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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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Immunoprecipitation of PC1 truncation proteins.To determine the region of PC1 responsible for interacting with PP1α, various IL2-PC1 fusion proteins were tested for their ability to co-immunoprecipitate with PP1α. The HA74 region of PC1 contains the putative PP1-binding motif, but lacks the coiled-coil domain. The AT120 region of PC1 lacks the putative PP1-binding motif, but contains the coiled-coil domain. Cell lysates from transfected 293T cells were immunoprecipitated and immunoblotted as described in Figure 2. Additional replicates of this experiment can be seen in Figures S3. ns  =  non-specific band.
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pone-0036798-g003: Immunoprecipitation of PC1 truncation proteins.To determine the region of PC1 responsible for interacting with PP1α, various IL2-PC1 fusion proteins were tested for their ability to co-immunoprecipitate with PP1α. The HA74 region of PC1 contains the putative PP1-binding motif, but lacks the coiled-coil domain. The AT120 region of PC1 lacks the putative PP1-binding motif, but contains the coiled-coil domain. Cell lysates from transfected 293T cells were immunoprecipitated and immunoblotted as described in Figure 2. Additional replicates of this experiment can be seen in Figures S3. ns  =  non-specific band.

Mentions: To determine the region of PC1 responsible for interacting with PP1α, IL2-PC1 fusion proteins (Figure 1B) encoding either the membrane distal 120 amino acids (IL2-AT120, a region which lacks the putative PP1-binding and PKA phosphorylation sites but contains the coiled-coil) or the membrane proximal 74 amino acids (IL2-HA74, a region which lacks the coiled-coil but contains the putative PP1-binding and PKA phosphorylation sites) of the C-terminal tail of PC1 were tested for their ability to co-immunoprecipitate with PP1α. As shown in Figure 3 (also Figure S3), both of these truncations (IL2-AT120 and -HA74) dramatically reduced, but failed to eliminate binding of PP1α to PC1, as compared to the longer IL2-HT193 protein. No binding of IL2-AT120 and HA74 occurred in the absence of co-expressed PP1α (data not shown). Weak non-specific binding of IL2-0 to PP1α could only be detected upon over-expression of IL2-0 (see Figure S3). These results suggest that multiple PC1 sites are involved in PP1α binding and that PP1α interacts with the conserved PP1-binding motif plus additional elements within the membrane distal portion of the PC1 C-tail. It is also possible that additional PC1-interacting proteins may be required to stabilize the interaction.


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

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

Immunoprecipitation of PC1 truncation proteins.To determine the region of PC1 responsible for interacting with PP1α, various IL2-PC1 fusion proteins were tested for their ability to co-immunoprecipitate with PP1α. The HA74 region of PC1 contains the putative PP1-binding motif, but lacks the coiled-coil domain. The AT120 region of PC1 lacks the putative PP1-binding motif, but contains the coiled-coil domain. Cell lysates from transfected 293T cells were immunoprecipitated and immunoblotted as described in Figure 2. Additional replicates of this experiment can be seen in Figures S3. ns  =  non-specific band.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0036798-g003: Immunoprecipitation of PC1 truncation proteins.To determine the region of PC1 responsible for interacting with PP1α, various IL2-PC1 fusion proteins were tested for their ability to co-immunoprecipitate with PP1α. The HA74 region of PC1 contains the putative PP1-binding motif, but lacks the coiled-coil domain. The AT120 region of PC1 lacks the putative PP1-binding motif, but contains the coiled-coil domain. Cell lysates from transfected 293T cells were immunoprecipitated and immunoblotted as described in Figure 2. Additional replicates of this experiment can be seen in Figures S3. ns  =  non-specific band.
Mentions: To determine the region of PC1 responsible for interacting with PP1α, IL2-PC1 fusion proteins (Figure 1B) encoding either the membrane distal 120 amino acids (IL2-AT120, a region which lacks the putative PP1-binding and PKA phosphorylation sites but contains the coiled-coil) or the membrane proximal 74 amino acids (IL2-HA74, a region which lacks the coiled-coil but contains the putative PP1-binding and PKA phosphorylation sites) of the C-terminal tail of PC1 were tested for their ability to co-immunoprecipitate with PP1α. As shown in Figure 3 (also Figure S3), both of these truncations (IL2-AT120 and -HA74) dramatically reduced, but failed to eliminate binding of PP1α to PC1, as compared to the longer IL2-HT193 protein. No binding of IL2-AT120 and HA74 occurred in the absence of co-expressed PP1α (data not shown). Weak non-specific binding of IL2-0 to PP1α could only be detected upon over-expression of IL2-0 (see Figure S3). These results suggest that multiple PC1 sites are involved in PP1α binding and that PP1α interacts with the conserved PP1-binding motif plus additional elements within the membrane distal portion of the PC1 C-tail. It is also possible that additional PC1-interacting proteins may be required to stabilize the interaction.

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