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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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Mutations within the RVxF motif prevent dephosphorylation of PC1 by PP1α.To determine whether mutations in the RVxF motif affect the ability of PP1α to dephosphorylate PC1, GST-HT193 fusion proteins with point mutations within and around this motif and GST-HA74 were analyzed in an in vitro kinase/phosphatase assay as described in Figure 4. The amount of phosphorylated protein (relative to input material at the start of the reaction) remaining after 2 h in the presence or absence of PP1α was determined by autoradiography and immunoblotting. Following detection of the fusion proteins, membranes were stripped and re-probed for the presence of PP1α. (A) Sequence of wild type (WT), and mutant RVxF constructs of PC1. The primary amino acid sequence spanning the various sites of mutagenesis is shown. The identity of the mutated residue is shown at left and underlined in the primary sequence. The RVxF motif is in bold. (B) Representative autoradiographs and immunoblots showing phosphorylation and total protein levels of GST-HT193 WT, V4143A, and F4145A PC1 fusion proteins. (C) Summary of the effects of PC1 mutations on PP1α-mediated dephosphorylation of PC1. On average, approximately 67% of WT input material was dephosphorylated over the 2 h assay period. Data (mean ± SE) represent the percent dephosphorylation of PC1 constructs by PP1α, relative to dephosphorylation of WT PC1 (set to 100%). n = 6 for WT and n = 5 for mutant constructs. *P<0.05 and **P<0.01, compared to the effect of PP1α on WT PC1, determined by one-way ANOVA and the Dunnett multiple comparison post-test. Representative autoradiographs and immunoblots for GST-HT193 V4136A, R4140A, H4141A, K4142A, R4144C, and V4143A/F4145A are shown in Figure S4A. Representative autoradiographs and immunoblots for additional mutants which lacked obvious defects in PP1α dephosphorylation are shown in Figure S4B. (D) Summary and representative autoradiographs and immunoblots showing phosphorylation and total protein levels of GST-HT193 WT and F4145V PC1 fusion proteins. Data (mean ± SE) represent the percent dephosphorylation of PC1 constructs by PP1α, relative to dephosphorylation of WT PC1 (set to 100%). n = 6 for WT and F4145V. ***P<0.001, compared to the effect of PP1α on WT PC1, determined by unpaired t test.
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pone-0036798-g006: Mutations within the RVxF motif prevent dephosphorylation of PC1 by PP1α.To determine whether mutations in the RVxF motif affect the ability of PP1α to dephosphorylate PC1, GST-HT193 fusion proteins with point mutations within and around this motif and GST-HA74 were analyzed in an in vitro kinase/phosphatase assay as described in Figure 4. The amount of phosphorylated protein (relative to input material at the start of the reaction) remaining after 2 h in the presence or absence of PP1α was determined by autoradiography and immunoblotting. Following detection of the fusion proteins, membranes were stripped and re-probed for the presence of PP1α. (A) Sequence of wild type (WT), and mutant RVxF constructs of PC1. The primary amino acid sequence spanning the various sites of mutagenesis is shown. The identity of the mutated residue is shown at left and underlined in the primary sequence. The RVxF motif is in bold. (B) Representative autoradiographs and immunoblots showing phosphorylation and total protein levels of GST-HT193 WT, V4143A, and F4145A PC1 fusion proteins. (C) Summary of the effects of PC1 mutations on PP1α-mediated dephosphorylation of PC1. On average, approximately 67% of WT input material was dephosphorylated over the 2 h assay period. Data (mean ± SE) represent the percent dephosphorylation of PC1 constructs by PP1α, relative to dephosphorylation of WT PC1 (set to 100%). n = 6 for WT and n = 5 for mutant constructs. *P<0.05 and **P<0.01, compared to the effect of PP1α on WT PC1, determined by one-way ANOVA and the Dunnett multiple comparison post-test. Representative autoradiographs and immunoblots for GST-HT193 V4136A, R4140A, H4141A, K4142A, R4144C, and V4143A/F4145A are shown in Figure S4A. Representative autoradiographs and immunoblots for additional mutants which lacked obvious defects in PP1α dephosphorylation are shown in Figure S4B. (D) Summary and representative autoradiographs and immunoblots showing phosphorylation and total protein levels of GST-HT193 WT and F4145V PC1 fusion proteins. Data (mean ± SE) represent the percent dephosphorylation of PC1 constructs by PP1α, relative to dephosphorylation of WT PC1 (set to 100%). n = 6 for WT and F4145V. ***P<0.001, compared to the effect of PP1α on WT PC1, determined by unpaired t test.

Mentions: Mutation of the critical hydrophobic residues in the RVxF PP1-binding motif to alanine frequently disrupts the interaction between the phosphatase and its regulatory proteins [5], [28], [29], [30]. To examine this, we tested whether a series of mutations (Figure 6A), including V4143A and F4145A in the RVxF motif, would disrupt the ability of PP1α to dephosphorylate PC1 using GST-HT193 fusion proteins containing these mutations in an in vitro phosphatase assay. As seen in Figure 6B, V4143A and F4145A mutations dramatically blocked the ability of PP1α to dephosphorylate PC1, thus indicating the importance of the RVxF motif in PP1α function. To determine if other residues in and around the RVxF motif also play a critical role, those residues in close proximity to the RVxF motif were mutated and tested (Figure 6C). These included R4140A, H4141A, K4142A, V4143A/F4145A, and R4144C. The R4144C mutation corresponds to a human disease-associated mutation in the “x” residue of the RVxF motif [31]. As a control, a V4136A mutation was also tested. V4136 lies in close proximity to the RVxF motif and within an amino acid sequence that is similar to but does not conform to an RVxF motif (KVKEF; V4136 underlined).


