Cross-talk between Two Essential Nutrient-sensitive Enzymes: O-GlcNAc TRANSFERASE (OGT) AND AMP-ACTIVATED PROTEIN KINASE (AMPK)*

Nutrient-sensitive pathways regulate both O-GlcNAc transferase (OGT) and AMP-activated protein kinase (AMPK), cooperatively connecting metabolic homeostasis to regulation of numerous intracellular processes essential for life. Similar to phosphorylation, catalyzed by kinases such as AMPK, O-GlcNAcylation is a highly dynamic Ser/Thr-specific post-translational modification of nuclear, cytoplasmic, and mitochondrial proteins catalyzed exclusively by OGT. OGT and AMPK target a multitude of intracellular proteins, with the net effect to protect cells from the damaging effects of metabolic stress. Despite hundreds of studies demonstrating significant overlap in upstream and downstream signaling processes, no study has investigated if OGT and AMPK can directly regulate each other. We show acute activation of AMPK alters the substrate selectivity of OGT in several cell lines and nuclear localization of OGT in C2C12 skeletal muscle myotubes. Nuclear localization of OGT affects O-GlcNAcylation of numerous nuclear proteins and acetylation of Lys-9 on histone 3 in myotubes. AMPK phosphorylates Thr-444 on OGT in vitro; phosphorylation of Thr-444 is tightly associated with AMPK activity and nuclear localization of OGT in myotubes, and phospho-mimetic T444E-OGT exhibits altered substrate selectivity. Conversely, the α- and γ-subunits of AMPK are O-GlcNAcylated, O-GlcNAcylation of the γ1-subunit increases with AMPK activity, and acute inhibition of O-GlcNAc cycling disrupts activation of AMPK. We have demonstrated significant cross-talk between the O-GlcNAc and AMPK systems, suggesting OGT and AMPK may cooperatively regulate nutrient-sensitive intracellular processes that mediate cellular metabolism, growth, proliferation, and/or tissue function.     

Knockdown of STIM1 inhibits 6-hydroxydopamine-induced oxidative stress through attenuating calcium-dependent ER stress and mitochondrial dysfunction in undifferentiated PC12 cells

Abstract

Stromal interaction molecule (STIM) proteins are parts of elaborate eukaryotic Ca²⁺ signaling systems and are considered to be important players in regulating neuronal Ca²⁺ homeostasis under normal ageing and pathological conditions. Here, we investigated the potential role of STIM1 in 6-hydroxydopamine (6-OHDA)-induced toxicity in undifferentiated PC12 cell lines. Cells exposed to 6-OHDA demonstrated alterations in the generation of reactive oxygen species (ROS) in a Ca²⁺-dependent manner. Downregulation of STIM1 expression by specific small interfering RNA (siRNA) attenuated apoptotic cell death, reduced intracellular ROS production, and partially prevented the impaired endogenous antioxidant enzyme activities after 6-OHDA treatment. Furthermore, STIM1 knockdown significantly attenuated 6-OHDA-induced intracellular Ca²⁺ overload by inhibiting endogenous store-operated calcium entry (SOCE). The effect of STIM1 siNRA on SOCE was related to orai1 and L-type Ca²⁺ channels, but not to transient receptor potential canonical type 1 (TRPC1) channel. In addition, silencing of STIM1 increased the Ca²⁺ buffering capacity of the endoplasmic reticulum (ER) in 6-OHDA-injured cells. ER vacuoles formed from the destruction of ER structural integrity and activation of ER-related apoptotic factors (CHOP and Caspase-12) were partially prevented by STIM1 knockdown. Moreover, STIM1 knockdown attenuated 6-OHDA-induced mitochondrial Ca²⁺ uptake and mitochondrial dysfunction, including the collapse of mitochondrial membrane potential (MMP) and the decrease of ATP generation. Taken together, our data provide the first evidence that inhibition of STIM1-meditated intracellular Ca²⁺ dyshomeostasis protects undifferentiated PC12 cells against 6-OHDA toxicity and indicate that STIM1 may be responsible for neuronal oxidative stress induced by ER stress and mitochondrial dysfunction in PD.     

