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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.


Glycogenic activity of different mutated forms of R6.(A) Measurement of glycogenic activity of different R6 mutated forms. N2a cells were transfected using 1 μg of pFLAG plasmid (negative control), pFLAG-R6 plasmid or its corresponding mutants. Forty-eight hours after transfection, the amount of glycogen was determined as described in Materials and Methods and represented as μg of glucose/mg of protein/relative amount of R6 respect to actin (wild type value considered as 1). Bars indicate standard deviation of three independent experiments (**p<0.01 or ***p<0.001, compared with control cells transfected with an empty plasmid; ##p<0.01, compared with cells expressing R6-WT). An inset with the mean values +/- standard deviation is included. (B) Protein levels of FLAG-R6 forms. A representative western blot analysis is shown. Cell extracts (30 μg) were analyzed using the corresponding anti-FLAG and anti-actin antibodies.
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pone.0131476.g004: Glycogenic activity of different mutated forms of R6.(A) Measurement of glycogenic activity of different R6 mutated forms. N2a cells were transfected using 1 μg of pFLAG plasmid (negative control), pFLAG-R6 plasmid or its corresponding mutants. Forty-eight hours after transfection, the amount of glycogen was determined as described in Materials and Methods and represented as μg of glucose/mg of protein/relative amount of R6 respect to actin (wild type value considered as 1). Bars indicate standard deviation of three independent experiments (**p<0.01 or ***p<0.001, compared with control cells transfected with an empty plasmid; ##p<0.01, compared with cells expressing R6-WT). An inset with the mean values +/- standard deviation is included. (B) Protein levels of FLAG-R6 forms. A representative western blot analysis is shown. Cell extracts (30 μg) were analyzed using the corresponding anti-FLAG and anti-actin antibodies.

Mentions: We have previously described that the expression of R6 in a neuroblastoma cell line (N2a) triggers de novo glycogen synthesis. In these cells glycogen production is fully dependent on the expression of functional PP1 glycogen targeting subunits since in its absence, glycogen production is very low. The expression of PP1 glycogen targeting subunits, as R6, induces the dephosphorylation of endogenous GS leading to its activation, resulting in glycogen production [17]. To assess the glycogenic activity (capacity to induce glycogen synthesis) of the different R6 mutants we have described above, we expressed FLAG-tagged versions of them in N2a cells and measured the glycogen levels after 48 h of transfection. Consistent with previous results, expression of wild type R6 promoted the accumulation of glycogen (expressed as μg glucose/mg protein/relative amount of FLAG-R6) (Fig 4A). Expression of the R6-RARA mutant, which cannot bind to PP1c but binds to PP1 glycogenic substrates (GS, GP; see above), did not support the promotion of glycogen production (similar levels of glycogen were measured as in cells transfected with an empty plasmid). Next, we analyzed the mutants that affected substrate binding. When R6-RAHA and R6-WANNA mutants were expressed in N2a cells (they do not bind to endogenous GS and GP enzymes; see above), the capacity to support glycogen production was impaired as well, resulting in undetectable levels of glycogen. The expression of the R6-WDNAD mutant, which interacts with PP1c and PP1 glycogenic substrates as wild type (see above), produced amounts of glycogen comparable to the wild type protein. In summary, mutations in R6 affecting the interaction with either PP1c or PP1 glycogenic substrates resulted in an impairment of the glycogenic activity of the mutated forms.


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)

Glycogenic activity of different mutated forms of R6.(A) Measurement of glycogenic activity of different R6 mutated forms. N2a cells were transfected using 1 μg of pFLAG plasmid (negative control), pFLAG-R6 plasmid or its corresponding mutants. Forty-eight hours after transfection, the amount of glycogen was determined as described in Materials and Methods and represented as μg of glucose/mg of protein/relative amount of R6 respect to actin (wild type value considered as 1). Bars indicate standard deviation of three independent experiments (**p<0.01 or ***p<0.001, compared with control cells transfected with an empty plasmid; ##p<0.01, compared with cells expressing R6-WT). An inset with the mean values +/- standard deviation is included. (B) Protein levels of FLAG-R6 forms. A representative western blot analysis is shown. Cell extracts (30 μg) were analyzed using the corresponding anti-FLAG and anti-actin antibodies.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4482762&req=5

pone.0131476.g004: Glycogenic activity of different mutated forms of R6.(A) Measurement of glycogenic activity of different R6 mutated forms. N2a cells were transfected using 1 μg of pFLAG plasmid (negative control), pFLAG-R6 plasmid or its corresponding mutants. Forty-eight hours after transfection, the amount of glycogen was determined as described in Materials and Methods and represented as μg of glucose/mg of protein/relative amount of R6 respect to actin (wild type value considered as 1). Bars indicate standard deviation of three independent experiments (**p<0.01 or ***p<0.001, compared with control cells transfected with an empty plasmid; ##p<0.01, compared with cells expressing R6-WT). An inset with the mean values +/- standard deviation is included. (B) Protein levels of FLAG-R6 forms. A representative western blot analysis is shown. Cell extracts (30 μg) were analyzed using the corresponding anti-FLAG and anti-actin antibodies.
Mentions: We have previously described that the expression of R6 in a neuroblastoma cell line (N2a) triggers de novo glycogen synthesis. In these cells glycogen production is fully dependent on the expression of functional PP1 glycogen targeting subunits since in its absence, glycogen production is very low. The expression of PP1 glycogen targeting subunits, as R6, induces the dephosphorylation of endogenous GS leading to its activation, resulting in glycogen production [17]. To assess the glycogenic activity (capacity to induce glycogen synthesis) of the different R6 mutants we have described above, we expressed FLAG-tagged versions of them in N2a cells and measured the glycogen levels after 48 h of transfection. Consistent with previous results, expression of wild type R6 promoted the accumulation of glycogen (expressed as μg glucose/mg protein/relative amount of FLAG-R6) (Fig 4A). Expression of the R6-RARA mutant, which cannot bind to PP1c but binds to PP1 glycogenic substrates (GS, GP; see above), did not support the promotion of glycogen production (similar levels of glycogen were measured as in cells transfected with an empty plasmid). Next, we analyzed the mutants that affected substrate binding. When R6-RAHA and R6-WANNA mutants were expressed in N2a cells (they do not bind to endogenous GS and GP enzymes; see above), the capacity to support glycogen production was impaired as well, resulting in undetectable levels of glycogen. The expression of the R6-WDNAD mutant, which interacts with PP1c and PP1 glycogenic substrates as wild type (see above), produced amounts of glycogen comparable to the wild type protein. In summary, mutations in R6 affecting the interaction with either PP1c or PP1 glycogenic substrates resulted in an impairment of the glycogenic activity of the mutated forms.

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.