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

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

Mutations within the RVxF motif prevent dephosphorylation of PC1 by PP1α.To determine whether mutations in the RVxF motif affect the ability of PP1α to dephosphorylate PC1, GST-HT193 fusion proteins with point mutations within and around this motif and GST-HA74 were analyzed in an in vitro kinase/phosphatase assay as described in Figure 4. The amount of phosphorylated protein (relative to input material at the start of the reaction) remaining after 2 h in the presence or absence of PP1α was determined by autoradiography and immunoblotting. Following detection of the fusion proteins, membranes were stripped and re-probed for the presence of PP1α. (A) Sequence of wild type (WT), and mutant RVxF constructs of PC1. The primary amino acid sequence spanning the various sites of mutagenesis is shown. The identity of the mutated residue is shown at left and underlined in the primary sequence. The RVxF motif is in bold. (B) Representative autoradiographs and immunoblots showing phosphorylation and total protein levels of GST-HT193 WT, V4143A, and F4145A PC1 fusion proteins. (C) Summary of the effects of PC1 mutations on PP1α-mediated dephosphorylation of PC1. On average, approximately 67% of WT input material was dephosphorylated over the 2 h assay period. Data (mean ± SE) represent the percent dephosphorylation of PC1 constructs by PP1α, relative to dephosphorylation of WT PC1 (set to 100%). n = 6 for WT and n = 5 for mutant constructs. *P<0.05 and **P<0.01, compared to the effect of PP1α on WT PC1, determined by one-way ANOVA and the Dunnett multiple comparison post-test. Representative autoradiographs and immunoblots for GST-HT193 V4136A, R4140A, H4141A, K4142A, R4144C, and V4143A/F4145A are shown in Figure S4A. Representative autoradiographs and immunoblots for additional mutants which lacked obvious defects in PP1α dephosphorylation are shown in Figure S4B. (D) Summary and representative autoradiographs and immunoblots showing phosphorylation and total protein levels of GST-HT193 WT and F4145V PC1 fusion proteins. Data (mean ± SE) represent the percent dephosphorylation of PC1 constructs by PP1α, relative to dephosphorylation of WT PC1 (set to 100%). n = 6 for WT and F4145V. ***P<0.001, compared to the effect of PP1α on WT PC1, determined by unpaired t test.
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pone-0036798-g006: Mutations within the RVxF motif prevent dephosphorylation of PC1 by PP1α.To determine whether mutations in the RVxF motif affect the ability of PP1α to dephosphorylate PC1, GST-HT193 fusion proteins with point mutations within and around this motif and GST-HA74 were analyzed in an in vitro kinase/phosphatase assay as described in Figure 4. The amount of phosphorylated protein (relative to input material at the start of the reaction) remaining after 2 h in the presence or absence of PP1α was determined by autoradiography and immunoblotting. Following detection of the fusion proteins, membranes were stripped and re-probed for the presence of PP1α. (A) Sequence of wild type (WT), and mutant RVxF constructs of PC1. The primary amino acid sequence spanning the various sites of mutagenesis is shown. The identity of the mutated residue is shown at left and underlined in the primary sequence. The RVxF motif is in bold. (B) Representative autoradiographs and immunoblots showing phosphorylation and total protein levels of GST-HT193 WT, V4143A, and F4145A PC1 fusion proteins. (C) Summary of the effects of PC1 mutations on PP1α-mediated dephosphorylation of PC1. On average, approximately 67% of WT input material was dephosphorylated over the 2 h assay period. Data (mean ± SE) represent the percent dephosphorylation of PC1 constructs by PP1α, relative to dephosphorylation of WT PC1 (set to 100%). n = 6 for WT and n = 5 for mutant constructs. *P<0.05 and **P<0.01, compared to the effect of PP1α on WT PC1, determined by one-way ANOVA and the Dunnett multiple comparison post-test. Representative autoradiographs and immunoblots for GST-HT193 V4136A, R4140A, H4141A, K4142A, R4144C, and V4143A/F4145A are shown in Figure S4A. Representative autoradiographs and immunoblots for additional mutants which lacked obvious defects in PP1α dephosphorylation are shown in Figure S4B. (D) Summary and representative autoradiographs and immunoblots showing phosphorylation and total protein levels of GST-HT193 WT and F4145V PC1 fusion proteins. Data (mean ± SE) represent the percent dephosphorylation of PC1 constructs by PP1α, relative to dephosphorylation of WT PC1 (set to 100%). n = 6 for WT and F4145V. ***P<0.001, compared to the effect of PP1α on WT PC1, determined by unpaired t test.
Mentions: Mutation of the critical hydrophobic residues in the RVxF PP1-binding motif to alanine frequently disrupts the interaction between the phosphatase and its regulatory proteins [5], [28], [29], [30]. To examine this, we tested whether a series of mutations (Figure 6A), including V4143A and F4145A in the RVxF motif, would disrupt the ability of PP1α to dephosphorylate PC1 using GST-HT193 fusion proteins containing these mutations in an in vitro phosphatase assay. As seen in Figure 6B, V4143A and F4145A mutations dramatically blocked the ability of PP1α to dephosphorylate PC1, thus indicating the importance of the RVxF motif in PP1α function. To determine if other residues in and around the RVxF motif also play a critical role, those residues in close proximity to the RVxF motif were mutated and tested (Figure 6C). These included R4140A, H4141A, K4142A, V4143A/F4145A, and R4144C. The R4144C mutation corresponds to a human disease-associated mutation in the “x” residue of the RVxF motif [31]. As a control, a V4136A mutation was also tested. V4136 lies in close proximity to the RVxF motif and within an amino acid sequence that is similar to but does not conform to an RVxF motif (KVKEF; V4136 underlined).

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