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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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Suppression of PP2A provokes shuttling and phosphorylation of GSK3β. F9 cells expressed Rfz1 were treated without (-) or with (+) OA for 1 hr and then stimulated without (time + 0) or with Wnt3a for the indicated time periods. At indicated time points, cell cultures were harvested, disrupted, and subjected to subcellular fractionation to plasma membrane (PM), cytoplasm (CY) and nuclei (NU) fractions, as described in the Methods. Samples of each fraction were subjected to protein determination, SDS-PAGE, and the resolved proteins blotted and stained with antibodies specific for GSK3β and with antibodies specific for Ser9 phospho-GSK3β. Left-handed panel shows the quantitative analysis of the blots for Wnt3a alone (blue line) or Wnt3a in the presence of OA (pink line). The data are displayed as fold of control (time zero set to 1). Right-handed panel displays immunoblots stained with GSK3β and phospho-GSK3β (Ser 9) antibodies as well as antibodies specific for subcellular fractions, (i.e., marker proteins). The results shown are derived from a single experiment, representative of two additional experiments.
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Figure 6: Suppression of PP2A provokes shuttling and phosphorylation of GSK3β. F9 cells expressed Rfz1 were treated without (-) or with (+) OA for 1 hr and then stimulated without (time + 0) or with Wnt3a for the indicated time periods. At indicated time points, cell cultures were harvested, disrupted, and subjected to subcellular fractionation to plasma membrane (PM), cytoplasm (CY) and nuclei (NU) fractions, as described in the Methods. Samples of each fraction were subjected to protein determination, SDS-PAGE, and the resolved proteins blotted and stained with antibodies specific for GSK3β and with antibodies specific for Ser9 phospho-GSK3β. Left-handed panel shows the quantitative analysis of the blots for Wnt3a alone (blue line) or Wnt3a in the presence of OA (pink line). The data are displayed as fold of control (time zero set to 1). Right-handed panel displays immunoblots stained with GSK3β and phospho-GSK3β (Ser 9) antibodies as well as antibodies specific for subcellular fractions, (i.e., marker proteins). The results shown are derived from a single experiment, representative of two additional experiments.

Mentions: The activity of GSK3β becomes inhibited when the N-terminal serine residue (Ser 9) of the enzyme is phosphorylated, an event catalyzed by members of the AGC family of protein kinases [25,26]. Phospho-GSK3β is a substrate for PP2A, dephosphorylation of GSK3β by PP2A restores enzymatic activity [10]. OA provoked accumulation of GSK3β and phospho-GSK3β in the PM and the NU subcellular fractions, mimicking Wnt3a action (fig. 6). In the NU subcellular fraction, content of GSK3β as well as phospho-GSK3β increased 3- and 4-fold, respectively, in response to OA alone. For cells in which PP2A was suppressed by siRNA, nuclear GSK3β accumulated more than 5-fold and phospho-GSK3β by up to 2-fold (see additional file: Supplementary fig. 2A).


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

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

Suppression of PP2A provokes shuttling and phosphorylation of GSK3β. F9 cells expressed Rfz1 were treated without (-) or with (+) OA for 1 hr and then stimulated without (time + 0) or with Wnt3a for the indicated time periods. At indicated time points, cell cultures were harvested, disrupted, and subjected to subcellular fractionation to plasma membrane (PM), cytoplasm (CY) and nuclei (NU) fractions, as described in the Methods. Samples of each fraction were subjected to protein determination, SDS-PAGE, and the resolved proteins blotted and stained with antibodies specific for GSK3β and with antibodies specific for Ser9 phospho-GSK3β. Left-handed panel shows the quantitative analysis of the blots for Wnt3a alone (blue line) or Wnt3a in the presence of OA (pink line). The data are displayed as fold of control (time zero set to 1). Right-handed panel displays immunoblots stained with GSK3β and phospho-GSK3β (Ser 9) antibodies as well as antibodies specific for subcellular fractions, (i.e., marker proteins). The results shown are derived from a single experiment, representative of two additional experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: Suppression of PP2A provokes shuttling and phosphorylation of GSK3β. F9 cells expressed Rfz1 were treated without (-) or with (+) OA for 1 hr and then stimulated without (time + 0) or with Wnt3a for the indicated time periods. At indicated time points, cell cultures were harvested, disrupted, and subjected to subcellular fractionation to plasma membrane (PM), cytoplasm (CY) and nuclei (NU) fractions, as described in the Methods. Samples of each fraction were subjected to protein determination, SDS-PAGE, and the resolved proteins blotted and stained with antibodies specific for GSK3β and with antibodies specific for Ser9 phospho-GSK3β. Left-handed panel shows the quantitative analysis of the blots for Wnt3a alone (blue line) or Wnt3a in the presence of OA (pink line). The data are displayed as fold of control (time zero set to 1). Right-handed panel displays immunoblots stained with GSK3β and phospho-GSK3β (Ser 9) antibodies as well as antibodies specific for subcellular fractions, (i.e., marker proteins). The results shown are derived from a single experiment, representative of two additional experiments.
Mentions: The activity of GSK3β becomes inhibited when the N-terminal serine residue (Ser 9) of the enzyme is phosphorylated, an event catalyzed by members of the AGC family of protein kinases [25,26]. Phospho-GSK3β is a substrate for PP2A, dephosphorylation of GSK3β by PP2A restores enzymatic activity [10]. OA provoked accumulation of GSK3β and phospho-GSK3β in the PM and the NU subcellular fractions, mimicking Wnt3a action (fig. 6). In the NU subcellular fraction, content of GSK3β as well as phospho-GSK3β increased 3- and 4-fold, respectively, in response to OA alone. For cells in which PP2A was suppressed by siRNA, nuclear GSK3β accumulated more than 5-fold and phospho-GSK3β by up to 2-fold (see additional file: Supplementary fig. 2A).

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