Advertisement

Fluctuation in O-GlcNAcylation inactivates STIM1 to reduce store-operated calcium ion entry via down-regulation of Ser621 phosphorylation

Open AccessPublished:October 06, 2020DOI:https://doi.org/10.1074/jbc.RA120.014271
      Stromal interaction molecule 1 (STIM1) plays a pivotal role in store-operated Ca2+ entry (SOCE), an essential mechanism in cellular calcium signaling and in maintaining cellular calcium balance. Because O-GlcNAcylation plays pivotal roles in various cellular function, we examined the effect of fluctuation in STIM1 O-GlcNAcylation on SOCE activity. We found that both increase and decrease in STIM1 O-GlcNAcylation impaired SOCE activity. To determine the molecular basis, we established STIM1-knockout HEK293 (STIM1-KO-HEK) cells using the CRISPR/Cas9 system and transfected STIM1 WT (STIM1-KO-WT-HEK), S621A (STIM1-KO-S621A-HEK), or T626A (STIM1-KO-T626A-HEK) cells. Using these cells, we examined the possible O-GlcNAcylation sites of STIM1 to determine whether the sites were O-GlcNAcylated. Co-immunoprecipitation analysis revealed that Ser621 and Thr626 were O-GlcNAcylated and that Thr626 was O-GlcNAcylated in the steady state but Ser621 was not. The SOCE activity in STIM1-KO-S621A-HEK and STIM1-KO-T626A-HEK cells was lower than that in STIM1-KO-WT-HEK cells because of reduced phosphorylation at Ser621. Treatment with the O-GlcNAcase inhibitor Thiamet G or O-GlcNAc transferase (OGT) transfection, which increases O-GlcNAcylation, reduced SOCE activity, whereas treatment with the OGT inhibitor ST045849 or siOGT transfection, which decreases O-GlcNAcylation, also reduced SOCE activity. Decrease in SOCE activity due to increase and decrease in O-GlcNAcylation was attributable to reduced phosphorylation at Ser621. These data suggest that both decrease in O-GlcNAcylation at Thr626 and increase in O-GlcNAcylation at Ser621 in STIM1 lead to impairment of SOCE activity through decrease in Ser621 phosphorylation. Targeting STIM1 O-GlcNAcylation could provide a promising treatment option for the related diseases, such as neurodegenerative diseases.
      O-Linked N-acetylglucosamine (O-GlcNAc) modification (O-GlcNAcylation) is a common posttranslational modification in numerous cytoplasmic and nuclear proteins (
      • Holt G.D.
      • Haltiwanger R.S.
      • Torres C.R.
      • Hart G.W.
      Erythrocytes contain cytoplasmic glycoproteins: O-linked GlcNAc on Band 4.1.
      ,
      • Hart G.W.
      • Housley M.P.
      • Slawson C.
      Cycling of O-linked β-N-acetylglucosamine on nucleocytoplasmic proteins.
      ,
      • Zachara N.E.
      • O'Donnell N.
      • Cheung W.D.
      • Mercer J.J.
      • Marth J.D.
      • Hart G.W.
      Dynamic O-GlcNAc modification of nucleocytoplasmic proteins in response to stress: a survival response of mammalian cells.
      ). Extracellular glucose enters the hexosamine biosynthesis pathway, leading to the production of UDP-GlcNAc, which serves as a substrate for O-GlcNAcylation (
      • McClain D.A.
      Hexosamines as mediators of nutrient sensing and regulation in diabetes.
      ). O-GlcNAcylation is controlled by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) and occurs on the Ser/Thr residues of proteins (
      • Gao Y.
      • Wells L.
      • Comer F.I.
      • Parker G.J.
      • Hart G.W.
      Dynamic O-glycosylation of nuclear and cytosolic proteins: cloning and characterization of a neutral, cytosolic β-N-acetylglucosaminidase from human brain.
      ).
      Fluctuation in O-GlcNAcylation is involved in various diseases (
      • Hart G.W.
      Nutrient regulation of signaling and transcription.
      ). Abnormal increase in O-GlcNAcylation has been implicated in diabetes mellitus (
      • Banerjee P.S.
      • Ma J.
      • Hart G.W.
      Diabetes-associated dysregulation of O-GlcNAcylation in rat cardiac mitochondria.
      ,
      • Lefebvre T.
      • Dehennaut V.
      • Guinez C.
      • Olivier S.
      • Drougat L.
      • Mir A.M.
      • Mortuaire M.
      • Vercoutter-Edouart A.S.
      • Michalski J.C.
      Dysregulation of the nutrient/stress sensor O-GlcNAcylation is involved in the etiology of cardiovascular disorders, type-2 diabetes and Alzheimer's disease.
      ,
      • Yang X.
      • Ongusaha P.P.
      • Miles P.D.
      • Havstad J.C.
      • Zhang F.
      • So W.V.
      • Kudlow J.E.
      • Michell R.H.
      • Olefsky J.M.
      • Field S.J.
      • Evans R.M.
      Phosphoinositide signalling links O-GlcNAc transferase to insulin resistance.
      ), neurodegenerative diseases (
      • Hwang H.
      • Rhim H.
      Acutely elevated O-GlcNAcylation suppresses hippocampal activity by modulating both intrinsic and synaptic excitability factors.
      ,
      • Kim C.
      • Nam D.W.
      • Park S.Y.
      • Song H.
      • Hong H.S.
      • Boo J.H.
      • Jung E.S.
      • Kim Y.
      • Baek J.Y.
      • Kim K.S.
      • Cho J.W.
      • Mook-Jung I.
      O-Linked β-N-acetylglucosaminidase inhibitor attenuates β-amyloid plaque and rescues memory impairment.
      ), heart failure (
      • Yokoe S.
      • Asahi M.
      • Takeda T.
      • Otsu K.
      • Taniguchi N.
      • Miyoshi E.
      • Suzuki K.
      Inhibition of phospholamban phosphorylation by O-GlcNAcylation: implications for diabetic cardiomyopathy.
      ,
      • Du X.L.
      • Edelstein D.
      • Dimmeler S.
      • Ju Q.
      • Sui C.
      • Brownlee M.
      Hyperglycemia inhibits endothelial nitric oxide synthase activity by posttranslational modification at the Akt site.
      ), and cancer (
      • Ishimura E.
      • Nakagawa T.
      • Moriwaki K.
      • Hirano S.
      • Matsumori Y.
      • Asahi M.
      Augmented O-GlcNAcylation of AMP-activated kinase promotes the proliferation of LoVo cells, a colon cancer cell line.
      ,
      • Moriwaki K.
      • Asahi M.
      Augmented TME O-GlcNAcylation promotes tumor proliferation through the inhibition of p38 MAPK.
      ). However, the role of O-GlcNAcylation in Alzheimer's disease (AD) or Parkinson's disease is controversial. Abnormal increase in O-GlcNAcylation slows neurodegeneration and stabilizes tau against aggregation (
      • Yuzwa S.A.
      • Shan X.
      • Macauley M.S.
      • Clark T.
      • Skorobogatko Y.
      • Vosseller K.
      • Vocadlo D.J.
      Increasing O-GlcNAc slows neurodegeneration and stabilizes tau against aggregation.
      ). On the other hand, abnormal decrease in O-GlcNAcylation has been observed in the AD brain, accompanying hyperphosphorylation of tau (
      • Wang A.C.
      • Jensen E.H.
      • Rexach J.E.
      • Vinters H.V.
      • Hsieh-Wilson L.C.
      Loss of O-GlcNAc glycosylation in forebrain excitatory neurons induces neurodegeneration.
      ). O-GlcNAcylation blocks the aggregation and toxicity of the protein α-synuclein associated with Parkinson's disease (
      • Marotta N.P.
      • Lin Y.H.
      • Lewis Y.E.
      • Ambroso M.R.
      • Zaro B.W.
      • Roth M.T.
      • Arnold D.B.
      • Langen R.
      • Pratt M.R.
      O-GlcNAc modification blocks the aggregation and toxicity of the protein α-synuclein associated with Parkinson's disease.
      ). On the other hand, excessive O-GlcNAcylation is detrimental to neurons because it leads to the inhibition of autophagy and increase in α-synuclein accumulation (
      • Wani W.Y.
      • Ouyang X.
      • Benavides G.A.
      • Redmann M.
      • Cofield S.S.
      • Shacka J.J.
      • Chatham J.C.
      • Darley-Usmar V.
      • Zhang J.
      O-GlcNAc regulation of autophagy and α-synuclein homeostasis; implications for Parkinson's disease.
      ).
      Despite the accumulation of knowledge regarding the role of O-GlcNAcylation in these diseases, the functional effects of O-GlcNAcylation remain poorly understood. Therefore, obtaining further data on the O-GlcNAcylation of diabetes-related and neuroregulatory molecules may have important implications in developing therapeutic strategies for these diseases.
      Stromal interaction molecule 1 (STIM1), one of the main sensors for Ca2+ in the endoplasmic reticulum (ER), is a critical regulator of store-operated calcium entry (SOCE) (
      • Zhang S.L.
      • Yu Y.
      • Roos J.
      • Kozak J.A.
      • Deerinck T.J.
      • Ellisman M.H.
      • Stauderman K.A.
      • Cahalan M.D.
      STIM1 is a Ca2+ sensor that activates CRAC channels and migrates from the Ca2+ store to the plasma membrane.
      ,
      • Feske S.
      • Picard C.
      • Fischer A.
      Immunodeficiency due to mutations in ORAI1 and STIM1.
      ,
      • Roos J.
      • DiGregorio P.J.
      • Yeromin A.V.
      • Ohlsen K.
      • Lioudyno M.
      • Zhang S.
      • Safrina O.
      • Kozak J.A.
      • Wagner S.L.
      • Cahalan M.D.
      • Veliçelebi G.
      • Stauderman K.A.
      STIM1, an essential and conserved component of store-operated Ca2+ channel function.
      ,
      • Liou J.
      • Kim M.L.
      • Heo W.D.
      • Jones J.T.
      • Myers J.W.
      • Ferrell Jr., J.E.
      • Meyer T.
      STIM is a Ca2+ sensor essential for Ca2+-store-depletion-triggered Ca2+ influx.
      ). SOCE is the process by which the emptying of ER Ca2+ stores causes influx of Ca2+ across the plasma membrane, and it plays pivotal roles in several physiological and pathological conditions, including neuronal excitability (
      • Moccia F.
      • Zuccolo E.
      • Soda T.
      • Tanzi F.
      • Guerra G.
      • Mapelli L.
      • Lodola F.
      • D'Angelo E.
      Stim and Orai proteins in neuronal Ca2+ signaling and excitability.
      ), hypoxic/ischemic neuronal injury (
      • Zhang H.
      • Clemens R.A.
      • Liu F.
      • Hu Y.
      • Baba Y.
      • Theodore P.
      • Kurosaki T.
      • Lowell C.A.
      STIM1 calcium sensor is required for activation of the phagocyte oxidase during inflammation and host defense.
      ), cardiac hypertrophy (
      • Bénard L.
      • Oh J.G.
      • Cacheux M.
      • Lee A.
      • Nonnenmacher M.
      • Matasic D.S.
      • Kohlbrenner E.
      • Kho C.
      • Pavoine C.
      • Hajjar R.J.
      • Hulot J.S.
      Cardiac Stim1 silencing impairs adaptive hypertrophy and promotes heart failure through inactivation of mTORC2/Akt signaling.
      ), proliferation of vascular smooth muscle cells (
      • Jia S.
      • Rodriguez M.
      • Williams A.G.
      • Yuan J.P.
      Homer binds to Orai1 and TRPC channels in the neointima and regulates vascular smooth muscle cell migration and proliferation.
      ), and carcinogenesis (
      • White C.
      The regulation of tumor cell invasion and metastasis by endoplasmic reticulum-to-mitochondrial Ca2+ Transfer.
      ). STIM1 oligomerizes upon sensing Ca2+ depletion within the ER and is translocated to the ER–plasma membrane junction (
      • Liou J.
      • Fivaz M.
      • Inoue T.
      • Meyer T.
      Live-cell imaging reveals sequential oligomerization and local plasma membrane targeting of stromal interaction molecule 1 after Ca2+ store depletion.
      ,
      • Wu M.M.
      • Buchanan J.
      • Luik R.M.
      • Lewis R.S.
      Ca2+ store depletion causes STIM1 to accumulate in ER regions closely associated with the plasma membrane.
      ). STIM1 oligomerization activates SOCE via binding to store-sensitive calcium channels, such as Orai1 (
      • Feske S.
      • Gwack Y.
      • Prakriya M.
      • Srikanth S.
      • Puppel S.H.
      • Tanasa B.
      • Hogan P.G.
      • Lewis R.S.
      • Daly M.
      • Rao A.
      A mutation in Orai1 causes immune deficiency by abrogating CRAC channel function.
      ), and transient receptor potential channels (TRPCs) (
      • Yuan J.P.
      • Zeng W.
      • Huang G.N.
      • Worley P.F.
      • Muallem S.
      STIM1 heteromultimerizes TRPC channels to determine their function as store-operated channels.
      ) present on the plasma membrane. STIM1 contains EF-hand and sterile α motif (SAM; EF-SAMs) domains in its N terminus as a Ca2+-sensing region, which is crucial for promoting SOCE activity (
      • Stathopulos P.B.
      • Zheng L.
      • Li G.Y.
      • Plevin M.J.
      • Ikura M.
      Structural and mechanistic insights into STIM1-mediated initiation of store-operated calcium entry.
      ). Recently, extracellular signal–regulated kinase 1/2 (ERK1/2) was found to phosphorylate STIM1 at Ser575, Ser608, and Ser621 residues during Ca2+ depletion in the ER, which is also crucial for regulating SOCE activity (
      • Pozo-Guisado E.
      • Campbell D.G.
      • Deak M.
      • Alvarez-Barrientos A.
      • Morrice N.A.
      • Alvarez I.S.
      • Alessi D.R.
      • Martín-Romero F.J.
      Phosphorylation of STIM1 at ERK1/2 target sites modulates store-operated calcium entry.
      ). STIM1 has been found to be a microtubule plus-end–tracking protein and is directly associated with terminal-binding protein 1 (EB1), which plays a key role in regulating cellular Ca2+ homeostasis via modulating SOCE activity (
      • Grigoriev I.
      • Gouveia S.M.
      • van der Vaart B.
      • Demmers J.
      • Smyth J.T.
      • Honnappa S.
      • Splinter D.
      • Steinmetz M.O.
      • Putney Jr., J.W.
      • Hoogenraad C.C.
      • Akhmanova A.
      STIM1 is a MT-plus-end-tracking protein involved in remodeling of the ER.
      ,
      • Pozo-Guisado E.
      • Casas-Rua V.
      • Tomas-Martin P.
      • Lopez-Guerrero A.M.
      • Alvarez-Barrientos A.
      • Martin-Romero F.J.
      Phosphorylation of STIM1 at ERK1/2 target sites regulates interaction with the microtubule plus-end binding protein EB1.
      ,
      • Chang C.L.
      • Chen Y.J.
      • Quintanilla C.G.
      • Hsieh T.S.
      • Liou J.
      EB1 binding restricts STIM1 translocation to ER-PM junctions and regulates store-operated Ca2+ entry.
      ). O-GlcNAcylation of STIM1 impairs SOCE activity, resulting in impaired Ca2+ homeostasis in rat neonatal cardiomyocytes (
      • Zhu-Mauldin X.
      • Marsh S.A.
      • Zou L.
      • Marchase R.B.
      • Chatham J.C.
      Modification of STIM1 by O-linked N-acetylglucosamine (O-GlcNAc) attenuates store-operated calcium entry in neonatal cardiomyocytes.
      ). Therefore, in the current study, we aimed to investigate the involvement of O-GlcNAcylation of STIM1 in the phosphorylation, oligomer formation, interaction with EB1 and Orai1, and SOCE activity.

