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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.


Characterization and structural modelling of R6 domains.(A) Multiple sequence alignment of PPP1R3D (R6), PPP1R3C (R5) and PPP1R3B (GL) proteins sequences from H. sapiens (Uniprot entries: O95685, Q9UQK1 and Q86XI6, respectively) was performed using the Clustal Omega program (see Materials and Methods). Invariance and conservation are highlighted by black and gray shadowing respectively. Amino acid sequences for the binding to 14-3-3 proteins, PP1 catalytic subunit and PP1 glycogenic substrates are enclosed in yellow, orange and cyan boxes respectively. Brackets indicate the modeled sequence of R6 shown in panel B. The CBM21 domain of R6 (according to Uniprot) is underlined in orange. Red triangles point at the mutated residues obtained in this study. (B) Homology model of the CBM21 domain of R6 was based on the template of GL (pdb entry: 2EEF). The amino acids corresponding to the PP1 glycogenic substrate binding domain (W267DNND) and to the putative RVXF (R252VHF) are shown in sticks and colored in cyan or orange, respectively (left panel). The N- and C-terminus of the model is also indicated. A representation of the surface of the R6 homology model to show the exposed amino acids to the solvent is presented in the right panel. Images were generated using PyMol (DeLano Scientific LLC, USA).
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pone.0131476.g001: Characterization and structural modelling of R6 domains.(A) Multiple sequence alignment of PPP1R3D (R6), PPP1R3C (R5) and PPP1R3B (GL) proteins sequences from H. sapiens (Uniprot entries: O95685, Q9UQK1 and Q86XI6, respectively) was performed using the Clustal Omega program (see Materials and Methods). Invariance and conservation are highlighted by black and gray shadowing respectively. Amino acid sequences for the binding to 14-3-3 proteins, PP1 catalytic subunit and PP1 glycogenic substrates are enclosed in yellow, orange and cyan boxes respectively. Brackets indicate the modeled sequence of R6 shown in panel B. The CBM21 domain of R6 (according to Uniprot) is underlined in orange. Red triangles point at the mutated residues obtained in this study. (B) Homology model of the CBM21 domain of R6 was based on the template of GL (pdb entry: 2EEF). The amino acids corresponding to the PP1 glycogenic substrate binding domain (W267DNND) and to the putative RVXF (R252VHF) are shown in sticks and colored in cyan or orange, respectively (left panel). The N- and C-terminus of the model is also indicated. A representation of the surface of the R6 homology model to show the exposed amino acids to the solvent is presented in the right panel. Images were generated using PyMol (DeLano Scientific LLC, USA).

Mentions: A multiple sequence protein alignment between PPP1R3B (GL), PPP1R3C (R5/PTG) and PPP1R3D (R6) of H. sapiens (Fig 1A) allowed the definition of different characteristic protein domains in R6. As PP1 glycogen-targeting subunits, all these proteins possess protein motifs for binding to both the PP1 catalytic subunit (PP1c) and the glycogenic PP1 substrates [i.e. glycogen synthase (GS), glycogen phosphorylase (GP)]. The named-RVXF- motif is the primary PP1-docking motif present in about 70% of all PP1 interacting proteins ([2], [3]). Two putative RVXF sequences can be found in R6, R102VRF and R252VHF. However, only the R102VRF sequence was conserved in the three mammalian subunits tested (Fig 1A, first orange box), being the second putative R252VHF motif only present in R6 (Fig 1A, second orange box at the C-terminus). It has been described that the binding of glycogenic targeting subunits to glycogen-related protein substrates occurs via a single highly conserved WDNNE/D motif ([10], [11]). This motif was located at the C-terminus of R6 (W267DNND) (Fig 1A, cyan box). In addition, glycogenic targeting subunits contain a carbohydrate binding module of the CBM21 type ([12], [13]) that allows binding of these subunits to glycogen. In the case of R6 this domain spans from residue 169 to 278 (underlined region in Fig 1A).


