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

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