      Results

      Both glucose depletion and high glucose induced decrease in SOCE activity

      We measured SOCE activity using HEK293 cells to determine whether the glucose concentration affected this activity. HEK293 cells were treated with different glucose concentrations (low (0 g/liter), normal (1 g/liter), or high (4.5 g/liter)) for 48 h, and SOCE activity was measured via treatment with a sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) inhibitor, thapsigargin (TG). Both low and high glucose lowered SOCE activity, indicating that normal glucose concentration exhibited sufficient SOCE activity (Fig. 1, A and B). Next, we examined O-GlcNAcylation levels of STIM1 for each glucose concentration because high glucose is known to induce O-GlcNAcylation of numerous proteins (
      • Walgren J.L.E.
      • Vincent T.S.
      • Schey K.L.
      • Buse M.G.
      High glucose and insulin promote O-GlcNAc modification of proteins, including α-tubulin.
      ,
      • Hu Y.
      • Suarez J.
      • Fricovsky E.
      • Wang H.
      • Scott B.T.
      • Trauger S.A.
      • Han W.
      • Hu Y.
      • Oyeleye M.O.
      • Dillmann W.H.
      Increased enzymatic O-GlcNAcylation of mitochondrial proteins impairs mitochondrial function in cardiac myocytes exposed to high glucose.
      ). O-GlcNAcylation of STIM1 in low and high glucose–treated cells was significantly lower and higher, respectively, than that in normal glucose–treated cells (p < 0.05) (Fig. 1, C and D).
      Figure thumbnail gr1
      Figure 1Effects of exposure to low (0 g/liter), normal (1 g/liter), and high (4.5 g/liter) doses of glucose on SOCE activity. A, HEK293 cells were treated with different glucose concentrations for 48 h, and the SOCE activity was measured with 1 μm TG treatment. The black, gray, and light gray lines indicate the low glucose–, normal glucose–, and high glucose–treated groups, respectively. B, the peak SOCE activity in HEK293 cells after Ca2+ addition was evaluated from the experiments, as shown in A. Dots, data points. The data were analyzed by using one-way ANOVA followed by Tukey's test (n = 30–40 cells). *, p < 0.05. C, O-GlcNAc and STIM1 expression levels for HEK293 cells treated with different glucose concentrations. O-GlcNAcylation of endogenous STIM1 for HEK293 cells treated with different glucose concentrations was examined using immunoprecipitation assays. α-Tubulin served as the loading control. D, relative abundance of O-GlcNAcylated STIM1 was quantified as shown in C. The data were analyzed using one-way ANOVA followed by Tukey's test. Data are represented as mean ± S.D. values (error bars) (n = 3). *, p < 0.05. TG, a SOCE inducer (a SERCA inhibitor).

