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Protein phosphatase-1α interacts with and dephosphorylates polycystin-1.

Parnell SC, Puri S, Wallace DP, Calvet JP - PLoS ONE (2012)

Bottom Line: We have now determined that polycystin-1 is a regulatory subunit for protein phosphatase-1α (PP1α).To determine whether PP1α dephosphorylates polycystin-1, a PKA-phosphorylated GST-polycystin-1 fusion protein was shown to be dephosphorylated by PP1α but not by PP2B (calcineurin).The results suggest that polycystin-1 forms a holoenzyme complex with PP1α via a conserved PP1-binding motif within the polycystin-1 C-tail, and that PKA-phosphorylated polycystin-1 serves as a substrate for the holoenzyme.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City, Kansas, United States of America. sparnell@kumc.edu

ABSTRACT
Polycystin signaling is likely to be regulated by phosphorylation. While a number of potential protein kinases and their target phosphorylation sites on polycystin-1 have been identified, the corresponding phosphatases have not been extensively studied. We have now determined that polycystin-1 is a regulatory subunit for protein phosphatase-1α (PP1α). Sequence analysis has revealed the presence of a highly conserved PP1-interaction motif in the cytosolic, C-terminal tail of polycystin-1; and we have shown that transfected PP1α specifically co-immunoprecipitates with a polycystin-1 C-tail construct. To determine whether PP1α dephosphorylates polycystin-1, a PKA-phosphorylated GST-polycystin-1 fusion protein was shown to be dephosphorylated by PP1α but not by PP2B (calcineurin). Mutations within the PP1-binding motif of polycystin-1, including an autosomal dominant polycystic kidney disease (ADPKD)-associated mutation, significantly reduced PP1α-mediated dephosphorylation of polycystin-1. The results suggest that polycystin-1 forms a holoenzyme complex with PP1α via a conserved PP1-binding motif within the polycystin-1 C-tail, and that PKA-phosphorylated polycystin-1 serves as a substrate for the holoenzyme.

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PC1 interacts with PP1α.To determine whether PC1 can interact with PP1, 293T cells were transfected with plasmids encoding hemeagglutin (HA) epitope-tagged PP1α (HAPP1α) and the C-terminal, cytosolic 193 amino acids of PC1 fused to the extracellular and transmembrane portion of the IL2 receptor (IL2-HT193). Empty plasmid and an IL2 construct lacking PC1 sequence (IL2-0) were used as controls for HAPP1α and IL2-HT193, respectively. Lysates from the transfected cells were immunoprecipitated (IP) with anti-HA antibodies to pull down PP1α. Antibody-bound and total fractions were resolved by SDS-PAGE and immunoblotted (IB) with anti-IL2 antibodies. Blots were then stripped and re-probed with anti-PP1α antibody. All IL2 and sIg fusion proteins (including IL2-0 and sIg-0) used in this study migrate as doublets, presumably due to a modification of the IL2- and sIg-portions of the fusion proteins. Asterisks are used to mark the IL2- and sIg-specific bands (or their positions were they to be present). Solid arrows are used to indicate the position of other proteins. Different parts of this same gel image (as well as other gel images represented in this manuscript) have been cut and rearranged for consistency and clarity. All other modifications, such as resizing or adjustments to contrast, are performed such that all groupings of images from different parts of the same gel are treated identically. Dashed lines indicate image borders that have been spliced together.
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pone-0036798-g002: PC1 interacts with PP1α.To determine whether PC1 can interact with PP1, 293T cells were transfected with plasmids encoding hemeagglutin (HA) epitope-tagged PP1α (HAPP1α) and the C-terminal, cytosolic 193 amino acids of PC1 fused to the extracellular and transmembrane portion of the IL2 receptor (IL2-HT193). Empty plasmid and an IL2 construct lacking PC1 sequence (IL2-0) were used as controls for HAPP1α and IL2-HT193, respectively. Lysates from the transfected cells were immunoprecipitated (IP) with anti-HA antibodies to pull down PP1α. Antibody-bound and total fractions were resolved by SDS-PAGE and immunoblotted (IB) with anti-IL2 antibodies. Blots were then stripped and re-probed with anti-PP1α antibody. All IL2 and sIg fusion proteins (including IL2-0 and sIg-0) used in this study migrate as doublets, presumably due to a modification of the IL2- and sIg-portions of the fusion proteins. Asterisks are used to mark the IL2- and sIg-specific bands (or their positions were they to be present). Solid arrows are used to indicate the position of other proteins. Different parts of this same gel image (as well as other gel images represented in this manuscript) have been cut and rearranged for consistency and clarity. All other modifications, such as resizing or adjustments to contrast, are performed such that all groupings of images from different parts of the same gel are treated identically. Dashed lines indicate image borders that have been spliced together.

Mentions: To determine whether PC1 can interact with PP1, 293T cells were transfected with plasmids encoding HA epitope-tagged PP1α and the C-terminal, cytosolic 193 amino acids of mouse PC1 fused to the extracellular and transmembrane portion of the IL2 receptor (IL2-HT193, Figure 1B). Cell lysates were immunoprecipitated with anti-HA antibodies, and co-precipitating proteins were detected by SDS-PAGE and immunoblotting. As can be seen in Figure 2, HAPP1α readily co-precipitated the PC1 IL2-HT193 protein but not the IL2 control protein (IL2-0). Reciprocal co-immunoprecipitation was demonstrated (see Figure S2) using HAPP1α and an identical region of the PC1 C-tail fused to the membrane targeting cassette, sIg.7 [22].


