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Structure-Function Analysis of PPP1R3D, a Protein Phosphatase 1 Targeting Subunit, Reveals a Binding Motif for 14-3-3 Proteins which Regulates its Glycogenic Properties.

Rubio-Villena C, Sanz P, Garcia-Gimeno MA - PLoS ONE (2015)

Bottom Line: We have found that the PP1 binding domain of R6 comprises a conserved RVXF motif (R102VRF) located at the N-terminus of the protein.Our results indicate that although binding to PP1 and glycogenic substrates are independent processes, impairment of any of them results in lack of glycogenic activity of R6.These results define binding to 14-3-3 proteins as an additional pathway in the control of the glycogenic properties of R6.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Biomedicina de Valencia, CSIC, and Centro de Investigación en Red de Enfermedades Raras (CIBERER), Jaime Roig 11, Valencia, Spain.

ABSTRACT
Protein phosphatase 1 (PP1) is one of the major protein phosphatases in eukaryotic cells. It plays a key role in regulating glycogen synthesis, by dephosphorylating crucial enzymes involved in glycogen homeostasis such as glycogen synthase (GS) and glycogen phosphorylase (GP). To play this role, PP1 binds to specific glycogen targeting subunits that, on one hand recognize the substrates to be dephosphorylated and on the other hand recruit PP1 to glycogen particles. In this work we have analyzed the functionality of the different protein binding domains of one of these glycogen targeting subunits, namely PPP1R3D (R6) and studied how binding properties of different domains affect its glycogenic properties. We have found that the PP1 binding domain of R6 comprises a conserved RVXF motif (R102VRF) located at the N-terminus of the protein. We have also identified a region located at the C-terminus of R6 (W267DNND) that is involved in binding to the PP1 glycogenic substrates. Our results indicate that although binding to PP1 and glycogenic substrates are independent processes, impairment of any of them results in lack of glycogenic activity of R6. In addition, we have characterized a novel site of regulation in R6 that is involved in binding to 14-3-3 proteins (RARS74LP). We present evidence indicating that when binding of R6 to 14-3-3 proteins is prevented, R6 displays hyper-glycogenic activity although is rapidly degraded by the lysosomal pathway. These results define binding to 14-3-3 proteins as an additional pathway in the control of the glycogenic properties of R6.

No MeSH data available.


Binding of 14-3-3 proteins to R6 prevents its lysosomal degradation.A) R6-S74A mutant possesses a shorter half-life than wild type protein. Hek293 cells were transfected with pFLAG-R6 wt or pFLAG-R6-S74A plasmids. 24 hours after transfection, cells were treated with cycloheximide (300 μM) to block protein synthesis. At the indicated times, cell extracts (30 μg) were analyzed by Western blotting using anti-FLAG and anti-tubulin (as loading control). A representative western blot is shown on the left panel. On the right panel, the intensity of the bands related to the levels of tubulin is plotted and normalized respect to the values at time 0 (bars indicate the standard deviation of at least three independent experiments; **p < 0.01). B) R6-S74A protein is degraded by the lysosomal pathway. Hek293 cells were transfected with pFLAG-R6-S74A plasmid. Eighteen hours after transfection, cells were treated with ammonium chloride (20 mM)/ leupeptin (100 μM) or MG132 (5 μM) for six hours. Then, cells were lysed and extracts (30 μg) were analyzed by immunoblotting using anti-FLAG antibody and anti-tubulin as loading control. The intensity of the bands related to the levels of tubulin is plotted (bars indicate standard deviation of at least three independent experiments; *p < 0.05).
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pone.0131476.g006: Binding of 14-3-3 proteins to R6 prevents its lysosomal degradation.A) R6-S74A mutant possesses a shorter half-life than wild type protein. Hek293 cells were transfected with pFLAG-R6 wt or pFLAG-R6-S74A plasmids. 24 hours after transfection, cells were treated with cycloheximide (300 μM) to block protein synthesis. At the indicated times, cell extracts (30 μg) were analyzed by Western blotting using anti-FLAG and anti-tubulin (as loading control). A representative western blot is shown on the left panel. On the right panel, the intensity of the bands related to the levels of tubulin is plotted and normalized respect to the values at time 0 (bars indicate the standard deviation of at least three independent experiments; **p < 0.01). B) R6-S74A protein is degraded by the lysosomal pathway. Hek293 cells were transfected with pFLAG-R6-S74A plasmid. Eighteen hours after transfection, cells were treated with ammonium chloride (20 mM)/ leupeptin (100 μM) or MG132 (5 μM) for six hours. Then, cells were lysed and extracts (30 μg) were analyzed by immunoblotting using anti-FLAG antibody and anti-tubulin as loading control. The intensity of the bands related to the levels of tubulin is plotted (bars indicate standard deviation of at least three independent experiments; *p < 0.05).

