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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.


Related in: MedlinePlus

Analysis of the interacting properties of different domains of R6 by yeast two-hybrid analyses.Upper panels: yeast THY-AP4 strain was transformed with plasmids pBTM-R6 wt (LexA-R6), pBTM-R6 RARA and pBTM-R6 RAHA (A), pBTM-R6 WDNAD, and pBTM-WANNA (B) or pBTM-R6 S25A and pBTM-R6 S74A (C) and with pACT2 (GAD), pACT2-PP1α (GAD-PP1α), pACT2-laforin (GAD-laforin) or pACT2-14-3-3ε (GAD-14-3-3ε). Protein interaction was estimated by measuring the β-galactosidase activity. Values correspond to means from at least 6 different transformants (bars indicate standard deviation). Lower panels: protein expression in yeast transformants was analyzed by Western blotting using anti-HA antibodies (for the GAD-fusions) and anti-LexA (for the LexA-fusions) in several transformants from each condition. A representative western blot of some of these transformants is shown.
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pone.0131476.g002: Analysis of the interacting properties of different domains of R6 by yeast two-hybrid analyses.Upper panels: yeast THY-AP4 strain was transformed with plasmids pBTM-R6 wt (LexA-R6), pBTM-R6 RARA and pBTM-R6 RAHA (A), pBTM-R6 WDNAD, and pBTM-WANNA (B) or pBTM-R6 S25A and pBTM-R6 S74A (C) and with pACT2 (GAD), pACT2-PP1α (GAD-PP1α), pACT2-laforin (GAD-laforin) or pACT2-14-3-3ε (GAD-14-3-3ε). Protein interaction was estimated by measuring the β-galactosidase activity. Values correspond to means from at least 6 different transformants (bars indicate standard deviation). Lower panels: protein expression in yeast transformants was analyzed by Western blotting using anti-HA antibodies (for the GAD-fusions) and anti-LexA (for the LexA-fusions) in several transformants from each condition. A representative western blot of some of these transformants is shown.

Mentions: To assess the functionality of the different domains of R6 that we have described above, we generated different mutants in the corresponding protein motifs and checked whether the interaction with different partners was affected. We analyzed first the putative RVXF motifs involved in the interaction with PP1c. The hydrophobic residues valine and phenylalanine within the RVXF motifs were substituted for alanine, resulting in R102ARA and R252AHA mutants. By yeast two-hybrid analyses we checked the interaction of these mutated forms with the PP1 catalytic subunit (PP1c). In addition, we checked their interaction with laforin, a glucan phosphatase involved in Lafora disease, and with 14-3-3ε proteins, as they are potential interaction partners of R6 [18]. Unfortunately the interaction of R6 with glycogenic substrates, such as glycogen synthase (GS), was undetectable by yeast two-hybrid, what precluded the analysis of the effect of the generated mutations on the interaction with this substrate using this technique. As shown in Fig 2A, mutations in the R102VRF motif (R6-RARA mutant) disrupted the binding to PP1c, although the binding to other interacting partners (laforin and 14-3-3ε) remained as wild type. However, mutations in the R252VHF motif (R6-RAHA mutant) impaired the binding to PP1c and also to the other interaction partners, despite the construct was successfully produced, suggesting that the mutations had affected the overall structure of the protein when expressed in yeast (Fig 2A). In order to confirm these results, we performed an immunoprecipitation analysis in mammalian Hek293 cells expressing YFP-tagged versions of either R6-RARA or R6-RAHA mutants and compared the results with the wild type form of R6. As shown in Fig 3A, using the GFP-Trap technique, we confirmed that YFP-R6-RARA had completely lost the interaction to endogenous PP1c. This mammalian system allowed us to study the interaction of the different mutants with endogenous glycogenic substrates. In this sense, we observed that the YFP-R6-RARA mutant was still able to interact with endogenous GS and GP (although to at a lesser extend in the latter case), thus indicating that binding to these PP1 substrates was independent to the binding to PP1c. Binding of YFP-R6-RARA to endogenous 14-3-3ε protein was also not affected (Fig 3A). We were not able to check the binding to endogenous laforin as the levels of this protein were very low in these cells (not shown). On the contrary, we found that the YFP-R6-RAHA mutant was able to interact with the endogenous PP1c catalytic subunit (Fig 3A), therefore discarding the R252VHF motif as an area of contact to PP1c. The discrepancy between these results and the data obtained by yeast two-hybrid could be due to the different expression systems, yeast vs mammalian cells, being the latter more complete as it has endogenous levels of all glycogenic enzymes. Although the R6-RAHA mutant interacted with endogenous 14-3-3ε proteins, it had an impaired interaction with the endogenous PP1 substrates GS and GP (Fig 3A). Probably, the introduced mutations could have altered the conformation of the protein and affected the functionality of the W267DNND substrate binding motif present in the near vicinity (Fig 1B) (see below).