Protein phosphatase-1α interacts with and dephosphorylates polycystin-1.

Parnell SC, Puri S, Wallace DP, Calvet JP - PLoS ONE (2012)

PC1 interacts with PP1α.To determine whether PC1 can interact with PP1, 293T cells were transfected with plasmids encoding hemeagglutin (HA) epitope-tagged PP1α (HAPP1α) and the C-terminal, cytosolic 193 amino acids of PC1 fused to the extracellular and transmembrane portion of the IL2 receptor (IL2-HT193). Empty plasmid and an IL2 construct lacking PC1 sequence (IL2-0) were used as controls for HAPP1α and IL2-HT193, respectively. Lysates from the transfected cells were immunoprecipitated (IP) with anti-HA antibodies to pull down PP1α. Antibody-bound and total fractions were resolved by SDS-PAGE and immunoblotted (IB) with anti-IL2 antibodies. Blots were then stripped and re-probed with anti-PP1α antibody. All IL2 and sIg fusion proteins (including IL2-0 and sIg-0) used in this study migrate as doublets, presumably due to a modification of the IL2- and sIg-portions of the fusion proteins. Asterisks are used to mark the IL2- and sIg-specific bands (or their positions were they to be present). Solid arrows are used to indicate the position of other proteins. Different parts of this same gel image (as well as other gel images represented in this manuscript) have been cut and rearranged for consistency and clarity. All other modifications, such as resizing or adjustments to contrast, are performed such that all groupings of images from different parts of the same gel are treated identically. Dashed lines indicate image borders that have been spliced together.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3366979&req=5

pone-0036798-g002: PC1 interacts with PP1α.To determine whether PC1 can interact with PP1, 293T cells were transfected with plasmids encoding hemeagglutin (HA) epitope-tagged PP1α (HAPP1α) and the C-terminal, cytosolic 193 amino acids of PC1 fused to the extracellular and transmembrane portion of the IL2 receptor (IL2-HT193). Empty plasmid and an IL2 construct lacking PC1 sequence (IL2-0) were used as controls for HAPP1α and IL2-HT193, respectively. Lysates from the transfected cells were immunoprecipitated (IP) with anti-HA antibodies to pull down PP1α. Antibody-bound and total fractions were resolved by SDS-PAGE and immunoblotted (IB) with anti-IL2 antibodies. Blots were then stripped and re-probed with anti-PP1α antibody. All IL2 and sIg fusion proteins (including IL2-0 and sIg-0) used in this study migrate as doublets, presumably due to a modification of the IL2- and sIg-portions of the fusion proteins. Asterisks are used to mark the IL2- and sIg-specific bands (or their positions were they to be present). Solid arrows are used to indicate the position of other proteins. Different parts of this same gel image (as well as other gel images represented in this manuscript) have been cut and rearranged for consistency and clarity. All other modifications, such as resizing or adjustments to contrast, are performed such that all groupings of images from different parts of the same gel are treated identically. Dashed lines indicate image borders that have been spliced together.
Mentions: To determine whether PC1 can interact with PP1, 293T cells were transfected with plasmids encoding HA epitope-tagged PP1α and the C-terminal, cytosolic 193 amino acids of mouse PC1 fused to the extracellular and transmembrane portion of the IL2 receptor (IL2-HT193, Figure 1B). Cell lysates were immunoprecipitated with anti-HA antibodies, and co-precipitating proteins were detected by SDS-PAGE and immunoblotting. As can be seen in Figure 2, HAPP1α readily co-precipitated the PC1 IL2-HT193 protein but not the IL2 control protein (IL2-0). Reciprocal co-immunoprecipitation was demonstrated (see Figure S2) using HAPP1α and an identical region of the PC1 C-tail fused to the membrane targeting cassette, sIg.7 [22].

Bottom Line: We have now determined that polycystin-1 is a regulatory subunit for protein phosphatase-1α (PP1α).To determine whether PP1α dephosphorylates polycystin-1, a PKA-phosphorylated GST-polycystin-1 fusion protein was shown to be dephosphorylated by PP1α but not by PP2B (calcineurin).The results suggest that polycystin-1 forms a holoenzyme complex with PP1α via a conserved PP1-binding motif within the polycystin-1 C-tail, and that PKA-phosphorylated polycystin-1 serves as a substrate for the holoenzyme.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City, Kansas, United States of America. sparnell@kumc.edu

ABSTRACT
Polycystin signaling is likely to be regulated by phosphorylation. While a number of potential protein kinases and their target phosphorylation sites on polycystin-1 have been identified, the corresponding phosphatases have not been extensively studied. We have now determined that polycystin-1 is a regulatory subunit for protein phosphatase-1α (PP1α). Sequence analysis has revealed the presence of a highly conserved PP1-interaction motif in the cytosolic, C-terminal tail of polycystin-1; and we have shown that transfected PP1α specifically co-immunoprecipitates with a polycystin-1 C-tail construct. To determine whether PP1α dephosphorylates polycystin-1, a PKA-phosphorylated GST-polycystin-1 fusion protein was shown to be dephosphorylated by PP1α but not by PP2B (calcineurin). Mutations within the PP1-binding motif of polycystin-1, including an autosomal dominant polycystic kidney disease (ADPKD)-associated mutation, significantly reduced PP1α-mediated dephosphorylation of polycystin-1. The results suggest that polycystin-1 forms a holoenzyme complex with PP1α via a conserved PP1-binding motif within the polycystin-1 C-tail, and that PKA-phosphorylated polycystin-1 serves as a substrate for the holoenzyme.

Show MeSH
Related in: MedlinePlus