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)

Characterization and structural modelling of R6 domains.(A) Multiple sequence alignment of PPP1R3D (R6), PPP1R3C (R5) and PPP1R3B (GL) proteins sequences from H. sapiens (Uniprot entries: O95685, Q9UQK1 and Q86XI6, respectively) was performed using the Clustal Omega program (see Materials and Methods). Invariance and conservation are highlighted by black and gray shadowing respectively. Amino acid sequences for the binding to 14-3-3 proteins, PP1 catalytic subunit and PP1 glycogenic substrates are enclosed in yellow, orange and cyan boxes respectively. Brackets indicate the modeled sequence of R6 shown in panel B. The CBM21 domain of R6 (according to Uniprot) is underlined in orange. Red triangles point at the mutated residues obtained in this study. (B) Homology model of the CBM21 domain of R6 was based on the template of GL (pdb entry: 2EEF). The amino acids corresponding to the PP1 glycogenic substrate binding domain (W267DNND) and to the putative RVXF (R252VHF) are shown in sticks and colored in cyan or orange, respectively (left panel). The N- and C-terminus of the model is also indicated. A representation of the surface of the R6 homology model to show the exposed amino acids to the solvent is presented in the right panel. Images were generated using PyMol (DeLano Scientific LLC, USA).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4482762&req=5

pone.0131476.g001: Characterization and structural modelling of R6 domains.(A) Multiple sequence alignment of PPP1R3D (R6), PPP1R3C (R5) and PPP1R3B (GL) proteins sequences from H. sapiens (Uniprot entries: O95685, Q9UQK1 and Q86XI6, respectively) was performed using the Clustal Omega program (see Materials and Methods). Invariance and conservation are highlighted by black and gray shadowing respectively. Amino acid sequences for the binding to 14-3-3 proteins, PP1 catalytic subunit and PP1 glycogenic substrates are enclosed in yellow, orange and cyan boxes respectively. Brackets indicate the modeled sequence of R6 shown in panel B. The CBM21 domain of R6 (according to Uniprot) is underlined in orange. Red triangles point at the mutated residues obtained in this study. (B) Homology model of the CBM21 domain of R6 was based on the template of GL (pdb entry: 2EEF). The amino acids corresponding to the PP1 glycogenic substrate binding domain (W267DNND) and to the putative RVXF (R252VHF) are shown in sticks and colored in cyan or orange, respectively (left panel). The N- and C-terminus of the model is also indicated. A representation of the surface of the R6 homology model to show the exposed amino acids to the solvent is presented in the right panel. Images were generated using PyMol (DeLano Scientific LLC, USA).
Mentions: A multiple sequence protein alignment between PPP1R3B (GL), PPP1R3C (R5/PTG) and PPP1R3D (R6) of H. sapiens (Fig 1A) allowed the definition of different characteristic protein domains in R6. As PP1 glycogen-targeting subunits, all these proteins possess protein motifs for binding to both the PP1 catalytic subunit (PP1c) and the glycogenic PP1 substrates [i.e. glycogen synthase (GS), glycogen phosphorylase (GP)]. The named-RVXF- motif is the primary PP1-docking motif present in about 70% of all PP1 interacting proteins ([2], [3]). Two putative RVXF sequences can be found in R6, R102VRF and R252VHF. However, only the R102VRF sequence was conserved in the three mammalian subunits tested (Fig 1A, first orange box), being the second putative R252VHF motif only present in R6 (Fig 1A, second orange box at the C-terminus). It has been described that the binding of glycogenic targeting subunits to glycogen-related protein substrates occurs via a single highly conserved WDNNE/D motif ([10], [11]). This motif was located at the C-terminus of R6 (W267DNND) (Fig 1A, cyan box). In addition, glycogenic targeting subunits contain a carbohydrate binding module of the CBM21 type ([12], [13]) that allows binding of these subunits to glycogen. In the case of R6 this domain spans from residue 169 to 278 (underlined region in Fig 1A).

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.