Ca signaling and STIM1

An increase in the intracellular calcium ion concentration ([Ca²⁺]) impacts a diverse range of cell functions, including adhesion, motility, gene expression and proliferation. Elevation of intracellular calcium ion (Ca²⁺) regulates various cellular events after the stimulation of cells. Initial increase in Ca²⁺ comes from the endoplasmic reticulum (ER), intracellular storage space. However, the continuous influx of extracellular Ca²⁺ is required to maintain the increased level of Ca²⁺ inside cells. Store-operated Ca²⁺ entry (SOCE) manages this process, and STIM1, a newly discovered molecule, has a unique and essential role in SOCE. STIM1 can sense the exhaustion of Ca²⁺ in the ER, and activate the SOC channel in the plasma membrane, leading to the continuous influx of extracellular Ca²⁺. STIM1 senses the status of the intracellular Ca²⁺ stores via a luminal N-terminal Ca²⁺-binding EF-hand domain. Dissociation of Ca²⁺ from this domain induces the clustering of STIM1 to regions of the ER that lie close to the plasma membrane, where it regulates the activity of the store-operated Ca²⁺ channels/entry (calcium-release-activated calcium channels/entry). In this review, we summarize the mechanism by which STIM1 regulates SOCE, and also its role in the control of mast cell functions and allergic responses.     

O-GlcNAcylation of Cofilin Promotes Breast Cancer Cell Invasion

O-GlcNAcylation is a post-translational modification that regulates a broad range of nuclear and cytoplasmic proteins and is emerging as a key regulator of various biological processes. Previous studies have shown that increased levels of global O-GlcNAcylation and O-GlcNAc transferase (OGT) are linked to the incidence of metastasis in breast cancer patients, but the molecular basis behind this is not fully known. In this study, we have determined that the actin-binding protein cofilin is O-GlcNAcylated by OGT and mainly, if not completely, mediates OGT modulation of cell mobility. O-GlcNAcylation at Ser-108 of cofilin is required for its proper localization in invadopodia at the leading edge of breast cancer cells during three-dimensional cell invasion. Loss of O-GlcNAcylation of cofilin leads to destabilization of invadopodia and impairs cell invasion, although the actin-severing activity or lamellipodial localization is not affected. Our study provides insights into the mechanism of post-translational modification in fine-tuning the regulation of cofilin activity and suggests its important implications in cancer metastasis.     

The N-terminal portion of autoinhibitory element modulates human endothelial nitric-oxide synthase activity through coordinated controls of phosphorylation at Thr495 and Ser1177

NO production catalysed by eNOS (endothelial nitric-oxide synthase) plays an important role in the cardiovascular system. A variety of agonists activate eNOS through the Ser¹¹⁷⁷ phosphorylation concomitant with Thr⁴⁹⁵ dephosphorylation, resulting in increased ·NO production with a basal level of calcium. To date, the underlying mechanism remains unclear. We have previously demonstrated that perturbation of the AIE (autoinhibitory element) in the FMN-binding subdomain can also lead to eNOS activation with a basal level of calcium, implying that the AIE might regulate eNOS activation through modulating phosphorylation at Thr⁴⁹⁵ and Ser¹¹⁷⁷. Here we generated stable clones in HEK-293 (human embryonic kidney 293) cells with a series of deletion mutants in both the AIE (Δ594–604, Δ605–612 and Δ626–634) and the C-terminal tail (Δ14; deletion of 1164–1177). The expression of Δ594–604 and Δ605–612 mutants in non-stimulated HEK-293 cells substantially increased nitrate/nitrite release into the culture medium; the other two mutants, Δ626–634 and Δ1164–1177, displayed no significant difference when compared with WTeNOS (wild-type eNOS). Intriguingly, mutant Δ594–604 showed close correlation between Ser¹¹⁷⁷ phosphorylation and Thr⁴⁹⁵ dephosphorylation, and NO production. Our results have indicated that N-terminal portion of AIE (residues 594–604) regulates eNOS activity through coordinated phosphorylation on Ser¹¹⁷⁷ and Thr⁴⁹⁵.     