      Fluctuation in STIM1 O-GlcNAcylation reduced SOCE activity

      For in-depth analysis of the role of O-GlcNAcylation of STIM1 in SOCE activity, we generated STIM1-knockout HEK293 (STIM1-KO-HEK) cells by using CRISPR/Cas9 gene-editing technology. STIM1-KO-HEK cells showed almost no SOCE activity, but this activity was rescued by transient STIM1-WT overexpression (Fig. S1, A and B). STIM1-KO-HEK cells transfected with the STIM1-mKATE-WT plasmid (STIM1-KO-WT-HEK cells) showed decreased SOCE activity after Thiamet G (TMG) treatment (Fig. 2, A and B). STIM1-KO-WT-HEK cells showed decreased SOCE activity upon treatment with the OGT inhibitor ST045849 (Fig. 2, C and D) and on cotransfection with OGT (Fig. 2, E and F) or with siOGT (Fig. 2, G and H). Furthermore, STIM1-KO-WT-HEK cells showed decreased SOCE activity upon treatment with siOGA (Fig. S2, A and B). As expected, O-GlcNAcylation of STIM1 increased after TMG treatment and decreased after ST045849 treatment (Fig. S3, A and B). These data indicate that fluctuation in STIM1 O-GlcNAcylation reduces SOCE activity.
      Figure thumbnail gr2
      Figure 2Dual role played by STIM1 O-GlcNAcylation in SOCE activity. A, C, E, and G, STIM1-KO-HEK cells transfected with the STIM1-mKATE-WT plasmid were treated with 10 μm TMG (gray line) or DMSO (black line) as a solvent control (A), 50 μm OGT inhibitor ST045849 (gray line) or DMSO (black line) (C), the OGT plasmid (gray line) or mock control (black line) (E), or siOGT (gray line) or siControl (black line) (G) for 24–48 h before the experiments. Subsequently, the SOCE activity was measured. B, D, F, and H, peak SOCE activity in HEK293 cells after Ca2+ addition, which was evaluated from the results shown in A, C, E, and G, respectively. Dots, data points. The data were analyzed using the F-test followed by Student's t test. Data are represented as mean ± S.D. values (error bars) (n = 30–40 cells). *, p < 0.05.

      Fluctuation in STIM1 O-GlcNAcylation reduced phosphorylation at Ser621

      Phosphorylation of STIM1 at the ERK1/2 target sites Ser575, Ser608, and Ser621 plays a pivotal role in SOCE activity (
      • Pozo-Guisado E.
      • Casas-Rua V.
      • Tomas-Martin P.
      • Lopez-Guerrero A.M.
      • Alvarez-Barrientos A.
      • Martin-Romero F.J.
      Phosphorylation of STIM1 at ERK1/2 target sites regulates interaction with the microtubule plus-end binding protein EB1.
      ). To evaluate the effect of the fluctuation in STIM1 O-GlcNAcylation on phosphorylation at these sites, Western blotting analyses were performed using antibodies specific to phosphorylated STIM1. To examine the effect of increased O-GlcNAcylation on phosphorylation, STIM1-KO-WT-HEK cells cotransfected with OGT were treated with 1 μm TG for 30 min, and the phosphorylation level at each site was subsequently evaluated. STIM1 phosphorylation at Ser621 in OGT-overexpressing cells was markedly lower than that in mock-overexpressing cells, whereas STIM1 phosphorylation at Ser575 and Ser608 did not change significantly (Fig. 3, A and B). To examine the effect of decrease in O-GlcNAcylation on phosphorylation, STIM1-KO-WT-HEK cells cotransfected with siOGT were treated with 1 μm TG for 30 min. STIM1 phosphorylation at Ser621 in siOGT-transfected cells was markedly lower than that in siControl-transfected cells, whereas STIM1 phosphorylation at Ser575 and Ser608 did not change significantly (Fig. 3, C and D). A siOGT gene-silencing efficiency of nearly 90% was detected by Western blotting analysis (Fig. 3E). Furthermore, STIM1 phosphorylation at Ser621 in siOGA-transfected cells was markedly lower than that in siControl-transfected cells, whereas STIM1 phosphorylation at Ser575 and Ser608 did not change significantly (Fig. S2, C and D). An siOGA gene-silencing efficiency of about 58% was detected by Western blotting analyses (Fig. S2E). These data indicate that fluctuation in O-GlcNAcylation of STIM1 reduced Ser621 phosphorylation.
      Figure thumbnail gr3
      Figure 3Effects of OGT overexpression or OGT deficiency on the phosphorylation of STIM1 at ERK1/2 target sites. A, STIM1-KO-HEK cells cotransfected with mock/STIM1-mKATE-WT or OGT/STIM1-mKATE-WT plasmids were treated with 1 μm TG for 30 min. The cell lysates were subjected to Western blotting analyses with antibodies against OGT, O-GlcNAc, phospho-Ser575-STIM1, phospho-Ser608-STIM1, phospho-Ser621-STIM1, and total-STIM1. α-Tubulin served as the loading control. B, relative abundance of STIM1 phosphorylation at Ser575, Ser608, and Ser621 in relation to total STIM1 was quantified as shown in A, and the control levels (Mock) were set at 1.0. Dots, data points. The data were analyzed using one-way ANOVA, followed by Tukey's test. Data are represented as mean ± S.D. values (error bars) (n = 3). *, p < 0.05. C, STIM1-KO-HEK cells cotransfected with siControl/STIM1-mKATE-WT or siOGT/STIM1-mKATE-WT plasmids were treated with 1 μm TG for 30 min. The cell lysates were subjected to Western blotting analyses using antibodies against OGT, O-GlcNAc, phospho-Ser575-STIM1, phospho-Ser608-STIM1, phospho-Ser621-STIM1, and total-STIM1. α-Tubulin served as the loading control. D, relative abundance of STIM1 phosphorylation at Ser575, Ser608, and Ser621 in relation to total STIM1 was quantified as shown in C, and the control levels (siControl) were set at 1.0. Dots, data points. The data were analyzed using one-way ANOVA, followed by Tukey's test. Data are represented as mean ± S.D. values (n = 3). *, p < 0.05. E, relative abundance of OGT in siOGT-transfected cells was quantified after normalization to the loading control, α-tubulin, as shown in A, and the control levels (siControl) were set at 1.0. The data were analyzed using the F-test followed by Student's t test. Data are represented as mean ± S.D. values (error bars) (n = 3). *, p < 0.05.

      Ser621 and Thr626 were O-GlcNAcylated, but only Thr626, and not Ser621, was O-GlcNAcylated in the steady state