Mentions: In the course of the subcellular localization experiments described above, we noticed that the YFP-R6-S74A protein was expressed at much lower levels than the wild type or the R6-S25A mutant (Fig 5). Similarly, lower levels of FLAG-R6-S74A were observed in Fig 4B (lane 5). In order to analyze if mutation at Ser74 was affecting R6 stability, we performed an assay to compare the half-life of this mutated form to the wild type protein. We expressed in Hek293 cells either the FLAG-R6 wild type or the FLAG-R6-S74A mutant and treated the cells with cycloheximide to block de novo protein synthesis. Then, protein levels were measured by western blotting at different times after the treatment. As observed in Fig 6A, the R6-S74A protein had a shorter half-life than the wild type protein. After 24h of treatment, the R6-S74A mutant was degraded almost completely in comparison to the wild type form, which was rather stable (Fig 6A). To elucidate which mechanism of degradation was taking place, we treated the cells with either MG132, to inhibit proteasome function, or with leupeptin and NH4Cl to inhibit lysosomal degradation [36]. We observed that treatment with MG132 did not affect the degradation of R6-S74A protein (Fig 6B). On the contrary, treatment with leupeptin and NH4Cl (to block the lysosome) prevented the degradation of the R6-S74A mutated form (Fig 6B). Therefore, disrupting the binding of 14-3-3 proteins to R6 accelerated its degradation by the lysosomal pathway.


Structure-Function Analysis of PPP1R3D, a Protein Phosphatase 1 Targeting Subunit, Reveals a Binding Motif for 14-3-3 Proteins which Regulates its Glycogenic Properties.

Rubio-Villena C, Sanz P, Garcia-Gimeno MA - PLoS ONE (2015)

