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

Barrera SP, Castrejon-Tellez V, Trinidad M, Robles-Escajeda E, Vargas-Medrano J, Varela-Ramirez A, Miranda M - PLoS ONE (2015)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences and Border Biomedical Research Center, University of Texas at El Paso, El Paso, TX, 79968, United States of America.

ABSTRACT
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 N-and C-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.

No MeSH data available.


Related in: MedlinePlus

Localization of GlyT1 in mouse amacrine neurons.A) 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) 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 Fig 1D. Scale bars, 10 μm. C) 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 Fig 1D and analyzed by Student’s t-test.
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pone.0138897.g002: Localization of GlyT1 in mouse amacrine neurons.A) 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) 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 Fig 1D. Scale bars, 10 μm. C) 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 Fig 1D and analyzed by Student’s t-test.

Mentions: Several studies have described the regions in the mouse and rat central nervous systems that are enriched in glycinergic neurons such as the spinal cord, cerebellum and brain stem but the projections and connections are still not well defined. [6,27,28]. By contrast, the precise localization of glycinergic cell bodies, dendrites and connection are better characterized in the mammalian retina [29,30]. Therefore, to investigate whether endocytosis is a molecular event that takes place in glycinergic neurons, we used sections of mouse retina and primary cultures of mouse glycinergic amacrine neurons to study GlyT1 trafficking to early endosomes. To this extent, we obtained 15 μm retinal vertical sections and stained them with DAPI and antibodies specific for GlyT1 or EEA1. As shown in Fig 2A, three layers of cell bodies were clearly demarcated, corresponding to 1) the cell bodies of cones and bipolar cells (ONL, outer nuclear layer); 2) the middle layer accounting for cell bodies of horizontal and amacrine cells (INL, inner nuclear layer), and 3) the cell bodies of ganglion cells (GCL). Strong GlyT1 immunoreactivity was observed most prominently around cell bodies in the inner nuclear layer and descending dendrites to the inner plexiform layer that contact the ganglion cells. This arrangement clearly demonstrates the integrity of the sections and specific GlyT1 staining within the glycinergic amacrine neurons. As observed in Fig 2A, bottom panel, EEA1 staining appeared as puncta spread throughout all the retinal layers, which corresponded to early endosomes. Interestingly, a few discrete puncta showed co-localization between GlyT1 and EEA1 in 0.65 μm confocal optical sections and demonstrated by quantitative overlap coefficient analysis (Fig 2C), suggesting that GlyT1 uses early endosomes for endocytosis.


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

Barrera SP, Castrejon-Tellez V, Trinidad M, Robles-Escajeda E, Vargas-Medrano J, Varela-Ramirez A, Miranda M - PLoS ONE (2015)

Localization of GlyT1 in mouse amacrine neurons.A) 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) 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 Fig 1D. Scale bars, 10 μm. C) 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 Fig 1D and analyzed by Student’s t-test.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4587969&req=5

pone.0138897.g002: Localization of GlyT1 in mouse amacrine neurons.A) 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) 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 Fig 1D. Scale bars, 10 μm. C) 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 Fig 1D and analyzed by Student’s t-test.
Mentions: Several studies have described the regions in the mouse and rat central nervous systems that are enriched in glycinergic neurons such as the spinal cord, cerebellum and brain stem but the projections and connections are still not well defined. [6,27,28]. By contrast, the precise localization of glycinergic cell bodies, dendrites and connection are better characterized in the mammalian retina [29,30]. Therefore, to investigate whether endocytosis is a molecular event that takes place in glycinergic neurons, we used sections of mouse retina and primary cultures of mouse glycinergic amacrine neurons to study GlyT1 trafficking to early endosomes. To this extent, we obtained 15 μm retinal vertical sections and stained them with DAPI and antibodies specific for GlyT1 or EEA1. As shown in Fig 2A, three layers of cell bodies were clearly demarcated, corresponding to 1) the cell bodies of cones and bipolar cells (ONL, outer nuclear layer); 2) the middle layer accounting for cell bodies of horizontal and amacrine cells (INL, inner nuclear layer), and 3) the cell bodies of ganglion cells (GCL). Strong GlyT1 immunoreactivity was observed most prominently around cell bodies in the inner nuclear layer and descending dendrites to the inner plexiform layer that contact the ganglion cells. This arrangement clearly demonstrates the integrity of the sections and specific GlyT1 staining within the glycinergic amacrine neurons. As observed in Fig 2A, bottom panel, EEA1 staining appeared as puncta spread throughout all the retinal layers, which corresponded to early endosomes. Interestingly, a few discrete puncta showed co-localization between GlyT1 and EEA1 in 0.65 μm confocal optical sections and demonstrated by quantitative overlap coefficient analysis (Fig 2C), suggesting that GlyT1 uses early endosomes for endocytosis.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences and Border Biomedical Research Center, University of Texas at El Paso, El Paso, TX, 79968, United States of America.

ABSTRACT
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 N-and C-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.

No MeSH data available.


Related in: MedlinePlus