PKC-Dependent GlyT1 Ubiquitination Occurs Independent of Phosphorylation: Inespecificity in Lysine Selection for Ubiquitination

<div><p>Neurotransmitter transporter ubiquitination is emerging as the main mechanism for endocytosis and sorting of cargo into lysosomes. In this study, we demonstrate PKC-dependent ubiquitination of three different isoforms of the glycine transporter 1 (GlyT1). Incubation of cells expressing transporter with the PKC activator phorbol ester induced a dramatic, time-dependent increase in GlyT1 ubiquitination, followed by accumulation of GlyT1 in EEA1 positive early endosomes. This occurred via a mechanism that was abolished by inhibition of PKC. GlyT1 endocytosis was confirmed in both retinal sections and primary cultures of mouse amacrine neurons. Replacement of only all lysines in the <i>N</i>-and <i>C</i>-termini to arginines prevented ubiquitination and endocytosis, displaying redundancy in the mechanism of ubiquitination. Interestingly, a 40–50% reduction in glycine uptake was detected in phorbol-ester stimulated cells expressing the WT-GlyT1, whereas no significant change was for the mutant protein, demonstrating that endocytosis participates in the reduction of uptake. Consistent with previous findings for the dopamine transporter DAT, ubiquitination of GlyT1 tails functions as sorting signal to deliver transporter into the lysosome and removal of ubiquitination sites dramatically attenuated the rate of GlyT1 degradation. Finally, we showed for the first time that PKC-dependent GlyT1 phosphorylation was not affected by removal of ubiquitination sites, suggesting separate PKC-dependent signaling events for these posttranslational modifications.</p></div

GlyT1 phosphorylation and glycine uptake.

<p><b>A)</b> PAE cells stably expressing FH-GlyT1 were labeled with 50 μCi ³²P-orthophosphate/ml followed by incubation with DMSO or 1 μM PMA for 0 to 120 min. Labeled GlyT1 was purified by tandem affinity chromatography and analyzed by autoradiography and Western blotting with GlyT1 antibodies. <b>B)</b> PAE cells expressing WT FH-GlyT1c, or the mutants NTK-1c, CTK-1c, and NTK-CTK-1c were labeled with 50 μCi ³²P-orthophosphate/ml followed by incubation with DMSO or 1 μM PMA for 60 min and treated as described in A. The autoradiography and GlyT1 blots were subjected to densitometry analysis and the resulting values are expressed as mean ± SEM, n = 3, <b>C)</b> For uptake experiments, cells were incubated with vehicle (DMSO) or 1μM PMA for 30 min followed by a 10 min incubation with 400 μM of [³H]-Gly at 37°C. Values are represented as % of control DMSO for each cell line, calculated from the following average specific activities in nmol/min/mg of protein: WT-1c, 41.3+/-3; NTK-1c,51.2+/-6; CTK-1c 39.4+/-3: NTK-CTK-1c, 56.8+/- 4;. Error bars represent the mean ± SE, <i>n</i> = 3, *<i>p</i> = <i>0</i>.<i>002</i>, **<i>p</i> <0.001. <b>D)</b> PAE cells expressing WT-DAT, and the mutant DAT were labeled with 50 μCi ³²P-orthophosphate/ml followed by incubation with DMSO or 1 μM PMA for 60 min. Total DAT was purified by tandem affinity chromatography and analyzed by autoradiography and Western blotting with DAT antibodies. Values are expressed as mean + SEM, n = 3. A value of <i>p</i><0.05 was obtained when each experimental sample was compared with untreated control cells <i>via</i> one-way analysis of variance (ANOVA) and Student’s <i>t</i>-test.</p

Ubiquitination and endocytosis of multi-lysine GlyT1c mutants.

