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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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Dephosphorylation of human PC1 by PP1α.To determine whether PP1α can also dephosphorylate PKA-phosphorylated human PC1 (hPC1), GST-hPC1 C-tail fusion proteins (GST-hHT193 and GST-hHT193 S4168A) were purified, phosphorylated, and detected as described in Figure 4. GST-hHT193 S4168A was used in this analysis to be certain that phosphorylation and dephosphorylation was occurring on a residue other than S4168, which is equivalent to the site of PKA phosphorylation on mouse PC1 (S4159).
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pone-0036798-g005: Dephosphorylation of human PC1 by PP1α.To determine whether PP1α can also dephosphorylate PKA-phosphorylated human PC1 (hPC1), GST-hPC1 C-tail fusion proteins (GST-hHT193 and GST-hHT193 S4168A) were purified, phosphorylated, and detected as described in Figure 4. GST-hHT193 S4168A was used in this analysis to be certain that phosphorylation and dephosphorylation was occurring on a residue other than S4168, which is equivalent to the site of PKA phosphorylation on mouse PC1 (S4159).

Mentions: In contrast to mouse PC1, which is phosphorylated by PKA on S4159, human PC1 is phosphorylated by PKA at a different position on a non-conserved serine [27]. To determine whether PP1α dephosphorylates PKA-phosphorylated human PC1, a GST fusion protein containing the C-terminal 193 amino acid residues of human PC1 (GST-hHT193, Figure 1B) was tested in the in vitro phosphatase assay. As shown in Figure 5, the GST-hHT193 fusion protein was readily dephosphorylated by PP1α. A fusion protein in which hS4168 (equivalent to the S4159 PKA phosphorylation site of mouse PC1) was mutated to alanine was also readily phosphorylated and dephosphorylated. These results confirm that PKA phosphorylation occurs on distinct residues at different locations in mouse and human PC1 but that both mouse and human PC1 are dephosphorylated by PP1α.


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

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

Dephosphorylation of human PC1 by PP1α.To determine whether PP1α can also dephosphorylate PKA-phosphorylated human PC1 (hPC1), GST-hPC1 C-tail fusion proteins (GST-hHT193 and GST-hHT193 S4168A) were purified, phosphorylated, and detected as described in Figure 4. GST-hHT193 S4168A was used in this analysis to be certain that phosphorylation and dephosphorylation was occurring on a residue other than S4168, which is equivalent to the site of PKA phosphorylation on mouse PC1 (S4159).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3366979&req=5

pone-0036798-g005: Dephosphorylation of human PC1 by PP1α.To determine whether PP1α can also dephosphorylate PKA-phosphorylated human PC1 (hPC1), GST-hPC1 C-tail fusion proteins (GST-hHT193 and GST-hHT193 S4168A) were purified, phosphorylated, and detected as described in Figure 4. GST-hHT193 S4168A was used in this analysis to be certain that phosphorylation and dephosphorylation was occurring on a residue other than S4168, which is equivalent to the site of PKA phosphorylation on mouse PC1 (S4159).
Mentions: In contrast to mouse PC1, which is phosphorylated by PKA on S4159, human PC1 is phosphorylated by PKA at a different position on a non-conserved serine [27]. To determine whether PP1α dephosphorylates PKA-phosphorylated human PC1, a GST fusion protein containing the C-terminal 193 amino acid residues of human PC1 (GST-hHT193, Figure 1B) was tested in the in vitro phosphatase assay. As shown in Figure 5, the GST-hHT193 fusion protein was readily dephosphorylated by PP1α. A fusion protein in which hS4168 (equivalent to the S4159 PKA phosphorylation site of mouse PC1) was mutated to alanine was also readily phosphorylated and dephosphorylated. These results confirm that PKA phosphorylation occurs on distinct residues at different locations in mouse and human PC1 but that both mouse and human PC1 are dephosphorylated by PP1α.

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