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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

GlyT1 phosphorylation and glycine uptake.A) 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) 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, C) 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, n = 3, *p = 0.002, **p <0.001. D) 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 p<0.05 was obtained when each experimental sample was compared with untreated control cells via one-way analysis of variance (ANOVA) and Student’s t-test.
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pone.0138897.g007: GlyT1 phosphorylation and glycine uptake.A) 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) 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, C) 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, n = 3, *p = 0.002, **p <0.001. D) 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 p<0.05 was obtained when each experimental sample was compared with untreated control cells via one-way analysis of variance (ANOVA) and Student’s t-test.

Mentions: We previously demonstrated that GlyT1 is phosphorylated in response to PKCβ activation along with a reduction in glycine uptake capacity. This reduction in uptake is likely due to endocytosis, as described below. To analyze the relationship between phosphorylation, ubiquitination and further reduction in glycine uptake, we next studied the effects of lysine substitutions on GlyT1c phosphorylation and uptake to define the relationship between phosphorylation, ubiquitination, and glycine uptake. In control experiments, we subjected PAE cells transfected with the vector or expressing the WT GlyT1c to 32P-ATP metabolic labeling, followed by DMSO or PMA stimulation. After incubation, the cells were lysed, and the transporter purified and subjected to SDS-PAGE and autoradiography. Consistent with our previous published findings, incubation of PAE cells transfected with pCDNA3.1 vector with DMSO or PMA for 60 min failed to identify in any radiolabeled band or immunoreactivitiy to GlyT1 antibodies (Fig 7A, lanes 1 and 2). In the other hand, a faint smear could be detected when GlyT1 was purified from DMSO-treated cells. By contrast, in PAE cells expressing WT GlyT1c stimulated with PMA for varying periods of time (15–120 min), we observed a dramatic increase in GlyT1 phosphorylation that decayed after 2 h (lanes 5–8). When cells were treated with BIM to inhibit PKC and stimulated with PMA, this increase in phosphorylation of WT GlyT1was lost (Fig 7A, lane 4), demonstrating again its PKC dependence.


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)

GlyT1 phosphorylation and glycine uptake.A) 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) 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, C) 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, n = 3, *p = 0.002, **p <0.001. D) 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 p<0.05 was obtained when each experimental sample was compared with untreated control cells via one-way analysis of variance (ANOVA) and Student’s t-test.
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pone.0138897.g007: GlyT1 phosphorylation and glycine uptake.A) 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) 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, C) 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, n = 3, *p = 0.002, **p <0.001. D) 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 p<0.05 was obtained when each experimental sample was compared with untreated control cells via one-way analysis of variance (ANOVA) and Student’s t-test.
Mentions: We previously demonstrated that GlyT1 is phosphorylated in response to PKCβ activation along with a reduction in glycine uptake capacity. This reduction in uptake is likely due to endocytosis, as described below. To analyze the relationship between phosphorylation, ubiquitination and further reduction in glycine uptake, we next studied the effects of lysine substitutions on GlyT1c phosphorylation and uptake to define the relationship between phosphorylation, ubiquitination, and glycine uptake. In control experiments, we subjected PAE cells transfected with the vector or expressing the WT GlyT1c to 32P-ATP metabolic labeling, followed by DMSO or PMA stimulation. After incubation, the cells were lysed, and the transporter purified and subjected to SDS-PAGE and autoradiography. Consistent with our previous published findings, incubation of PAE cells transfected with pCDNA3.1 vector with DMSO or PMA for 60 min failed to identify in any radiolabeled band or immunoreactivitiy to GlyT1 antibodies (Fig 7A, lanes 1 and 2). In the other hand, a faint smear could be detected when GlyT1 was purified from DMSO-treated cells. By contrast, in PAE cells expressing WT GlyT1c stimulated with PMA for varying periods of time (15–120 min), we observed a dramatic increase in GlyT1 phosphorylation that decayed after 2 h (lanes 5–8). When cells were treated with BIM to inhibit PKC and stimulated with PMA, this increase in phosphorylation of WT GlyT1was lost (Fig 7A, lane 4), demonstrating again its PKC dependence.

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