      We focused on the potential O-GlcNAcylation sites within the Ser/Pro-rich domain of STIM1 because STIM1-dependent SOCE activity is accomplished by phosphorylation of STIM1 at ERK1/2 target sites within the Ser/Pro-rich domain of STIM1 (
      • Pozo-Guisado E.
      • Casas-Rua V.
      • Tomas-Martin P.
      • Lopez-Guerrero A.M.
      • Alvarez-Barrientos A.
      • Martin-Romero F.J.
      Phosphorylation of STIM1 at ERK1/2 target sites regulates interaction with the microtubule plus-end binding protein EB1.
      ). The computational prediction program YinOYang 1.2 was used to identify potential O-GlcNAcylation sites (
      • Jochmann R.
      • Holz P.
      • Sticht H.
      • Stürzl M.
      Validation of the reliability of computational O-GlcNAc prediction.
      ). The program provided two potential O-GlcNAcylation sites (Ser621 and Thr626; YinOYang threshold >0.5; Fig. 4A). To confirm whether Ser621 and/or Thr626 sites are O-GlcNAcylated, we performed a co-immunoprecipitation assay using S621A and/or T626A mutants. The overall O-GlcNAcylation in T626A and S621A/T626A reduced considerably with or without siOGT treatment, and O-GlcNAcylation decreased in WT and S621A after siOGT treatment (Fig. 4, B and C). Moreover, O-GlcNAcylation increased in WT and T626A after TMG treatment, whereas S621A and S621A/T626A did not show changes (Fig. 4, D and E). These data suggest that Thr626 is O-GlcNAcylated in the steady state and that the O-GlcNAcylation is down-regulated with decrease in O-GlcNAcylation. Ser621 is not O-GlcNAcylated in the steady state, and the O-GlcNAcylation is up-regulated with increase in O-GlcNAcylation.
      Figure thumbnail gr4
      Figure 4Identification of potential O-GlcNAcylated sites within the Ser/Pro-rich domain of STIM1. A, schematic illustration of the STIM1 domains. The potential O-GlcNAcylation sites (Ser621 and Thr626) on the STIM1 Ser/Pro-rich domain are shown by black arrowheads. The ERK1/2 target sites (Ser575, Ser608, and Ser621) are shown by white arrowheads. EF, EF-hand; SAM, sterile α motif; TM, transmembrane; SOAR, STIM-Orai–activated region; S/P, serine/proline-rich; TRIP, threonine-arginine-isoleucine-proline region. B, STIM1-KO-HEK cells cotransfected with STIM1-mKATE-WT, STIM1-mKATE-S621A, STIM1-mKATE-T626A, or STIM1-mKATE-S621A/T626A—with or without siOGT—were incubated for 48 h at 37 °C. The cell lysates were subjected to co-immunoprecipitation with anti-STIM1 antibody. The OGT, O-GlcNAc, and STIM1 expression levels in the cell lysates, and the O-GlcNAc and STIM1 expression levels in the immunoprecipitates were then determined by Western blotting analyses performed using OGT, O-GlcNAc, and STIM1 antibodies. α-Tubulin served as the loading control. C, relative abundance of O-GlcNAcylated STIM1 was quantified as shown in B. Dots, data points. The data were analyzed using one-way ANOVA followed by Tukey's test. Data are represented as mean ± S.D. values (error bars) (n = 3). *, p < 0.05. D, STIM1-KO-HEK cells cotransfected with STIM1-mKATE-WT, STIM1-mKATE-S621A, STIM1-mKATE-T626A, or STIM1-mKATE-S621A/T626A—with or without TMG—were incubated for 48 h at 37 °C. The cell lysates were subjected to co-immunoprecipitation with anti-STIM1 antibody. The O-GlcNAc, STIM1, and phospho-Ser621-STIM1 expression levels in the cell lysates and the O-GlcNAc and STIM1 expression levels in the immunoprecipitates were then determined by Western blotting analyses performed using O-GlcNAc, STIM1 and phospho-Ser621-STIM1 antibodies, respectively. GAPDH served as the loading control. E, relative abundance of O-GlcNAcylated STIM1 was quantified as shown in D. Dots, data points. The data were analyzed using one-way ANOVA followed by Tukey's test. Data are represented as mean ± S.D. values (n = 3). *, p < 0.05. F, relative abundance of phosphorylated STIM1 at Ser621 in relation to total STIM1 was quantified as shown in D, and the control levels (WT with TMG) were set at 1.0. Dots, data points. The data were analyzed using one-way ANOVA, followed by Tukey's test. Data are represented as mean ± S.D. values (n = 3). *, p < 0.05.

      SOCE activity decreased on decrease in STIM1 O-GlcNAcylation at Thr626 via decrease in Ser621 phosphorylation

      The phosphorylation of STIM1 at Ser621 markedly decreased in T626A compared with WT, as well as S621A and S621A/T626A in the steady state (Fig. 4, D and F). The phosphorylation decreased in WT after TMG treatment, whereas S621A, T626A, and S621A/T626A did not show changes after TMG treatment (Fig. 4, D and F). Given that T626A lacks O-GlcNAcylation at Thr626, O-GlcNAcylation might be involved in the phosphorylation at Ser621. To evaluate the effect of STIM1 O-GlcNAcylation at Thr626 on SOCE activity, STIM1-KO-HEK cells transfected with S621A (STIM1-KO-S621A-HEK cells) or T626A (STIM1-KO-T626A-HEK cells) were prepared, and SOCE activity was measured. Both STIM1-KO-S621A-HEK and STIM1-KO-T626A-HEK cells showed significantly lower SOCE activity than STIM1-KO-WT-HEK cells (Fig. 5, A and B). Moreover, the SOCE activity of STIM1-KO-T626A-HEK cells was not significantly affected by TMG treatment (Fig. 5, C and D). To determine whether O-GlcNAcylation of STIM1 at Thr626 regulates SOCE activity by decreasing the phosphorylation of STIM1 at Ser621, we introduced phosphomimetic mutations (S575E, S608E, and S621E) in the Ser/Pro-rich domain of STIM1 in the T626A mutants and transfected them into STIM1-KO-HEK (STIM1-KO-S575E/T626A-HEK, STIM1-KO-S608E/T626A-HEK, and STIM1-KO-S621E/T626A-HEK, respectively) cells. There were no significant differences in expression levels among these mutants (Fig. S4, A and B). STIM1-KO-S621E/T626A-HEK cells showed significantly higher SOCE activity than STIM1-KO-T626A-HEK cells, whereas STIM1-KO-S575E/T626A-HEK and STIM1-KO-S608E/T626A-HEK cells did not show significant changes, indicating that the phosphorylated mimetic STIM1 at Ser621 (S621E) overcomes the effect of T626A on SOCE activity (Fig. 5, E and F). These data suggest that decrease in STIM1 O-GlcNAcylation at Thr626 lowers SOCE activity by decreasing the phosphorylation of STIM1 at Ser621.
      Figure thumbnail gr5
      Figure 5Effects of non-O-GlcNAcylation of the STIM1 Ser/Pro-rich domain on SOCE activity. A, STIM1-KO-HEK cells transfected with the STIM1-mKATE-WT, STIM1-mKATE-S621A, or STIM1-mKATE-T626A plasmids were incubated at 37 °C for 48 h, and the SOCE activity was measured. The black, gray, and light gray lines indicate STIM1-mKATE-WT–, STIM1-mKATE-S621A–, and STIM1-mKATE-T626A–transfected cells, respectively. B, peak SOCE activity in each cell type after Ca2+ addition was evaluated from the results shown in A. Dots, data points. The data were analyzed using one-way ANOVA followed by Tukey's test (n = 30–40 cells). *, p < 0.05. C, STIM1-KO-HEK293 cells transfected with the STIM1-mKATE-T626A plasmid were incubated with or without 5 μm TMG for 48 h at 37 °C, and the SOCE activity was measured. Black and gray lines indicate DMSO- and TMG-treated cells, respectively. D, peak SOCE activity in each cell type after Ca2+ addition, as evaluated from the results shown in C. Dots, data points. The data were analyzed using one-way ANOVA followed by Tukey's test (n = 30–40 cells). *, p < 0.05. E, STIM1-KO-HEK cells transfected with the STIM1-mKATE-WT, STIM1-mKATE-T626A, STIM1-mKATE-S575E/T626A, STIM1-mKATE-S608E/T626A, or STIM1-mKATE-S621E/T626A plasmids were incubated at 37 °C for 48 h, and the SOCE activity was measured. Black, deep gray, dark gray, gray, and light gray lines indicate STIM1-mKATE-WT–, STIM1-mKATE-T626A–, STIM1-mKATE-S575E/T626A–, STIM1-mKATE-S608E/T626A–, and STIM1-mKATE-S621E/T626A–transfected cells, respectively. F, peak SOCE activity in each cell type after Ca2+ addition, evaluated from the results shown in E. Dots, data points. The data were analyzed using one-way ANOVA followed by Tukey's test (n = 30–40 cells). *, p < 0.05.