Binding of 14-3-3 proteins to R6 prevents its lysosomal degradation.A) R6-S74A mutant possesses a shorter half-life than wild type protein. Hek293 cells were transfected with pFLAG-R6 wt or pFLAG-R6-S74A plasmids. 24 hours after transfection, cells were treated with cycloheximide (300 μM) to block protein synthesis. At the indicated times, cell extracts (30 μg) were analyzed by Western blotting using anti-FLAG and anti-tubulin (as loading control). A representative western blot is shown on the left panel. On the right panel, the intensity of the bands related to the levels of tubulin is plotted and normalized respect to the values at time 0 (bars indicate the standard deviation of at least three independent experiments; **p < 0.01). B) R6-S74A protein is degraded by the lysosomal pathway. Hek293 cells were transfected with pFLAG-R6-S74A plasmid. Eighteen hours after transfection, cells were treated with ammonium chloride (20 mM)/ leupeptin (100 μM) or MG132 (5 μM) for six hours. Then, cells were lysed and extracts (30 μg) were analyzed by immunoblotting using anti-FLAG antibody and anti-tubulin as loading control. The intensity of the bands related to the levels of tubulin is plotted (bars indicate standard deviation of at least three independent experiments; *p < 0.05).
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pone.0131476.g006: Binding of 14-3-3 proteins to R6 prevents its lysosomal degradation.A) R6-S74A mutant possesses a shorter half-life than wild type protein. Hek293 cells were transfected with pFLAG-R6 wt or pFLAG-R6-S74A plasmids. 24 hours after transfection, cells were treated with cycloheximide (300 μM) to block protein synthesis. At the indicated times, cell extracts (30 μg) were analyzed by Western blotting using anti-FLAG and anti-tubulin (as loading control). A representative western blot is shown on the left panel. On the right panel, the intensity of the bands related to the levels of tubulin is plotted and normalized respect to the values at time 0 (bars indicate the standard deviation of at least three independent experiments; **p < 0.01). B) R6-S74A protein is degraded by the lysosomal pathway. Hek293 cells were transfected with pFLAG-R6-S74A plasmid. Eighteen hours after transfection, cells were treated with ammonium chloride (20 mM)/ leupeptin (100 μM) or MG132 (5 μM) for six hours. Then, cells were lysed and extracts (30 μg) were analyzed by immunoblotting using anti-FLAG antibody and anti-tubulin as loading control. The intensity of the bands related to the levels of tubulin is plotted (bars indicate standard deviation of at least three independent experiments; *p < 0.05).
Mentions: In the course of the subcellular localization experiments described above, we noticed that the YFP-R6-S74A protein was expressed at much lower levels than the wild type or the R6-S25A mutant (Fig 5). Similarly, lower levels of FLAG-R6-S74A were observed in Fig 4B (lane 5). In order to analyze if mutation at Ser74 was affecting R6 stability, we performed an assay to compare the half-life of this mutated form to the wild type protein. We expressed in Hek293 cells either the FLAG-R6 wild type or the FLAG-R6-S74A mutant and treated the cells with cycloheximide to block de novo protein synthesis. Then, protein levels were measured by western blotting at different times after the treatment. As observed in Fig 6A, the R6-S74A protein had a shorter half-life than the wild type protein. After 24h of treatment, the R6-S74A mutant was degraded almost completely in comparison to the wild type form, which was rather stable (Fig 6A). To elucidate which mechanism of degradation was taking place, we treated the cells with either MG132, to inhibit proteasome function, or with leupeptin and NH4Cl to inhibit lysosomal degradation [36]. We observed that treatment with MG132 did not affect the degradation of R6-S74A protein (Fig 6B). On the contrary, treatment with leupeptin and NH4Cl (to block the lysosome) prevented the degradation of the R6-S74A mutated form (Fig 6B). Therefore, disrupting the binding of 14-3-3 proteins to R6 accelerated its degradation by the lysosomal pathway.

Bottom Line: We have found that the PP1 binding domain of R6 comprises a conserved RVXF motif (R102VRF) located at the N-terminus of the protein.Our results indicate that although binding to PP1 and glycogenic substrates are independent processes, impairment of any of them results in lack of glycogenic activity of R6.These results define binding to 14-3-3 proteins as an additional pathway in the control of the glycogenic properties of R6.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Biomedicina de Valencia, CSIC, and Centro de Investigación en Red de Enfermedades Raras (CIBERER), Jaime Roig 11, Valencia, Spain.

ABSTRACT
Protein phosphatase 1 (PP1) is one of the major protein phosphatases in eukaryotic cells. It plays a key role in regulating glycogen synthesis, by dephosphorylating crucial enzymes involved in glycogen homeostasis such as glycogen synthase (GS) and glycogen phosphorylase (GP). To play this role, PP1 binds to specific glycogen targeting subunits that, on one hand recognize the substrates to be dephosphorylated and on the other hand recruit PP1 to glycogen particles. In this work we have analyzed the functionality of the different protein binding domains of one of these glycogen targeting subunits, namely PPP1R3D (R6) and studied how binding properties of different domains affect its glycogenic properties. We have found that the PP1 binding domain of R6 comprises a conserved RVXF motif (R102VRF) located at the N-terminus of the protein. We have also identified a region located at the C-terminus of R6 (W267DNND) that is involved in binding to the PP1 glycogenic substrates. Our results indicate that although binding to PP1 and glycogenic substrates are independent processes, impairment of any of them results in lack of glycogenic activity of R6. In addition, we have characterized a novel site of regulation in R6 that is involved in binding to 14-3-3 proteins (RARS74LP). We present evidence indicating that when binding of R6 to 14-3-3 proteins is prevented, R6 displays hyper-glycogenic activity although is rapidly degraded by the lysosomal pathway. These results define binding to 14-3-3 proteins as an additional pathway in the control of the glycogenic properties of R6.

No MeSH data available.