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)

Analysis of the interacting properties of different domains of R6 by yeast two-hybrid analyses.Upper panels: yeast THY-AP4 strain was transformed with plasmids pBTM-R6 wt (LexA-R6), pBTM-R6 RARA and pBTM-R6 RAHA (A), pBTM-R6 WDNAD, and pBTM-WANNA (B) or pBTM-R6 S25A and pBTM-R6 S74A (C) and with pACT2 (GAD), pACT2-PP1α (GAD-PP1α), pACT2-laforin (GAD-laforin) or pACT2-14-3-3ε (GAD-14-3-3ε). Protein interaction was estimated by measuring the β-galactosidase activity. Values correspond to means from at least 6 different transformants (bars indicate standard deviation). Lower panels: protein expression in yeast transformants was analyzed by Western blotting using anti-HA antibodies (for the GAD-fusions) and anti-LexA (for the LexA-fusions) in several transformants from each condition. A representative western blot of some of these transformants is shown.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0131476.g002: Analysis of the interacting properties of different domains of R6 by yeast two-hybrid analyses.Upper panels: yeast THY-AP4 strain was transformed with plasmids pBTM-R6 wt (LexA-R6), pBTM-R6 RARA and pBTM-R6 RAHA (A), pBTM-R6 WDNAD, and pBTM-WANNA (B) or pBTM-R6 S25A and pBTM-R6 S74A (C) and with pACT2 (GAD), pACT2-PP1α (GAD-PP1α), pACT2-laforin (GAD-laforin) or pACT2-14-3-3ε (GAD-14-3-3ε). Protein interaction was estimated by measuring the β-galactosidase activity. Values correspond to means from at least 6 different transformants (bars indicate standard deviation). Lower panels: protein expression in yeast transformants was analyzed by Western blotting using anti-HA antibodies (for the GAD-fusions) and anti-LexA (for the LexA-fusions) in several transformants from each condition. A representative western blot of some of these transformants is shown.
Mentions: To assess the functionality of the different domains of R6 that we have described above, we generated different mutants in the corresponding protein motifs and checked whether the interaction with different partners was affected. We analyzed first the putative RVXF motifs involved in the interaction with PP1c. The hydrophobic residues valine and phenylalanine within the RVXF motifs were substituted for alanine, resulting in R102ARA and R252AHA mutants. By yeast two-hybrid analyses we checked the interaction of these mutated forms with the PP1 catalytic subunit (PP1c). In addition, we checked their interaction with laforin, a glucan phosphatase involved in Lafora disease, and with 14-3-3ε proteins, as they are potential interaction partners of R6 [18]. Unfortunately the interaction of R6 with glycogenic substrates, such as glycogen synthase (GS), was undetectable by yeast two-hybrid, what precluded the analysis of the effect of the generated mutations on the interaction with this substrate using this technique. As shown in Fig 2A, mutations in the R102VRF motif (R6-RARA mutant) disrupted the binding to PP1c, although the binding to other interacting partners (laforin and 14-3-3ε) remained as wild type. However, mutations in the R252VHF motif (R6-RAHA mutant) impaired the binding to PP1c and also to the other interaction partners, despite the construct was successfully produced, suggesting that the mutations had affected the overall structure of the protein when expressed in yeast (Fig 2A). In order to confirm these results, we performed an immunoprecipitation analysis in mammalian Hek293 cells expressing YFP-tagged versions of either R6-RARA or R6-RAHA mutants and compared the results with the wild type form of R6. As shown in Fig 3A, using the GFP-Trap technique, we confirmed that YFP-R6-RARA had completely lost the interaction to endogenous PP1c. This mammalian system allowed us to study the interaction of the different mutants with endogenous glycogenic substrates. In this sense, we observed that the YFP-R6-RARA mutant was still able to interact with endogenous GS and GP (although to at a lesser extend in the latter case), thus indicating that binding to these PP1 substrates was independent to the binding to PP1c. Binding of YFP-R6-RARA to endogenous 14-3-3ε protein was also not affected (Fig 3A). We were not able to check the binding to endogenous laforin as the levels of this protein were very low in these cells (not shown). On the contrary, we found that the YFP-R6-RAHA mutant was able to interact with the endogenous PP1c catalytic subunit (Fig 3A), therefore discarding the R252VHF motif as an area of contact to PP1c. The discrepancy between these results and the data obtained by yeast two-hybrid could be due to the different expression systems, yeast vs mammalian cells, being the latter more complete as it has endogenous levels of all glycogenic enzymes. Although the R6-RAHA mutant interacted with endogenous 14-3-3ε proteins, it had an impaired interaction with the endogenous PP1 substrates GS and GP (Fig 3A). Probably, the introduced mutations could have altered the conformation of the protein and affected the functionality of the W267DNND substrate binding motif present in the near vicinity (Fig 1B) (see below).

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.


Related in: MedlinePlus