Role of STIM1 in the surface expression of SARAF

The store-operated Ca²⁺ entry-associated regulatory factor (SARAF), a protein expressed both in the endoplasmic reticulum and the plasma membrane, has been presented as a STIM1-interacting protein with the ability to modulate intracellular Ca²⁺ homeostasis. SARAF negatively modulates store-operated Ca²⁺ entry (SOCE) by preventing STIM1 spontaneous activation and regulating STIM1-Orai1 complex formation. In addition, SARAF is a negative regulator of Ca²⁺ entry through the arachidonate-regulated Ca²⁺ (ARC) channels. Here we explored the possible role of the surface expression of SARAF on the location of STIM1 in the plasma membrane. In NG115-401L cells, lacking a detectable expression of native STIM1, transfection with pHluorin-STIM1, which is able to translocate to the cell surface, enhances the plasma membrane location of SARAF as compared to cells transfected with YFP-STIM1, lacking the ability to translocate to the cell surface. These findings suggest that the surface location of SARAF is dependent on the expression of STIM1 in the plasma membrane.     

3,3'-Diindolylmethane induces gastric cancer cells death via STIM1 mediated store-operated calcium entry

3,3''-Diindolylmethane (DIM), a natural phytochemicals isolated from cruciferous vegetables, has been reported to inhibit human gastric cancer cells proliferation and induce cells apoptosis as well as autophagy, but its mechanisms are still unclear. Store-operated calcium entry (SOCE) is a main Ca²⁺ influx pathway in various of cancers, which is activated by the depletion of endoplasmic reticulum (ER) Ca²⁺ store. Stromal interaction molecular 1 (STIM1) is the necessary component of SOCE. In this study, we focus on to examine the regulatory mechanism of SOCE on DIM-induced death in gastric cancer. After treating the human BGC-823 and SGC-7901 gastric cancer cells with DIM, cellular proliferation was determined by MTT, apoptosis and autophagy were detected by flow cytometry or Hoechst 33342 staining. The expression levels of related proteins were evaluated by Western blotting. Free cytosolilc Ca²⁺ level was assessed by fluorescence monitoring under a laser scanning confocal microscope. The data have shown that DIM could significantly inhibit proliferation and induce apoptosis as well as autophagy in two gastric cancer cell lines. After DIM treatment, the STIM1-mediated SOCE was activated by upregulating STIM1 and decreasing ER Ca²⁺ level. Knockdown STIM1 with siRNA or pharmacological inhibition of SOCE attenuated DIM induced apoptosis and autophagy by inhibiting p-AMPK mediated ER stress pathway. Our data highlighted that the potential of SOCE as a promising target for treating cancers. Developing effective and selective activators targeting STIM1-mediated SOCE pathway will facilitate better therapeutic sensitivity of phytochemicals acting on SOCE in gastric cancer. Moreover, more research should be performed to validate the efficacy of combination chemotherapy of anti-cancer drugs targeting SOCE for clinical application.     

A Highlights from MBoC Selection: O-GlcNAc Cycling Enzymes Associate with the Translational Machinery and Modify Core Ribosomal Proteins

Protein synthesis is globally regulated through posttranslational modifications of initiation and elongation factors. Recent high-throughput studies have identified translation factors and ribosomal proteins (RPs) as substrates for the O-GlcNAc modification. Here we determine the extent and abundance of O-GlcNAcylated proteins in translational preparations. O-GlcNAc is present on many proteins that form active polysomes. We identify twenty O-GlcNAcylated core RPs, of which eight are newly reported. We map sites of O-GlcNAc modification on four RPs (L6, L29, L32, and L36). RPS6, a component of the mammalian target of rapamycin (mTOR) signaling pathway, follows different dynamics of O-GlcNAcylation than nutrient-induced phosphorylation. We also show that both O-GlcNAc cycling enzymes OGT and OGAse strongly associate with cytosolic ribosomes. Immunofluorescence experiments demonstrate that OGAse is present uniformly throughout the nucleus, whereas OGT is excluded from the nucleolus. Moreover, nucleolar stress only alters OGAse nuclear staining, but not OGT staining. Lastly, adenovirus-mediated overexpression of OGT, but not of OGAse or GFP control, causes an accumulation of 60S subunits and 80S monosomes. Our results not only establish that O-GlcNAcylation extensively modifies RPs, but also suggest that O-GlcNAc play important roles in regulating translation and ribosome biogenesis.     