      Puncta formation decreased in both STIM1-KO-S621A-HEK and STIM1-KO-T626A-HEK cells

      STIM1 forms oligomers and interacts with and activates the calcium ion channel Orai1 (
      • Soboloff J.
      • Spassova M.A.
      • Tang X.D.
      • Hewavitharana T.
      • Xu W.
      • Gill D.L.
      Orai1 and STIM reconstitute store-operated calcium channel function.
      ,
      • Deng X.
      • Wang Y.
      • Zhou Y.
      • Soboloff J.
      • Gill D.L.
      STIM and Orai: dynamic intermembrane coupling to control cellular calcium signals.
      ,
      • Wang Y.
      • Deng X.
      • Zhou Y.
      • Hendron E.
      • Mancarella S.
      • Ritchie M.F.
      • Tang X.D.
      • Baba Y.
      • Kurosaki T.
      • Mori Y.
      • Soboloff J.
      • Gill D.L.
      STIM protein coupling in the activation of Orai channels.
      ). STIM1 oligomer formation can be observed as puncta formation with confocal microscopy. To evaluate the effect of increased O-GlcNAcylation at Thr626 on puncta formation, STIM1-KO-WT-HEK, STIM1-KO-S621A-HEK, and STIM1-KO-T626A-HEK cells were incubated with or without 10 μm TMG at 37 °C for 48 h. Puncta formation increased after TG treatment in STIM1-KO-WT-HEK cells; however, it was eliminated by TMG pretreatment (Fig. 6A). STIM1-KO-S621A-HEK and STIM1-KO-T626A-HEK cells showed no puncta formation even after TG treatment (Fig. 6, B and C). These results suggest that the STIM1 activity was mainly triggered by the phosphorylation of STIM1 at Ser621, which was negatively regulated by O-GlcNAcylation of STIM1 at Thr626.
      Figure thumbnail gr6
      Figure 6Effects of non-O-GlcNAcylation of the STIM1 Ser/Pro-rich domain on puncta formation of STIM1. A, B, and C, STIM1-KO-HEK cells transfected with STIM1-mKATE-WT, STIM1-mKATE-S621A, or STIM1-mKATE-T626A plasmids were incubated at 37 °C for 48 h—with or without 10 μm TMG—and the cells were treated with 1 μm TG for 10 min, fixed, and stained with anti-STIM1 antibody. Representative images of the cells are shown (n = 10–15 cells). Scale bar, 20 μm.

      O-GlcNAcylation of STIM1 may be involved in its dissociation from EB1 and interaction with Orai1 via STIM1 phosphorylation at Ser621

      Phosphorylation of STIM1 at ERK1/2 target sites triggers its dissociation from EB1 during Ca2+ store depletion and increases its interaction with Orai1 (
      • Pozo-Guisado E.
      • Casas-Rua V.
      • Tomas-Martin P.
      • Lopez-Guerrero A.M.
      • Alvarez-Barrientos A.
      • Martin-Romero F.J.
      Phosphorylation of STIM1 at ERK1/2 target sites regulates interaction with the microtubule plus-end binding protein EB1.
      ). STIM1-KO-WT-HEK, STIM1-KO-S621A-HEK, and STIM1-KO-T626A-HEK cells were incubated with or without 10 μm TMG for 48 h at 37 °C, and the effect of O-GlcNAcylation of STIM1 at Ser621 and Thr626 on the interaction with EB1 was examined with a co-immunoprecipitation assay. The dissociation of STIM1 from EB1 after TG treatment was reduced by TMG pretreatment in STIM1-KO-WT-HEK cells (Fig. 7, A and B). Conversely, STIM1 did not dissociate from EB1 after TG treatment in STIM1-KO-S621A-HEK and STIM1-KO-T626A-HEK cells. STIM1 activates SOCE via interaction with Orai1 (
      • Feske S.
      • Gwack Y.
      • Prakriya M.
      • Srikanth S.
      • Puppel S.H.
      • Tanasa B.
      • Hogan P.G.
      • Lewis R.S.
      • Daly M.
      • Rao A.
      A mutation in Orai1 causes immune deficiency by abrogating CRAC channel function.
      ); therefore, we examined the effect of O-GlcNAcylation of STIM1 at Ser621 and Thr626 on interaction with Orai1. The increased interaction between STIM1 and Orai1 after TG treatment was reduced by TMG pretreatment in STIM1-KO-WT-HEK cells (Fig. 7, A and C). Conversely, interaction with Orai1 did not significantly increase after TG treatment in STIM1-KO-S621A-HEK and STIM1-KO-T626A-HEK cells. These data suggest that O-GlcNAcylation of STIM1 at Thr626 plays a pivotal role in TG-stimulated STIM1 dissociation from EB1 and the increased interaction with Orai1, probably through regulation of Ser621 phosphorylation. To prove that O-GlcNAc does not modify the other protein kinases and cellular pathways that indirectly affect STIM1 phosphorylation, we examined whether ERK, upstream of STIM phospho-Ser621, was O-GlcNAcylated and affected the phosphorylation. ERK was not O-GlcNAcylated (data not shown), and the activity (phosphorylated ERK) was not changed after TMG and ST045849 treatment in HEK293 cells (Fig. S5, A and B), indicating that the upstream signaling pathway may not indirectly affect STIM1 Ser621 phosphorylation.
      Figure thumbnail gr7
      Figure 7Effects of non-O-GlcNAcylation of the STIM1 Ser/Pro-rich domain on interaction of STIM1 with EB1 and Orai1. A, STIM1-KO cells transfected with the STIM1-mKATE-WT, STIM1-mKATE-S621A, or STIM1-mKATE-T626A plasmids were grown with 5 μm TMG for 24 h and treated with 1 μm TG for another 30 min. The cell lysates were subjected to co-immunoprecipitation with the anti-STIM1 antibody. The EB1, STIM1, and O-GlcNAc expression levels in the cell lysates and the EB1, Orai1, and STIM1 expression levels in immunoprecipitates were then determined by Western blotting analyses. α-Tubulin served as the loading control. B, relative abundance of co-precipitated EB1 was quantified as shown in A. Dots, data points. The data were analyzed using one-way ANOVA followed by Tukey's test. Data are represented as mean ± S.D. values (error bars) (n = 3). *, p < 0.05. C, relative abundance of co-precipitated Orai1 was quantified as shown in A. Dots, data points. The data were analyzed using one-way ANOVA followed by Tukey's test. Data are represented as mean ± S.D. values (error bars) (n = 3). *, p < 0.05.

      Discussion

      In the current study, we examined the effect of STIM1 O-GlcNAcylation on SOCE activity. Both increase and decrease in O-GlcNAcylation resulted in impaired activity, implying that fluctuation in O-GlcNAcylation regulates the function (Fig. 8). We had expected that STIM1 O-GlcNAcylation would either increase or decrease at the same Ser/Thr residue, and therefore we performed further in-depth analysis to determine how both increase and decrease in O-GlcNAcylation caused the same effect (i.e. STIM1 inactivation). We found two sites of O-GlcNAcylation (i.e. Ser621 and Thr626). Our results showed that Thr626 was O-GlcNAcylated in the steady state, whereas Ser621 was not, suggesting that O-GlcNAcylation of Thr626 was easier than that of Ser621. When overall O-GlcNAcylation decreased, the O-GlcNAcylated Thr626 was reduced, resulting in a decrease in SOCE activity. Therefore, it is thought that this Thr626 O-GlcNAcylation in the steady state is important for STIM1 function. When overall O-GlcNAcylation increased, the intact Ser621 in the steady state was O-GlcNAcylated, leading to a decrease in SOCE activity. Both decrease in O-GlcNAcylation at Thr626 and increase in O-GlcNAcylation at Ser621 decreased SOCE activity through reduction of phosphorylation at Ser621.
      Figure thumbnail gr8
      Figure 8Cartoon of the proposed mechanism by which SOCE function is regulated by fluctuation in STIM1 O-GlcNAcylation. At a normal O-GlcNAc level, STIM1 is dissociated from EB1 and interacts with Orai1 to induce SOCE activation by ER Ca2+ depletion because of O-GlcNAcylation at the Thr626 residue of STIM1, which promotes Ser621 phosphorylation. At a low O-GlcNAc level, the ER Ca2+ depletion–induced dissociation of STIM1 from EB1 and interaction with Orai1 are reduced because of decreased O-GlcNAcylation at the Thr626 residue of STIM1. At a high O-GlcNAc level, the ER Ca2+ depletion–induced dissociation of STIM1 from EB1 and interaction with Orai1 are decreased because of increased O-GlcNAcylation at the Ser621 residue of STIM1, which competes with Ser621 phosphorylation.
      Reduced O-GlcNAcylation of STIM1 at Thr626 lowered SOCE activity by decreasing the phosphorylation of STIM1 at Ser621. We speculated that O-GlcNAcylation of Ser621 competes with its phosphorylation. We could not obtain direct evidence to show how O-GlcNAcylation of Thr626 affects phosphorylation of Ser621 and are planning to investigate this further in our future studies.
      A previous study had shown that SOCE activity is triggered via STIM1 phosphorylation at the Ser575, Ser608, and Ser621 residues (
      • Pozo-Guisado E.
      • Campbell D.G.
      • Deak M.
      • Alvarez-Barrientos A.
      • Morrice N.A.
      • Alvarez I.S.
      • Alessi D.R.
      • Martín-Romero F.J.
      Phosphorylation of STIM1 at ERK1/2 target sites modulates store-operated calcium entry.
      ). However, we found that decrease in STIM1 phosphorylation at Ser621, but not at Ser575 or Ser608, led to its dissociation from EB1 and subsequent interaction with Orai1 to decrease SOCE activity, indicating that STIM1 phosphorylation at Ser621 could be sufficient for increasing SOCE activity. Phosphorylation at Ser621 might play the most important role in STIM1 activity and should be investigated further in future studies, along with the effect of STIM1 phosphorylation at Ser575 and Ser608 on STIM1 activity.
      STIM1 abnormality is closely related to the etiology of various diseases; for instance, STIM1 malfunction is linked to neurodegeneration and heart disease (
      • Bénard L.
      • Oh J.G.
      • Cacheux M.
      • Lee A.
      • Nonnenmacher M.
      • Matasic D.S.
      • Kohlbrenner E.
      • Kho C.
      • Pavoine C.
      • Hajjar R.J.
      • Hulot J.S.
      Cardiac Stim1 silencing impairs adaptive hypertrophy and promotes heart failure through inactivation of mTORC2/Akt signaling.
      ,
      • Pascual-Caro C.
      • Espinosa-Bermejo N.
      • Pozo-Guisado E.
      • Martin-Romero F.J.
      Role of STIM1 in neurodegeneration.
      ). Familial AD–associated presenilin 1 mutants promote γ-secretase cleavage of STIM1, leading to impairment of SOCE (
      • Tong B.C.
      • Lee C.S.
      • Cheng W.H.
      • Lai K.O.
      • Foskett J.K.
      • Cheung K.H.
      Familial Alzheimer's disease-associated presenilin 1 mutants promote γ-secretase cleavage of STIM1 to impair store-operated Ca2+ entry.
      ). Cardiac STIM1 silencing impairs adaptive hypertrophy and promotes heart failure through inactivation of mTORC2/Akt signaling (
      • Bénard L.
      • Oh J.G.
      • Cacheux M.
      • Lee A.
      • Nonnenmacher M.
      • Matasic D.S.
      • Kohlbrenner E.
      • Kho C.
      • Pavoine C.
      • Hajjar R.J.
      • Hulot J.S.
      Cardiac Stim1 silencing impairs adaptive hypertrophy and promotes heart failure through inactivation of mTORC2/Akt signaling.
      ). Targeting STIM1 itself might not be a promising therapy for these diseases, because the STIM1 activator is not available at the moment. Given that the fluctuation in STIM1 O-GlcNAcylation regulates STIM1 function, this modification could be involved in various diseases, such as AD and heart failure. TMG increases O-GlcNAcylation, although it is not specific for STIM1. Therefore, targeting STIM1 O-GlcNAcylation could provide a promising treatment option for these diseases if the specific modulator for STIM1 O-GlcNAcylation can be found.

