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Phosphoprotein phosphatase-2A docks to Dishevelled and counterregulates Wnt3a/beta-catenin signaling.

Yokoyama N, Malbon CC - J Mol Signal (2007)

Bottom Line: Although mimicking effects of Wnt3a on the cellular abundance and trafficking of key signaling elements in the Wnt canonical pathway, suppression of phosphatase-2A alone did not provoke activation of the Lef/Tcf-sensitive transcriptional response, but did potentiate its activation by Wnt3a.In current study, we showed new roles of phosphoprotein phosphatase-2A in Wnt/beta-catenin signaling pathway: effect on protein expression, effect on protein trafficking, retention of molecules in subcellular compartments, and regulation of enzymatic activity of several key players.Docking of phosphoprotein phosphatase-2A by Dishevelled-2 suppresses phosphatase activity and explains in part the central role of this phosphatase in the counterregulation of the Wnt/beta-catenin signaling pathway.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pharmacology, Health Sciences Center, State University of New York at Stony Brook, Stony Brook, NY 11794-8651, USA. noriko@pharm.stonybrook.edu.

ABSTRACT

Background: Wnt3a stimulates cellular trafficking of key signaling elements (e.g., Axin, Dishevelled-2, beta-catenin, and glycogen synthase kinase-3beta) and primitive endoderm formation in mouse F9 embryonic teratocarcinoma cells.

Results: The role of phosphoprotein phosphatase-2A in signaling of the Wnt/beta-catenin/Lef-Tcf-sensitive gene activation pathway was investigated. Wnt3a action attenuates phosphoprotein phosphatase-2A activity and stimulates the Lef/Tcf-sensitive gene transcription. Inhibiting phosphoprotein phosphatase-2A by okadaic acid, by treatment with siRNA (targeting the C-subunit of the enzyme), or by expression of SV40 small t antigen mimics Wnt3a action, increasing the cellular abundance of Axin and phospho-glycogen synthase kinase-3beta as well as the trafficking of signaling elements in the Wnt/beta-catenin pathway. Although mimicking effects of Wnt3a on the cellular abundance and trafficking of key signaling elements in the Wnt canonical pathway, suppression of phosphatase-2A alone did not provoke activation of the Lef/Tcf-sensitive transcriptional response, but did potentiate its activation by Wnt3a. Phosphoprotein phosphatase-2A and the scaffold phosphoprotein Dishevelled-2 display similarities in cellular trafficking in response to either Wnt3a or suppression of the phosphatase. A docking site for phosphoprotein phosphatase-2A in the DEP domain of Dishevelled-2 was identified.

Conclusion: In current study, we showed new roles of phosphoprotein phosphatase-2A in Wnt/beta-catenin signaling pathway: effect on protein expression, effect on protein trafficking, retention of molecules in subcellular compartments, and regulation of enzymatic activity of several key players. Docking of phosphoprotein phosphatase-2A by Dishevelled-2 suppresses phosphatase activity and explains in part the central role of this phosphatase in the counterregulation of the Wnt/beta-catenin signaling pathway.

No MeSH data available.


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Dvl2 interacts with PP2A and PP2A activity is attenuated in response to Wnt3a. Panel A, Association of Dvl2 and PP2A was investigated in the cytosolic fraction. Cell lysates (1 mg) were immunoprecipitated with Anti-Dvl2 antibody. Bound proteins were resolved by SDS-PAGE, immunoblotting, and made visible by staining with either anti-Dvl2 or anti-PP2A C-subunit antibodies. Dvl2 and PP2AC association is displayed as "fold" (time = 0, set to "1"). Representative blots are displayed. The results are shown as mean values ± S.E. from 5 independent experiments. Panel B, F9 cells expressing Rfz1 were stimulated with purified Wnt3a for the indicated time. Cells were washed with PBS and lysed. Cell lysates were applied to a small Sephadex G-50 column to remove small molecular substances that interfere with the assay, and PP2A activity determined. Results were shown as the mean values ± S.E. from 8 independent experiments.
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Figure 9: Dvl2 interacts with PP2A and PP2A activity is attenuated in response to Wnt3a. Panel A, Association of Dvl2 and PP2A was investigated in the cytosolic fraction. Cell lysates (1 mg) were immunoprecipitated with Anti-Dvl2 antibody. Bound proteins were resolved by SDS-PAGE, immunoblotting, and made visible by staining with either anti-Dvl2 or anti-PP2A C-subunit antibodies. Dvl2 and PP2AC association is displayed as "fold" (time = 0, set to "1"). Representative blots are displayed. The results are shown as mean values ± S.E. from 5 independent experiments. Panel B, F9 cells expressing Rfz1 were stimulated with purified Wnt3a for the indicated time. Cells were washed with PBS and lysed. Cell lysates were applied to a small Sephadex G-50 column to remove small molecular substances that interfere with the assay, and PP2A activity determined. Results were shown as the mean values ± S.E. from 8 independent experiments.

Mentions: Dvl2 is a phosphoprotein, transiently phosphorylated in cells in response to stimulation by Wnt3a [28]. The demonstration of a direct association of Dvl2 with PP2A (via DEP domain, fig. 8) provokes the question if Wnt3a stimulates protein-protein interactions between PP2A and Dvl2. Association of Dvl2 and PP2A was assayed in pull-downs employing anti-Dvl2 antibody, followed by immunoblotting of the pull downs with anti-Dvl2 and anti-PP2A C subunit antibodies. PP2A binding to Dvl2 increased in response to stimulation by Wnt3a and sustained over 2 hr (fig. 9A).