Differential Regulation of the Ten-Eleven Translocation (TET) Family of Dioxygenases by O-Linked β-N-Acetylglucosamine Transferase (OGT)

The ten-eleven translocation (TET) family of dioxygenases (TET1/2/3) converts 5-methylcytosine to 5-hydroxymethylcytosine and provides a vital mechanism for DNA demethylation. However, how TET proteins are regulated is largely unknown. Here we report that the O-linked β-GlcNAc (O-GlcNAc) transferase (OGT) is not only a major TET3-interacting protein but also regulates TET3 subcellular localization and enzymatic activity. OGT catalyzes the O-GlcNAcylation of TET3, promotes TET3 nuclear export, and, consequently, inhibits the formation of 5-hydroxymethylcytosine catalyzed by TET3. Although TET1 and TET2 also interact with and can be O-GlcNAcylated by OGT, neither their subcellular localization nor their enzymatic activity are affected by OGT. Furthermore, we show that the nuclear localization and O-GlcNAcylation of TET3 are regulated by glucose metabolism. Our study reveals the differential regulation of TET family proteins by OGT and a novel link between glucose metabolism and DNA epigenetic modification.     

Altered O-GlcNAc modification and phosphorylation of mitochondrial proteins in myoblast cells exposed to high glucose

Hyperglycemia induced increased posttranslational modification of proteins by O-linked-β-N-acetyl glucosamine (O-GlcNAcylation) and mitochondrial dysfunction has been independently implicated in the development of insulin resistance. It is not known whether repertoire of O-GlcNAcylated proteins includes mitochondrial proteins and their altered O-GlcNAcylation impinges on their phosphorylation mediated normal functioning thus contribute to mitochondrial dysfunction and insulin resistance. We have explored the O-GlcNAcylation of mitochondrial proteins from myoblast cells under basal (4 mM) and high glucose (30 mM) conditions using a combination of proteomic approaches. Furthermore, we have assessed the accompanied changes in the phosphorylation of mitochondrial proteins. We report that a number of mitochondrial proteins are O-GlcNAcylated under basal condition which is altered under high glucose condition. In addition, we report that exposure to high glucose not only changes the O-GlcNAcylation of mitochondrial proteins but also changes their phosphorylation profiles. The dynamic and complex interplay between O-GlcNAcylation and phosphorylation of mitochondrial proteins was further validated by immunoblot analysis of HSP60, prohibitin, and voltage-dependent anion channel 1 as candidate proteins. O-GlcNAcylation of mitochondrial proteins may play a role in normal functioning of mitochondria. High glucose induced changes in O-GlcNAcylation and phosphorylation of mitochondrial proteins may be associated with mitochondrial dysfunction and insulin resistance.     

Orai1 contributes to the establishment of an apoptosis-resistant phenotype in prostate cancer cells

The molecular nature of calcium (Ca²⁺)-dependent mechanisms and the ion channels having a major role in the apoptosis of cancer cells remain a subject of debate. Here, we show that the recently identified Orai1 protein represents the major molecular component of endogenous store-operated Ca²⁺ entry (SOCE) in human prostate cancer (PCa) cells, and constitutes the principal source of Ca²⁺ influx used by the cell to trigger apoptosis. The downregulation of Orai1, and consequently SOCE, protects the cells from diverse apoptosis-inducing pathways, such as those induced by thapsigargin (Tg), tumor necrosis factor α, and cisplatin/oxaliplatin. The transfection of functional Orai1 mutants, such as R91W, a selectivity mutant, and L273S, a coiled-coil mutant, into the cells significantly decreased both SOCE and the rate of Tg-induced apoptosis. This suggests that the functional coupling of STIM1 to Orai1, as well as Orai1 Ca²⁺-selectivity as a channel, is required for its pro-apoptotic effects. We have also shown that the apoptosis resistance of androgen-independent PCa cells is associated with the downregulation of Orai1 expression as well as SOCE. Orai1 rescue, following Orai1 transfection of steroid-deprived cells, re-established the store-operated channel current and restored the normal rate of apoptosis. Thus, Orai1 has a pivotal role in the triggering of apoptosis, irrespective of apoptosis-inducing stimuli, and in the establishment of an apoptosis-resistant phenotype in PCa cells.     

