrane permeabilization by osmotic swelling (i.e., via the removal of sucrose) or complete lysis with Triton X-100 revealed that MICU1 behaved similarly to the inner mitochondrial membrane (IMM)–intermembrane space (IMS) protein TIM23 (both of which became susceptible to proteolysis after outer membrane permeabilization), in contrast to the matrix proteins HSP60 and MCU, which only became digested when the detergent was added (Fig 2F). This finding indicates that MICU1 is located at the outer surface of the IMM, as previously suggested (Csordas et al, 2013; Tsai et al, 2016). Importantly, in response to rapamycin, active Akt located predominantly at the IMS and, to a lesser extent, in the matrix compartment (Fig 2F). Alkaline carbonate extraction of isolated HEK293T cell mitochondria revealed that active Akt is loosely attached to the IMM, sharing this characteristic with cytochrome c (Fig 2G). Taken together, these results demonstrate that active Akt localizes in the mitochondria in a membrane-unbound state and accumulates in the same submitochondrial compartment as MICU1. Figure 2. Mitochondrial Akt phosphorylates MICU1 at the Ser124 position Sequence alignment of the MICU1 protein from nine vertebrate species. The Akt consensus phosphorylation motif, R-X-R-X-X-S/T, is marked in yellow. HeLa cells treated with vehicle or 1 μM rapamycin for 4 h were stained for phosphorylated (S473) Akt (p-Akt) or HSP60 (mitochondrial marker). Merged images are indicated (merge). Scale bar 10 μm. Analysis of the number of cells, expressed as a percentage, showing obvious mitochondrial staining of activated (S473 phosphorylated) Akt (p-Akt) (n = 3 independent experiments; 350-355 cells). Means ± SEM. ****P < 0.0001 (Student's t-test). HEK293T cells treated with vehicle or 1 μM rapamycin (Rapa.) for 4 h were fractionated into cytosol (Cyt.) or mitochondrial (Mito.) extracts and analyzed by Western blotting. E-cadherin: plasma membrane marker; β-tubulin: cytosolic marker; VDAC: mitochondrial marker; Homo: cell homogenate. Mouse livers treated with vehicle or 1 μM rapamycin (Rapa.) for 16 h by an intraperitoneal injection were fractionated into cytosol (Cyt.) or mitochondrial (Mito.) extracts and analyzed by Western blotting. Phosphorylated (S2468) mTOR (p-mTOR) was used to assess the rapamycin activity. E-cadherin: plasma membrane marker; β-tubulin: cytosolic marker; VDAC: mitochondrial marker; Homo: cell homogenate. Mitochondria isolated from HEK293T cells were subjected to the indicated treatments and analyzed by Western blotting against Akt, phosphorylated (S473) Akt (p-Akt), and mitochondrial proteins with known localiza
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