Phosphoprotein phosphatase-2A docks to Dishevelled and counterregulates Wnt3a/beta-catenin signaling.

Yokoyama N, Malbon CC - J Mol Signal (2007)

Dvl2 interacts with PP2A and PP2A activity is attenuated in response to Wnt3a. Panel A, Association of Dvl2 and PP2A was investigated in the cytosolic fraction. Cell lysates (1 mg) were immunoprecipitated with Anti-Dvl2 antibody. Bound proteins were resolved by SDS-PAGE, immunoblotting, and made visible by staining with either anti-Dvl2 or anti-PP2A C-subunit antibodies. Dvl2 and PP2AC association is displayed as "fold" (time = 0, set to "1"). Representative blots are displayed. The results are shown as mean values ± S.E. from 5 independent experiments. Panel B, F9 cells expressing Rfz1 were stimulated with purified Wnt3a for the indicated time. Cells were washed with PBS and lysed. Cell lysates were applied to a small Sephadex G-50 column to remove small molecular substances that interfere with the assay, and PP2A activity determined. Results were shown as the mean values ± S.E. from 8 independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2211464&req=5

Figure 9: Dvl2 interacts with PP2A and PP2A activity is attenuated in response to Wnt3a. Panel A, Association of Dvl2 and PP2A was investigated in the cytosolic fraction. Cell lysates (1 mg) were immunoprecipitated with Anti-Dvl2 antibody. Bound proteins were resolved by SDS-PAGE, immunoblotting, and made visible by staining with either anti-Dvl2 or anti-PP2A C-subunit antibodies. Dvl2 and PP2AC association is displayed as "fold" (time = 0, set to "1"). Representative blots are displayed. The results are shown as mean values ± S.E. from 5 independent experiments. Panel B, F9 cells expressing Rfz1 were stimulated with purified Wnt3a for the indicated time. Cells were washed with PBS and lysed. Cell lysates were applied to a small Sephadex G-50 column to remove small molecular substances that interfere with the assay, and PP2A activity determined. Results were shown as the mean values ± S.E. from 8 independent experiments.
Mentions: Dvl2 is a phosphoprotein, transiently phosphorylated in cells in response to stimulation by Wnt3a [28]. The demonstration of a direct association of Dvl2 with PP2A (via DEP domain, fig. 8) provokes the question if Wnt3a stimulates protein-protein interactions between PP2A and Dvl2. Association of Dvl2 and PP2A was assayed in pull-downs employing anti-Dvl2 antibody, followed by immunoblotting of the pull downs with anti-Dvl2 and anti-PP2A C subunit antibodies. PP2A binding to Dvl2 increased in response to stimulation by Wnt3a and sustained over 2 hr (fig. 9A).

Bottom Line: Although mimicking effects of Wnt3a on the cellular abundance and trafficking of key signaling elements in the Wnt canonical pathway, suppression of phosphatase-2A alone did not provoke activation of the Lef/Tcf-sensitive transcriptional response, but did potentiate its activation by Wnt3a.In current study, we showed new roles of phosphoprotein phosphatase-2A in Wnt/beta-catenin signaling pathway: effect on protein expression, effect on protein trafficking, retention of molecules in subcellular compartments, and regulation of enzymatic activity of several key players.Docking of phosphoprotein phosphatase-2A by Dishevelled-2 suppresses phosphatase activity and explains in part the central role of this phosphatase in the counterregulation of the Wnt/beta-catenin signaling pathway.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pharmacology, Health Sciences Center, State University of New York at Stony Brook, Stony Brook, NY 11794-8651, USA. noriko@pharm.stonybrook.edu.

ABSTRACT

Background: Wnt3a stimulates cellular trafficking of key signaling elements (e.g., Axin, Dishevelled-2, beta-catenin, and glycogen synthase kinase-3beta) and primitive endoderm formation in mouse F9 embryonic teratocarcinoma cells.

Results: The role of phosphoprotein phosphatase-2A in signaling of the Wnt/beta-catenin/Lef-Tcf-sensitive gene activation pathway was investigated. Wnt3a action attenuates phosphoprotein phosphatase-2A activity and stimulates the Lef/Tcf-sensitive gene transcription. Inhibiting phosphoprotein phosphatase-2A by okadaic acid, by treatment with siRNA (targeting the C-subunit of the enzyme), or by expression of SV40 small t antigen mimics Wnt3a action, increasing the cellular abundance of Axin and phospho-glycogen synthase kinase-3beta as well as the trafficking of signaling elements in the Wnt/beta-catenin pathway. Although mimicking effects of Wnt3a on the cellular abundance and trafficking of key signaling elements in the Wnt canonical pathway, suppression of phosphatase-2A alone did not provoke activation of the Lef/Tcf-sensitive transcriptional response, but did potentiate its activation by Wnt3a. Phosphoprotein phosphatase-2A and the scaffold phosphoprotein Dishevelled-2 display similarities in cellular trafficking in response to either Wnt3a or suppression of the phosphatase. A docking site for phosphoprotein phosphatase-2A in the DEP domain of Dishevelled-2 was identified.

Conclusion: In current study, we showed new roles of phosphoprotein phosphatase-2A in Wnt/beta-catenin signaling pathway: effect on protein expression, effect on protein trafficking, retention of molecules in subcellular compartments, and regulation of enzymatic activity of several key players. Docking of phosphoprotein phosphatase-2A by Dishevelled-2 suppresses phosphatase activity and explains in part the central role of this phosphatase in the counterregulation of the Wnt/beta-catenin signaling pathway.

No MeSH data available.


Related in: MedlinePlus