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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

Multi-lysine mutations affect the levels of cell-surface GlyT1.PAE cells expressing A) WT or 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. C) 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.
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pone.0138897.g005: Multi-lysine mutations affect the levels of cell-surface GlyT1.PAE cells expressing A) WT or 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. C) 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.

Mentions: To determine whether mutations at ubiquitination sites prevented endocytosis and degradation of GlyT1, we incubated cells expressing the WT GlyT1c isoform or the mutant NTK-CTK with vehicle DMSO or PMA for 30 min. We then labeled the total cell surface proteins by incubation of the cells with the impermeant reagent sulfo-N-hydroxysuccinimido biotin. Total biotinylated cell surface proteins were precipitated with Neutravidin beads and biotinylated proteins subjected to western blotting with GlyT1-specific antibodies. As shown in Fig 5A, PMA induced a consistent 50–70% reduction in WT transporter at the cell surface when compared to vehicle-treated cells. In the other hand, PMA treatment of cells expressing the NTK-CTK mutant showed no change in the amount of cell surface transporter, as compared to non-treated cells, suggesting that ubiquitination is critical for accelerated endocytosis (Fig 5B). This data is consistent with a poor localization of the mutant transporter NTKCTK into early endosomes in PMA-stimulated cells (Fig 4B).


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)

Multi-lysine mutations affect the levels of cell-surface GlyT1.PAE cells expressing A) WT or 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. C) 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.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0138897.g005: Multi-lysine mutations affect the levels of cell-surface GlyT1.PAE cells expressing A) WT or 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. C) 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.
Mentions: To determine whether mutations at ubiquitination sites prevented endocytosis and degradation of GlyT1, we incubated cells expressing the WT GlyT1c isoform or the mutant NTK-CTK with vehicle DMSO or PMA for 30 min. We then labeled the total cell surface proteins by incubation of the cells with the impermeant reagent sulfo-N-hydroxysuccinimido biotin. Total biotinylated cell surface proteins were precipitated with Neutravidin beads and biotinylated proteins subjected to western blotting with GlyT1-specific antibodies. As shown in Fig 5A, PMA induced a consistent 50–70% reduction in WT transporter at the cell surface when compared to vehicle-treated cells. In the other hand, PMA treatment of cells expressing the NTK-CTK mutant showed no change in the amount of cell surface transporter, as compared to non-treated cells, suggesting that ubiquitination is critical for accelerated endocytosis (Fig 5B). This data is consistent with a poor localization of the mutant transporter NTKCTK into early endosomes in PMA-stimulated cells (Fig 4B).

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