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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.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.

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.


Related in: MedlinePlus

Subcellular localization of R6 S25A and R6 S74A mutated forms.N2a cells were transfected with pYFP empty plasmid, pYFP-R6 wild type, pYFP-R6 S25A or pYFP-R6 S74A plasmids. The subcellular localization of R6 forms and glycogen granules was carried out as described in Materials and Methods. Images were obtained by using confocal microscopy (bars indicate 10 μm). Images corresponding to visible, YFP (in yellow) and glycogen (in red) fluorescences are shown.
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pone.0131476.g005: Subcellular localization of R6 S25A and R6 S74A mutated forms.N2a cells were transfected with pYFP empty plasmid, pYFP-R6 wild type, pYFP-R6 S25A or pYFP-R6 S74A plasmids. The subcellular localization of R6 forms and glycogen granules was carried out as described in Materials and Methods. Images were obtained by using confocal microscopy (bars indicate 10 μm). Images corresponding to visible, YFP (in yellow) and glycogen (in red) fluorescences are shown.

Mentions: We further investigated whether the binding of R6 to 14-3-3 proteins could affect the glycogenic properties of R6. We found that R6-S25A mutant was as glycogenic as wild type (Fig 4A). Surprisingly, the expression of R6-S74A mutant, which is not able to bind to 14-3-3ε proteins (see above), produced around 9 fold increase on glycogen accumulation (Fig 4A). In order to explain the hyper-glycogenic properties of the R6-S74A mutant and since it has been reported that binding to 14-3-3 proteins can affect the subcellular localization of a particular protein [19], we investigated whether the lack of 14-3-3 protein binding present in R6-S74A could have modified the subcellular localization of this protein. So, we expressed in N2a cells the YFP-R6-S25A and YFP-R6-S74A mutants and assessed the subcellular distribution of these proteins. As shown in Fig 5, R6-WT, R6-S25A and R6-S74A located in similar granular structures in the cytoplasm of N2a which contained glycogen, confirming previous results [17]. So, we did not observe any change in the localization of the different mutants in comparison to the wild type protein (Fig 5).


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)

Subcellular localization of R6 S25A and R6 S74A mutated forms.N2a cells were transfected with pYFP empty plasmid, pYFP-R6 wild type, pYFP-R6 S25A or pYFP-R6 S74A plasmids. The subcellular localization of R6 forms and glycogen granules was carried out as described in Materials and Methods. Images were obtained by using confocal microscopy (bars indicate 10 μm). Images corresponding to visible, YFP (in yellow) and glycogen (in red) fluorescences are shown.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4482762&req=5

pone.0131476.g005: Subcellular localization of R6 S25A and R6 S74A mutated forms.N2a cells were transfected with pYFP empty plasmid, pYFP-R6 wild type, pYFP-R6 S25A or pYFP-R6 S74A plasmids. The subcellular localization of R6 forms and glycogen granules was carried out as described in Materials and Methods. Images were obtained by using confocal microscopy (bars indicate 10 μm). Images corresponding to visible, YFP (in yellow) and glycogen (in red) fluorescences are shown.
Mentions: We further investigated whether the binding of R6 to 14-3-3 proteins could affect the glycogenic properties of R6. We found that R6-S25A mutant was as glycogenic as wild type (Fig 4A). Surprisingly, the expression of R6-S74A mutant, which is not able to bind to 14-3-3ε proteins (see above), produced around 9 fold increase on glycogen accumulation (Fig 4A). In order to explain the hyper-glycogenic properties of the R6-S74A mutant and since it has been reported that binding to 14-3-3 proteins can affect the subcellular localization of a particular protein [19], we investigated whether the lack of 14-3-3 protein binding present in R6-S74A could have modified the subcellular localization of this protein. So, we expressed in N2a cells the YFP-R6-S25A and YFP-R6-S74A mutants and assessed the subcellular distribution of these proteins. As shown in Fig 5, R6-WT, R6-S25A and R6-S74A located in similar granular structures in the cytoplasm of N2a which contained glycogen, confirming previous results [17]. So, we did not observe any change in the localization of the different mutants in comparison to the wild type protein (Fig 5).

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.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.

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.


Related in: MedlinePlus