Inhibition of mTOR affects protein stability of OGT

Autophagy regulates cellular homeostasis through degradation of aged or damaged subcellular organelles and components. Interestingly, autophagy-deficient beta cells, for example Atg7-mutant mice, exhibited hypoinsulinemia and hyperglycemia. Also, autophagy response is diminished in heart of diabetic mice. These results implied that autophagy and diabetes are closely connected and affect each other. Although protein O-GlcNAcylation is up-regulated in hyperglycemia and diabetes, and O-GlcNAcylated proteins play an important role in metabolism and nutrient sensing, little is known whether autophagy affects O-GlcNAc modification and vice versa. In this study, we suppressed the action of mTOR by treatment of mTOR catalytic inhibitors (PP242 and Torin1) to induce autophagic flux. Results showed a decrease in global O-GlcNAcylation, which is due to decreased OGT protein and increased OGA protein. Interestingly, knockdown of ATG genes or blocking of lysosomal degradation enhanced protein stability of OGT. In addition, when proteasomal inhibitor was treated together with mTOR inhibitor, protein level of OGT almost recovered to control level. These data suggest that mTOR inhibition is a more efficient way to reduce protein level of OGT rather than that of CHX treatment. We also showed that not only proteasomal degradation regulated OGT stability but autophagic degradation also affected OGT stability in part. We concluded that mTOR signaling regulates protein O-GlcNAc modification through adjustment of OGT stability.     

Sp1 modulates ncOGT activity to alter target recognition and enhanced thermotolerance in E. coli

cDNAs encoding three isoforms of OGT (ncOGT, mOGT, and sOGT) were expressed in Escherichia coli in which the coexpression system of OGT with target substrates was established in vivo. No endogenous bacterial proteins were significantly O-GlcNAcylated by any type of OGT isoform while co-expressed p62 and Sp1 were strongly O-GlcNAcylated by ncOGT. These results suggest that most of bacterial proteins appear not to be recognized as right substrates by mammalian OGT whereas cytosolic environments may supply UDP-GlcNAc enough to proceed to O-GlcNAcylation in E. coli. Under these conditions, sOGT was auto-O-GlcNAcylated whereas ncOGT and mOGT were not. Importantly, we found that when Sp1 was coexpressed, ncOGT can O-GlcNAcylate not only Sp1 but also many bacterial proteins. Our findings suggest that Sp1 may modulate the capability of target recognition of ncOGT by which ncOGT can be led to newly recognize bacterial proteins as target substrates, finally generating the O-glyco-bacteria. Our results demonstrate that the O-glyco-bacteria showed enhanced thermal resistance to allow cell survival at a temperature as high as 52 °C.     

Store-operated Ca2+ Entry (SOCE) Induced by Protease-activated Receptor-1 Mediates STIM1 Protein Phosphorylation to Inhibit SOCE in Endothelial Cells through AMP-activated Protein Kinase and p38β Mitogen-activated Protein Kinase

The Ca²⁺ sensor STIM1 is crucial for activation of store-operated Ca²⁺ entry (SOCE) through transient receptor potential canonical and Orai channels. STIM1 phosphorylation serves as an “off switch” for SOCE. However, the signaling pathway for STIM1 phosphorylation is unknown. Here, we show that SOCE activates AMP-activated protein kinase (AMPK); its effector p38β mitogen-activated protein kinase (p38β MAPK) phosphorylates STIM1, thus inhibiting SOCE in human lung microvascular endothelial cells. Activation of AMPK using 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR) resulted in STIM1 phosphorylation on serine residues and prevented protease-activated receptor-1 (PAR-1)-induced Ca²⁺ entry. Furthermore, AICAR pretreatment blocked PAR-1-induced increase in the permeability of mouse lung microvessels. Activation of SOCE with thrombin caused phosphorylation of isoform α1 but not α2 of the AMPK catalytic subunit. Moreover, knockdown of AMPKα1 augmented SOCE induced by thrombin. Interestingly, SB203580, a selective inhibitor of p38 MAPK, blocked STIM1 phosphorylation and led to sustained STIM1-puncta formation and Ca²⁺ entry. Of the three p38 MAPK isoforms expressed in endothelial cells, p38β knockdown prevented PAR-1-mediated STIM1 phosphorylation and potentiated SOCE. In addition, inhibition of the SOCE downstream target CaM kinase kinase β (CaMKKβ) or knockdown of AMPKα1 suppressed PAR-1-mediated phosphorylation of p38β and hence STIM1. Thus, our findings demonstrate that SOCE activates CaMKKβ-AMPKα1-p38β MAPK signaling to phosphorylate STIM1, thereby suppressing endothelial SOCE and permeability responses.     