      Experimental procedures

      Reagents and antibodies

      The OGA inhibitor TMG, the OGT inhibitor ST045849, and doxycycline (DOX) were purchased from Carbosynth (Compton, UK), TimTec (Newark, DE, USA), and Tokyo Chemical Industry (D4116, Tokyo, Japan), respectively. The SERCA inhibitor TG, the nonionic surfactant Pluronic-F127, and the prototypic inhibitor of organic anion transport, probenecid, were obtained from Sigma–Aldrich. The calcium-sensitive dye Fura-2-AM was purchased from Dojindo (Kumamoto, Japan). Anti-STIM1 (catalog no. MAB3602, Abnova (Taipei, Taiwan)), anti-O-GlcNAc (catalog no. NB300-524 (RL-2), Novus (Centennial, CO, USA)), anti-EB1 (catalog no. sc-15347, Santa Cruz Biotechnology, Inc. (Dallas, TX, USA)), anti-Orai1 (catalog no. SAB3500127, Sigma–Aldrich), and anti-α-tubulin (catalog no. PM054, MBL (Nagoya, Japan)) antibodies were used. Antibodies against phospho-Ser575-STIM1, phospho-Ser608-STIM1, and phospho-Ser621-STIM1 were raised against phosphopeptides corresponding to appropriate regions of the mouse STIM1, respectively (
      • Pozo-Guisado E.
      • Casas-Rua V.
      • Tomas-Martin P.
      • Lopez-Guerrero A.M.
      • Alvarez-Barrientos A.
      • Martin-Romero F.J.
      Phosphorylation of STIM1 at ERK1/2 target sites regulates interaction with the microtubule plus-end binding protein EB1.
      ).

      Cell culture

      HEK293 cells were obtained from the JCRB (Japanese Collection of Research Bioresources) Cell Bank (National Institute of Health Sciences, Kanagawa, Japan) and cultured in Dulbecco's modified Eagle's medium (Life Technologies, Inc.), supplemented with 10% (v/v) fetal bovine serum (Gibco), 100 units/ml penicillin, and 0.1 mg/ml streptomycin in a humidified atmosphere of air/CO2 at 37 °C. HEK293 cells were treated with different glucose concentrations (low (0 g/liter), normal (1 g/liter), or high (4.5 g/liter)), as shown in Fig. 1. Otherwise, Dulbecco's modified Eagle's medium containing 1 g/liter glucose was used.

      Generation of STIM1-KO-HEK cells

      The guide pair (sense 5′-CACCAGATGACAGACCGGAGTCAT and antisense 5′-AAACTGAGGTGATTATGGCGAGTC) was identified using the CRISPR.mit.edu-Zhang Lab web tool (https://zlab.bio/guide-design-resources). This pair targets exon 6 of the STIM1 (NM_003156, locus (ENSE00003492515)). The antisense dsDNA guide and the sense guide were cloned into eSPCAS9 1.1 constructs using BbsI restriction enzyme (New England Biolabs). Cells were cotransfected with 7 μg of guide RNA construct and 3 μg of pCW-hyPBase by electroporation in a 10-cm dish. Transfected cells were selected using blasticidin (10 μg/ml) for 48 h, and individual clones were analyzed by immunoblotting and sequencing. Genomic DNA was isolated, and the target site was amplified by PCR (primers: forward, 5′-AGCTTACTGTAATAGTGTATGGCAAGTGTGTATCT; reverse, 5′-CAGACTCTGCTCAGCTCGGTGTAAC). Sequencing of exon 6 in STIM1-KO-HEK cells revealed a bp 627–631 deletion of STIM1, confirming the successful KO of the STIM1 locus.

      Expression vectors, siRNAs, and transfection

      Mouse STIM1 (NCBI accession number: NM_009287.5) was cloned into PB-TET-CF-Bridge-2A-mKATE (kindly provided by Dr. Cody Kime; Retinal Regeneration, RIKEN Center for Biosystems Dynamics Research, Kobe, Japan) inducible by DOX (STIM1-mKATE). Cotransfection of STIM1-mKATE with OGT siRNA or OGA siRNA was performed using the TransIT-TKO reagent (Mirus Corp., Madison, WI) according to the manufacturer's instructions. The targeting sequences of the OGT siRNA were as follows: sense, 5′-GCAACAAACCUGACCACAUTT-3′; antisense, 3′-TTCGUUGUUUGGACUGGUGUA-5′. The targeting sequences of the OGA siRNA were as follows: sense, 5′-GCAACAAACCUGACCACAUTT-3′; antisense, 3′-TTCGUUGUUUGGACUGGUGUA-5′.

      Immunoblotting and co-immunoprecipitation

      DOX-treated cells were lysed in SDS-PAGE sample buffer and boiled for 10 min at 100 °C. After SDS-PAGE, the proteins were transferred to a polyvinylidene fluoride membrane (Merck Millipore, Billerica, MA, USA) and subjected to immunoblotting with anti-STIM1 (1:3000 dilution), anti-O-GlcNAc (1:2000 dilution), anti-EB1 (1:5000 dilution), anti-Orai1 (1:2000 dilution), anti-phospho-Ser575-STIM1 with nonphosphorylated peptide (1:3000 dilution), anti-phospho-Ser608-STIM1 with nonphosphorylated peptide (1:3000 dilution), anti-phospho-Ser621-STIM1 with nonphosphorylated peptide (1:3000 dilution), and anti-α-tubulin (1:5000 dilution) antibodies. The immunoblotted proteins were detected using Fusion FX7 (Vilber–Lourmat, Eberhardzell, Germany). For co-immunoprecipitation analysis, cells were lysed with lysis buffer composed of 50 mm Tris-HCl (pH 7.5), 1 mm EGTA, 1 mm EDTA, 1% (v/v) Nonidet P-40, 1 mm sodium orthovanadate, 50 mm sodium fluoride, 5 mm sodium pyrophosphate, and 0.1 mm phenylmethylsulfonyl fluoride. Subsequently, they were centrifuged for 10 min at 20,400 × g, and the supernatants obtained were incubated with anti-STIM1 antibody (Figs. 1C, 4 (B and D), and 7A) conjugated with SureBeads Magnetic Beads (Bio-Rad). The proteins eluted with SDS-PAGE sample buffer were subjected to SDS-PAGE.

      Confocal microscopy

      Cells on glass-bottomed dishes were fixed with 4% paraformaldehyde for 10 min and permeabilized with PBS containing 0.1% Triton X-100 for 15 min. After the cells were subjected to blocking in PBS with 10% BSA, they were treated with anti-STIM1 antibody (1:500 dilution) for 60 min, followed by treatment with Alexa Fluor 488–labeled secondary antibody (1:400 dilution). The fluorescently labeled cells were visualized using confocal microscopy (SP8; Leica, Wetzlar, Germany).