<p><b>A)</b> PAE cells expressing WT FH-GlyT1c, NTK-1c, CTK-1c, and NTK-CTK-1c were incubated with DMSO or PMA (1μM) for 30 min. After incubation, the proteins were solubilized and GlyT1 purified by tandem affinity chromatography using Ni-NTA-agarose and FLAG M2 gel. Purified transporter was subjected to SDS-PAGE and western blot with ubiquitin and GlyT1 antibodies. Densitometry analysis was performed as described for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138897#pone.0138897.g003" target="_blank">Fig 3</a> and expressed as a mean ± SEM, n = 4. <b>B)</b> PAE cells expressing WT FH-GlyT1c, NTK-1c, CTK-1c, and NTK-CTK-1c were incubated with DMSO or PMA (1 μM) for 30 min. at 37°C, fixed and immunostained with anti-GlyT1 and anti-EAA1 antibodies followed by incubation with a CY-3 and Alexa 488 labeled secondary antibodies, respectively. A z-stack of optical sections was acquired through YFP (green) and CY3 (red) filter channels. Single optical sections through the middle of the cells are shown. ‘Yellow’ in the merged images signifies co-localization of CY3 (GlyT1) and YFP (EEA1). Images were selected to represent the cell population. <i>Scale bars</i>, 10 μm. C) Manders’ overlap coefficient of merged images captured from doubly-labeled PAE cells with anti-GlyT1 (red) and anti-EEA1 (green) antibodies; a value of 1 represents 100% of both fluorescence signals co-localized in all the pixels involved in the regions of interest (ROIs). Values are presented for 15 randomly selected endosomes in different cells from wild-type and mutants. Statistical analysis was performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138897#pone.0138897.g001" target="_blank">Fig 1</a>.</p

Schematic representation of the predicted topology of GlyT1 isoforms and PKC- induced endocytosis of GlyT1.

<p><b>A)</b> The twelve membrane-spanning segments are depicted by cylinders, and intracellular <i>N</i> and <i>C</i> termini, loops and extracellular glycosylation sites by solid lines. The position of lysine residues in the three different <i>N</i>-terminal splice variants are presented by beads (GlyT1a, 1b and 1c). Conserved lysines are highlighted by gray beads. <b>B)</b> PAE cells stably expressing FH-GlyT1b and <b>C)</b> PAE cells stably expressing FH-GlyT1c were incubated with DMSO or PMA for 30–60 min at 37°C, fixed and immunostained with anti-GlyT1 and anti-EAA1 antibodies followed by incubation with a CY-3 and Alexa 488 labeled secondary antibodies. Images were selected to represent the cell population and acquired through YFP (green) and CY3 (red) filter channels. Single optical sections through the middle of the cells are shown. ‘Yellow’ in the merged images signifies co-localization of CY3 (GlyT1) and YFP (EEA1). D) Co-localization was quantified in pixel by pixel bases from images obtained by confocal microscopy using the Mander’s overlap coefficient of merged images. A value of 1 represents 100% co-localization of both fluorescence signals in 15 randomly selected endosomes, whereas a zero value denotes complete absence of co-localization. <i>p</i> values were determine by student’s <i>t</i>-test. <i>Scale bars</i>, 10 μm.</p

PKC-dependent GlyT1 degradation is impaired by lysine mutations.

<p>PAE cells expressing WT and mutant forms of GlyT1 were incubated with 50 μg/ml of cycloheximide (CHX) for 2 h followed by treatment with 1μM PMA for 0–6 h. In all conditions, cells were incubated in the presence of CHX for a total of 8 h. After incubations, the cells were lysed and total lysates subjected to SDS-PAGE and western blot with GlyT1 and actin antibodies. <b>A)</b> WT-FH-GlyT1c, and mutants <b>B)</b> NTK-1c, <b>C)</b> CTK-1c, and <b>D)</b> NTK-CTK-1c. Densitometry was as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138897#pone.0138897.g003" target="_blank">Fig 3</a> and values expressed as a mean of three to five independent experiments ± SEM, n = 3–5. A value of <i>p</i><0.05 (*) was obtained from each experimental sample, as compared with untreated control cells, <i>via</i> one-way analysis of variance (ANOVA) and Student’s <i>t</i>-test.</p

Time course of PKC-induced ubiquitination of GlyT1 isoforms.

<p><b>A)</b> PAE cells stably expressing FH-GlyT1a were incubated with PMA (1μM) for 0 to 120 min. After incubation, the proteins were solubilized and GlyT1 purified by tandem affinity chromatography using Ni-NTA-agarose and FLAG M2 gel. Purified GlyT1 transporter was subjected to SDS-PAGE and western blotting using ubiquitin and GlyT1 antibodies. <b>B)</b> FH-GlyT1b isoform. <b>C)</b> FH-GlyT1c isoform. Blots were subjected to densitometry analysis using image J software and the relative amount of ubiquitinated GlyT1 was normalized to the total GlyT1 transporter. The Y axes represent the relative amount of ubiquitinated GlyT1. Data are expressed as the mean ± SEM, n = 4. ** A value of <i>p</i><0.05 (*) was obtained when each experimental sample was compared with untreated control cells <i>via</i> one-way analysis of variance (ANOVA) and Student’s <i>t</i>-test. <i>GlyT1-Ub</i>, ubiquitinated glycine transporter; <i>FH-GlyT1</i>, Flag, His-tagged glycosylated glycine transporter; <i>ng-GlyT1</i>, non-glycosylated glycine transporter.</p

Localization of GlyT1 in mouse amacrine neurons.