Designing dynamical output feedback controllers for store-operated Ca entry

Store-operated calcium entry (SOCE) has been proposed as the main process controlling Ca²⁺ entry in non-excitable cells. Although recent breakthroughs in experimental studies of SOCE have been made, its mathematical modeling has not been developed. In the present work, SOCE is viewed as a feedback control system subject to an extracellular agonist disturbance and an extracellular calcium input. We then design a dynamic output feedback controller to reject the disturbance and track Ca²⁺ resting levels in the cytosol and the endoplasmic reticulum (ER). The constructed feedback control system is validated by published experimental data and its global asymptotic stability is proved by using the LaSalle’s invariance principle. We then simulate the dynamic responses of STIM1 and Orai1, two major components in the operation of the store-operated channels, to the depletion of Ca²⁺ in the ER with thapsigargin, which show that: (1) Upon the depletion of Ca²⁺ in the ER, the concentrations of activated STIM1 and STIM1-Orai1 cluster are elevated gradually, indicating that STIM1 is accumulating in the ER-PM junctions and that the cytosolic portion of the active STIM1 is binding to Orai1 and driving the opening of CRAC channels for Ca²⁺ entry; (2) after the extracellular Ca²⁺ addition, the concentrations of both STIM1 and STIM1-Orai1 cluster decrease but still much higher than the original levels. We also simulate the system responses to the agonist disturbance, which show that, when a sequence of periodic agonist pulses is applied, the system returns to its equilibrium after each pulse. This indicates that the designed feedback controller can reject the disturbance and track the equilibrium.     

Plasma Membrane Localized GCaMP-MS4A12 by Orai1 Co-Expression Shows Thapsigargin- and Ca2+-Dependent Fluorescence Increases

Uniquely expressed in the colon, MS4A12 exhibits store-operated Ca²⁺ entry (SOCE) activity. However, compared to MS4A1 (CD20), a Ca²⁺ channel and ideal target for successful leukaemia immunotherapy, MS4A12 has rarely been studied. In this study, we investigated the involvement of MS4A12 in Ca²⁺ influx and expression changes in MS4A12 in human colonic malignancy. Fluorescence of GCaMP-fused MS4A12 (GCaMP-M12) was evaluated to analyse MS4A12 activity in Ca²⁺ influx. Plasma membrane expression of GCaMP-M12 was achieved by homo- or hetero-complex formation with no-tagged MS4A12 (nt-M12) or Orai1, respectively. GCaMP-M12 fluorescence in plasma membrane increased only after thapsigargin-induced depletion of endoplasmic reticulum Ca²⁺ stores, and this fluorescence was inhibited by typical SOCE inhibitors and siRNA for Orai1. Furthermore, GCaMP-MS4A12 and Orai1 co-transfection elicited greater plasma membrane fluorescence than GCaMP-M12 co-transfected with nt-M12. Interestingly, the fluorescence of GCaMP-M12 was decreased by STIM1 over-expression, while increased by siRNA for STIM1 in the presence of thapsigargin and extracellular Ca²⁺. Moreover, immunoprecipitation assay revealed that Orai1 co-expression decreased protein interactions between MS4A12 and STIM1. In human colon tissue, MS4A12 was expressed in the apical region of the colonic epithelium, although its expression was dramatically decreased in colon cancer tissues. In conclusion, we propose that MS4A12 contributes to SOCE through complex formation with Orai1, but does not cooperate with STIM1. Additionally, we discovered that MS4A12 is expressed in the apical membrane of the colonic epithelium and that its expression is decreased with cancer progression.