      SOCE measurement

      Cells on glass-bottomed dishes were incubated with 2 mm Fura-2-AM along with 0.025% Pluronic-F127 and 12.5 mm probenecid for 50 min at 37 °C in loading buffer. Subsequently, the buffer was replaced with Ca2+-free loading buffer. Depletion of Ca2+ stores was triggered by adding 1 μm TG to the dishes. The composition of the Ca2+-free loading buffer was as follows: 20 mm HEPES, 115 mm NaCl, 5.4 mm KCl, 0.34 mm Na2HPO4, 0.44 mm KH2PO4, 4.17 mm NaHCO3, and 0.8 mm Mg2+. SOCE was measured by monitoring the increase in [Ca2+]i after the addition of 2 mm CaCl2 to the TG-containing medium. The [Ca2+]i was calculated by monitoring the F340 nm/F380 nm excitation ratio with an emission wavelength of 510 nm (Nikon, Japan). Because this ratio correlates with intracellular Ca2+ concentration, this method is generally used for the measurement of SOCE activity. Digital images were recorded with a Hamamatsu C9100 EM-CCD camera (Hamamatsu, Japan) with the Metafluor software. All measurements were performed at 37 °C (MATS-505RA20, Tokai Hit, Japan).

      Statistical analyses

      The data are expressed as the means ± S.D. for more than three determinations. Statistical analyses were performed using Student's t test (Figs. 2 (B, D, F, and H), 3E, and 5D and Fig. S2 (B, D, and E)) or one-way analysis of variance (ANOVA) followed by Tukey's test (Figs. 1 (B and D), 3 (B and D), 4 (C, E, and F), 5 (B and F), and 7 (B and C) and Figs. S1 (B and D), S3B, and S5B) or one-way ANOVA followed by Dunnett's test (Fig. S4B). p values <0.05 were considered significant.

      Data availability

      All data are contained within the article.

      Acknowledgments

      We thank Nozomi Tokuhara (Osaka Medical College) for expert technical assistance. We also thank the members of the Department of Pharmacology at Osaka Medical College for scientific discussions.