<p><b>A</b>) Vertical sections from adult C57BL/6J mouse retinas were stained for GlyT1, DAPI and EEA1, and analyzed by confocal microscopy. DAPI staining depicts the nuclei in cell bodies of the retina layers. Outer Nuclear Layer, ONL; Outer Plexiform Layer, OPL; Inner Nuclear Layer, INL; Inner Plexiform Layer, INL and Ganglion cell layer, GCL. <b>B)</b> Retinas from neonatal mouse were isolated, the tissue digested with papain and the cells plated on poly-L lysine and laminin- coated glass coverslips. Primary cultures were incubated with DMSO or 1 μM PMA for 1 h followed by detection of glycinergic amacrine neurons by immunostaining with GlyT1 and co-localization with EEA1. Single 0.65 μm optical sections were acquired by confocal microscopy and analyzed with ZEN 2009 software, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138897#pone.0138897.g001" target="_blank">Fig 1D</a>. <i>Scale bars</i>, 10 μm. <b>C)</b> Co-localization was measured for 10 EEA1- and 10 EEA1/GlyT1-positive endosomes from retinal sections depicted in panel A. Data is represented as described for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138897#pone.0138897.g001" target="_blank">Fig 1D</a> and analyzed by Student’s <i>t</i>-test.</p

Multi-lysine mutations affect the levels of cell-surface GlyT1.

<p>PAE cells expressing <b>A)</b> WT or <b>B)</b> NTKCTK-GlyT1 were incubated with DMSO or PMA (1 μM) for 30 min at 37°C. The cells were subjected to cell surface biotinylation, and biotinylated proteins were pulled down with Neutravidin (NeuAv) beads. Non-biotinylated proteins were purified from NeuAv supernatants using Ni-NTA agarose. NeuAv and Ni-NTA precipitates were separated on SDS-PAGE, transferred to nitrocellulose and the blots were probed with GlyT1 antibodies. <b>C)</b> Quantification of the amount of biotinylated and non-biotinylated GlyT1. The densitometry analysis was performed using ImageJ and the values are expressed as the mean ± SEM, n = 2.</p

Fenofibrate Protects Cardiomyocytes from Hypoxia/Reperfusion- and High Glucose-Induced Detrimental Effects

Lesions caused by high glucose (HG), hypoxia/reperfusion (H/R), and the coexistence of both conditions in cardiomyocytes are linked to an overproduction of reactive oxygen species (ROS), causing irreversible damage to macromolecules in the cardiomyocyte as well as its ultrastructure. Fenofibrate, a peroxisome proliferator-activated receptor alpha (PPARα) agonist, promotes beneficial activities counteracting cardiac injury. Therefore, the objective of this work was to determine the potential protective effect of fenofibrate in cardiomyocytes exposed to HG, H/R, and HG+H/R. Cardiomyocyte cultures were divided into four main groups: (1) control (CT), (2) HG (25 mM), (3) H/R, and (4) HG+H/R. Our results indicate that cell viability decreases in cardiomyocytes undergoing HG, H/R, and both conditions, while fenofibrate improves cell viability in every case. Fenofibrate also decreases ROS production as well as nicotinamide adenine dinucleotide phosphate oxidase (NADPH) subunit expression. Regarding the antioxidant defense, superoxide dismutase (SOD Cu2+/Zn2+ and SOD Mn2+), catalase, and the antioxidant capacity were decreased in HG, H/R, and HG+H/R-exposed cardiomyocytes, while fenofibrate increased those parameters. The expression of nuclear factor erythroid 2-related factor 2 (Nrf2) increased significantly in treated cells, while pathologies increased the expression of its inhibitor Keap1. Oxidative stress-induced mitochondrial damage was lower in fenofibrate-exposed cardiomyocytes. Endothelial nitric oxide synthase was also favored in cardiomyocytes treated with fenofibrate. Our results suggest that fenofibrate preserves the antioxidant status and the ultrastructure in cardiomyocytes undergoing HG, H/R, and HG+H/R preventing damage to essential macromolecules involved in the proper functioning of the cardiomyocyte