      Supplementary Material

      References

        • Holt G.D.
        • Haltiwanger R.S.
        • Torres C.R.
        • Hart G.W.
        Erythrocytes contain cytoplasmic glycoproteins: O-linked GlcNAc on Band 4.1.
        J. Biol. Chem. 1987; 262 (3117790): 14847-14850
        • Hart G.W.
        • Housley M.P.
        • Slawson C.
        Cycling of O-linked β-N-acetylglucosamine on nucleocytoplasmic proteins.
        Nature. 2007; 446 (17460662): 1017-1022
        • Zachara N.E.
        • O'Donnell N.
        • Cheung W.D.
        • Mercer J.J.
        • Marth J.D.
        • Hart G.W.
        Dynamic O-GlcNAc modification of nucleocytoplasmic proteins in response to stress: a survival response of mammalian cells.
        J. Biol. Chem. 2004; 279 (15138254): 30133-30142
        • McClain D.A.
        Hexosamines as mediators of nutrient sensing and regulation in diabetes.
        J. Diabetes Complications. 2002; 16 (11872372): 72-80
        • Gao Y.
        • Wells L.
        • Comer F.I.
        • Parker G.J.
        • Hart G.W.
        Dynamic O-glycosylation of nuclear and cytosolic proteins: cloning and characterization of a neutral, cytosolic β-N-acetylglucosaminidase from human brain.
        J. Biol. Chem. 2001; 276 (11148210): 9838-9845
        • Hart G.W.
        Nutrient regulation of signaling and transcription.
        J. Biol. Chem. 2019; 294 (30626734): 2211-2231
        • Banerjee P.S.
        • Ma J.
        • Hart G.W.
        Diabetes-associated dysregulation of O-GlcNAcylation in rat cardiac mitochondria.
        Proc. Natl. Acad. Sci. U. S. A. 2015; 112 (25918408): 6050-6055
        • Lefebvre T.
        • Dehennaut V.
        • Guinez C.
        • Olivier S.
        • Drougat L.
        • Mir A.M.
        • Mortuaire M.
        • Vercoutter-Edouart A.S.
        • Michalski J.C.
        Dysregulation of the nutrient/stress sensor O-GlcNAcylation is involved in the etiology of cardiovascular disorders, type-2 diabetes and Alzheimer's disease.
        Biochim. Biophys. Acta. 2010; 1800 (19732809): 67-79
        • Yang X.
        • Ongusaha P.P.
        • Miles P.D.
        • Havstad J.C.
        • Zhang F.
        • So W.V.
        • Kudlow J.E.
        • Michell R.H.
        • Olefsky J.M.
        • Field S.J.
        • Evans R.M.
        Phosphoinositide signalling links O-GlcNAc transferase to insulin resistance.
        Nature. 2008; 451 (18288188): 964-969
        • Hwang H.
        • Rhim H.
        Acutely elevated O-GlcNAcylation suppresses hippocampal activity by modulating both intrinsic and synaptic excitability factors.
        Sci. Rep. 2019; 9 (31086206)7287
        • Kim C.
        • Nam D.W.
        • Park S.Y.
        • Song H.
        • Hong H.S.
        • Boo J.H.
        • Jung E.S.
        • Kim Y.
        • Baek J.Y.
        • Kim K.S.
        • Cho J.W.
        • Mook-Jung I.
        O-Linked β-N-acetylglucosaminidase inhibitor attenuates β-amyloid plaque and rescues memory impairment.
        Neurobiol. Aging. 2013; 34 (22503002): 275-285
        • Yokoe S.
        • Asahi M.
        • Takeda T.
        • Otsu K.
        • Taniguchi N.
        • Miyoshi E.
        • Suzuki K.
        Inhibition of phospholamban phosphorylation by O-GlcNAcylation: implications for diabetic cardiomyopathy.
        Glycobiology. 2010; 20 (20484118): 1217-1226
        • Du X.L.
        • Edelstein D.
        • Dimmeler S.
        • Ju Q.
        • Sui C.
        • Brownlee M.
        Hyperglycemia inhibits endothelial nitric oxide synthase activity by posttranslational modification at the Akt site.
        J. Clin. Invest. 2001; 108 (11696579): 1341-1348
        • Ishimura E.
        • Nakagawa T.
        • Moriwaki K.
        • Hirano S.
        • Matsumori Y.
        • Asahi M.
        Augmented O-GlcNAcylation of AMP-activated kinase promotes the proliferation of LoVo cells, a colon cancer cell line.
        Cancer Sci. 2017; 108 (28973823): 2373-2382
        • Moriwaki K.
        • Asahi M.
        Augmented TME O-GlcNAcylation promotes tumor proliferation through the inhibition of p38 MAPK.
        Mol. Cancer Res. 2017; 15 (28536142): 1287-1298
        • Yuzwa S.A.
        • Shan X.
        • Macauley M.S.
        • Clark T.
        • Skorobogatko Y.
        • Vosseller K.
        • Vocadlo D.J.
        Increasing O-GlcNAc slows neurodegeneration and stabilizes tau against aggregation.
        Nat. Chem. Biol. 2012; 8 (22366723): 393-399
        • Wang A.C.
        • Jensen E.H.
        • Rexach J.E.
        • Vinters H.V.
        • Hsieh-Wilson L.C.
        Loss of O-GlcNAc glycosylation in forebrain excitatory neurons induces neurodegeneration.
        Proc. Natl. Acad. Sci. U. S. A. 2016; 113 (27956640): 15120-15125
        • Marotta N.P.
        • Lin Y.H.
        • Lewis Y.E.
        • Ambroso M.R.
        • Zaro B.W.
        • Roth M.T.
        • Arnold D.B.
        • Langen R.
        • Pratt M.R.
        O-GlcNAc modification blocks the aggregation and toxicity of the protein α-synuclein associated with Parkinson's disease.
        Nat. Chem. 2015; 7 (26492012): 913-920
        • Wani W.Y.
        • Ouyang X.
        • Benavides G.A.
        • Redmann M.
        • Cofield S.S.
        • Shacka J.J.
        • Chatham J.C.
        • Darley-Usmar V.
        • Zhang J.
        O-GlcNAc regulation of autophagy and α-synuclein homeostasis; implications for Parkinson's disease.
        Mol. Brain. 2017; 10 (28724388): 32
        • Zhang S.L.
        • Yu Y.
        • Roos J.
        • Kozak J.A.
        • Deerinck T.J.
        • Ellisman M.H.
        • Stauderman K.A.
        • Cahalan M.D.
        STIM1 is a Ca2+ sensor that activates CRAC channels and migrates from the Ca2+ store to the plasma membrane.
        Nature. 2005; 437 (16208375): 902-905
        • Feske S.
        • Picard C.
        • Fischer A.
        Immunodeficiency due to mutations in ORAI1 and STIM1.
        Clin. Immunol. 2010; 135 (20189884): 169-182
        • Roos J.
        • DiGregorio P.J.
        • Yeromin A.V.
        • Ohlsen K.
        • Lioudyno M.
        • Zhang S.
        • Safrina O.
        • Kozak J.A.
        • Wagner S.L.
        • Cahalan M.D.
        • Veliçelebi G.
        • Stauderman K.A.
        STIM1, an essential and conserved component of store-operated Ca2+ channel function.
        J. Cell Biol. 2005; 169 (15866891): 435-445
        • Liou J.
        • Kim M.L.
        • Heo W.D.
        • Jones J.T.
        • Myers J.W.
        • Ferrell Jr., J.E.
        • Meyer T.
        STIM is a Ca2+ sensor essential for Ca2+-store-depletion-triggered Ca2+ influx.
        Curr. Biol. 2005; 15 (16005298): 1235-1241
        • Moccia F.
        • Zuccolo E.
        • Soda T.
        • Tanzi F.
        • Guerra G.
        • Mapelli L.
        • Lodola F.
        • D'Angelo E.
        Stim and Orai proteins in neuronal Ca2+ signaling and excitability.
        Front. Cell Neurosci. 2015; 9 (25964739): 153
        • Zhang H.
        • Clemens R.A.
        • Liu F.
        • Hu Y.
        • Baba Y.
        • Theodore P.
        • Kurosaki T.
        • Lowell C.A.
        STIM1 calcium sensor is required for activation of the phagocyte oxidase during inflammation and host defense.
        Blood. 2014; 123 (24493668): 2238-2249
        • Bénard L.
        • Oh J.G.
        • Cacheux M.
        • Lee A.
        • Nonnenmacher M.
        • Matasic D.S.
        • Kohlbrenner E.
        • Kho C.
        • Pavoine C.
        • Hajjar R.J.
        • Hulot J.S.
        Cardiac Stim1 silencing impairs adaptive hypertrophy and promotes heart failure through inactivation of mTORC2/Akt signaling.
        Circulation. 2016; 133 (26936863): 1458-1471
        • Jia S.
        • Rodriguez M.
        • Williams A.G.
        • Yuan J.P.
        Homer binds to Orai1 and TRPC channels in the neointima and regulates vascular smooth muscle cell migration and proliferation.
        Sci. Rep. 2017; 7 (28698564)5075
        • White C.
        The regulation of tumor cell invasion and metastasis by endoplasmic reticulum-to-mitochondrial Ca2+ Transfer.
        Front. Oncol. 2017; 7 (28848710): 171
        • Liou J.
        • Fivaz M.
        • Inoue T.
        • Meyer T.
        Live-cell imaging reveals sequential oligomerization and local plasma membrane targeting of stromal interaction molecule 1 after Ca2+ store depletion.
        Proc. Natl. Acad. Sci. U. S. A. 2007; 104 (17517596): 9301-9306
        • Wu M.M.
        • Buchanan J.
        • Luik R.M.
        • Lewis R.S.
        Ca2+ store depletion causes STIM1 to accumulate in ER regions closely associated with the plasma membrane.
        J. Cell Biol. 2006; 174 (16966422): 803-813
        • Feske S.
        • Gwack Y.
        • Prakriya M.
        • Srikanth S.
        • Puppel S.H.
        • Tanasa B.
        • Hogan P.G.
        • Lewis R.S.
        • Daly M.
        • Rao A.
        A mutation in Orai1 causes immune deficiency by abrogating CRAC channel function.
        Nature. 2006; 441 (16582901): 179-185
        • Yuan J.P.
        • Zeng W.
        • Huang G.N.
        • Worley P.F.
        • Muallem S.
        STIM1 heteromultimerizes TRPC channels to determine their function as store-operated channels.
        Nat. Cell Biol. 2007; 9 (17486119): 636-645
        • Stathopulos P.B.
        • Zheng L.
        • Li G.Y.
        • Plevin M.J.
        • Ikura M.
        Structural and mechanistic insights into STIM1-mediated initiation of store-operated calcium entry.
        Cell. 2008; 135 (18854159): 110-122
        • Pozo-Guisado E.
        • Campbell D.G.
        • Deak M.
        • Alvarez-Barrientos A.
        • Morrice N.A.
        • Alvarez I.S.
        • Alessi D.R.
        • Martín-Romero F.J.
        Phosphorylation of STIM1 at ERK1/2 target sites modulates store-operated calcium entry.
        J. Cell Sci. 2010; 123 (20736304): 3084-3093
        • Grigoriev I.
        • Gouveia S.M.
        • van der Vaart B.
        • Demmers J.
        • Smyth J.T.
        • Honnappa S.
        • Splinter D.
        • Steinmetz M.O.
        • Putney Jr., J.W.
        • Hoogenraad C.C.
        • Akhmanova A.
        STIM1 is a MT-plus-end-tracking protein involved in remodeling of the ER.
        Curr. Biol. 2008; 18 (18249114): 177-182
        • Pozo-Guisado E.
        • Casas-Rua V.
        • Tomas-Martin P.
        • Lopez-Guerrero A.M.
        • Alvarez-Barrientos A.
        • Martin-Romero F.J.
        Phosphorylation of STIM1 at ERK1/2 target sites regulates interaction with the microtubule plus-end binding protein EB1.
        J. Cell Sci. 2013; 126 (23687376): 3170-3180
        • Chang C.L.
        • Chen Y.J.
        • Quintanilla C.G.
        • Hsieh T.S.
        • Liou J.
        EB1 binding restricts STIM1 translocation to ER-PM junctions and regulates store-operated Ca2+ entry.
        J. Cell Biol. 2018; 217 (29563214): 2047-2058
        • Zhu-Mauldin X.
        • Marsh S.A.
        • Zou L.
        • Marchase R.B.
        • Chatham J.C.
        Modification of STIM1 by O-linked N-acetylglucosamine (O-GlcNAc) attenuates store-operated calcium entry in neonatal cardiomyocytes.
        J. Biol. Chem. 2012; 287 (22992728): 39094-39106
        • Walgren J.L.E.
        • Vincent T.S.
        • Schey K.L.
        • Buse M.G.
        High glucose and insulin promote O-GlcNAc modification of proteins, including α-tubulin.
        Am. J. Physiol. Endocrinol. Metab. 2003; 284 (12397027): E424-E434
        • Hu Y.
        • Suarez J.
        • Fricovsky E.
        • Wang H.
        • Scott B.T.
        • Trauger S.A.
        • Han W.
        • Hu Y.
        • Oyeleye M.O.
        • Dillmann W.H.
        Increased enzymatic O-GlcNAcylation of mitochondrial proteins impairs mitochondrial function in cardiac myocytes exposed to high glucose.
        J. Biol. Chem. 2009; 284 (19004814): 547-555
        • Jochmann R.
        • Holz P.
        • Sticht H.
        • Stürzl M.
        Validation of the reliability of computational O-GlcNAc prediction.
        Biochim. Biophys. Acta. 2014; 1844 (24332980): 416-421
        • Soboloff J.
        • Spassova M.A.
        • Tang X.D.
        • Hewavitharana T.
        • Xu W.
        • Gill D.L.
        Orai1 and STIM reconstitute store-operated calcium channel function.
        J. Biol. Chem. 2006; 281 (16766533): 20661-20665
        • Deng X.
        • Wang Y.
        • Zhou Y.
        • Soboloff J.
        • Gill D.L.
        STIM and Orai: dynamic intermembrane coupling to control cellular calcium signals.
        J. Biol. Chem. 2009; 284 (19473984): 22501-22505
        • Wang Y.
        • Deng X.
        • Zhou Y.
        • Hendron E.
        • Mancarella S.
        • Ritchie M.F.
        • Tang X.D.
        • Baba Y.
        • Kurosaki T.
        • Mori Y.
        • Soboloff J.
        • Gill D.L.
        STIM protein coupling in the activation of Orai channels.
        Proc. Natl. Acad. Sci. U. S. A. 2009; 106 (19376967): 7391-7396
        • Pascual-Caro C.
        • Espinosa-Bermejo N.
        • Pozo-Guisado E.
        • Martin-Romero F.J.
        Role of STIM1 in neurodegeneration.
        World J. Biol. Chem. 2018; 9 (30568747): 16-24
        • Tong B.C.
        • Lee C.S.
        • Cheng W.H.
        • Lai K.O.
        • Foskett J.K.
        • Cheung K.H.
        Familial Alzheimer's disease-associated presenilin 1 mutants promote γ-secretase cleavage of STIM1 to impair store-operated Ca2+ entry.
        Sci. Signal. 2016; 9 (27601731): ra89