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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.


Related in: MedlinePlus

Abundance of Axin, Dvl2 and β-catenin in response to Wnt3a: effects of okadaic acid. F9 cells expressing Rfz1 were pretreated with or without 40 nM OA for 1 hr and then stimulated with Wnt3a in the presence or absence of OA for the indicated time periods. Cells were harvested and fractionated to subcellular fractions highly enriched in plasma membrane (PM), cytoplasm (CY), or nuclei (NU), as described in the Methods. Each fraction was separated by SDS-PAGE and the resolved protein transferred to blots stained with anti-Axin, anti-Dvl2 or anti-β-catenin antibodies. The enrichment of the subcellular fractions was established by staining immunoblots with antibodies to well known protein markers for PM, CY, and NU subcellular fractions. Left-handed panel displays 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 the mean values "% of control" (i.e., t = 0 then set to 100%). Right-handed panel displays immunoblots. The results shown are derived from a single experiment, representative of two additional experiments.
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Figure 5: Abundance of Axin, Dvl2 and β-catenin in response to Wnt3a: effects of okadaic acid. F9 cells expressing Rfz1 were pretreated with or without 40 nM OA for 1 hr and then stimulated with Wnt3a in the presence or absence of OA for the indicated time periods. Cells were harvested and fractionated to subcellular fractions highly enriched in plasma membrane (PM), cytoplasm (CY), or nuclei (NU), as described in the Methods. Each fraction was separated by SDS-PAGE and the resolved protein transferred to blots stained with anti-Axin, anti-Dvl2 or anti-β-catenin antibodies. The enrichment of the subcellular fractions was established by staining immunoblots with antibodies to well known protein markers for PM, CY, and NU subcellular fractions. Left-handed panel displays 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 the mean values "% of control" (i.e., t = 0 then set to 100%). Right-handed panel displays immunoblots. The results shown are derived from a single experiment, representative of two additional experiments.

Mentions: We investigated the effects of the PP2A suppression on the intracellular shuttling of these Wnt/β-catenin pathway signaling molecules. OA-treated cells were stimulated with Wnt3a for up to 2 hr and the cells disrupted and then subjected to subcellular fractionation. Plasma membrane- (PM), cytoplasm- (CY), and nuclei- (NU) enriched subcellular fractions were prepared and the distributions of Axin, Dvl2, and β-catenin quantified (fig. 5). Most notable, OA alone mimics with high fidelity the effects of Wnt3a on the trafficking of Axin, Dvl2, and β-catenin. In response to Wnt3a, Axin initially traffics to PM, CY and NU fractions (fueled by its increase in cellular abundance) and OA alone mimics each of these effects of Wnt3a. OA treatment stimulated an initial rise in the amount of Dvl2 localized to PM and NU subcellular fractions. β-catenin levels increase markedly in CY and NU subcellular fractions in responseβ to OA.


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

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

Abundance of Axin, Dvl2 and β-catenin in response to Wnt3a: effects of okadaic acid. F9 cells expressing Rfz1 were pretreated with or without 40 nM OA for 1 hr and then stimulated with Wnt3a in the presence or absence of OA for the indicated time periods. Cells were harvested and fractionated to subcellular fractions highly enriched in plasma membrane (PM), cytoplasm (CY), or nuclei (NU), as described in the Methods. Each fraction was separated by SDS-PAGE and the resolved protein transferred to blots stained with anti-Axin, anti-Dvl2 or anti-β-catenin antibodies. The enrichment of the subcellular fractions was established by staining immunoblots with antibodies to well known protein markers for PM, CY, and NU subcellular fractions. Left-handed panel displays 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 the mean values "% of control" (i.e., t = 0 then set to 100%). Right-handed panel displays immunoblots. 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 5: Abundance of Axin, Dvl2 and β-catenin in response to Wnt3a: effects of okadaic acid. F9 cells expressing Rfz1 were pretreated with or without 40 nM OA for 1 hr and then stimulated with Wnt3a in the presence or absence of OA for the indicated time periods. Cells were harvested and fractionated to subcellular fractions highly enriched in plasma membrane (PM), cytoplasm (CY), or nuclei (NU), as described in the Methods. Each fraction was separated by SDS-PAGE and the resolved protein transferred to blots stained with anti-Axin, anti-Dvl2 or anti-β-catenin antibodies. The enrichment of the subcellular fractions was established by staining immunoblots with antibodies to well known protein markers for PM, CY, and NU subcellular fractions. Left-handed panel displays 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 the mean values "% of control" (i.e., t = 0 then set to 100%). Right-handed panel displays immunoblots. The results shown are derived from a single experiment, representative of two additional experiments.
Mentions: We investigated the effects of the PP2A suppression on the intracellular shuttling of these Wnt/β-catenin pathway signaling molecules. OA-treated cells were stimulated with Wnt3a for up to 2 hr and the cells disrupted and then subjected to subcellular fractionation. Plasma membrane- (PM), cytoplasm- (CY), and nuclei- (NU) enriched subcellular fractions were prepared and the distributions of Axin, Dvl2, and β-catenin quantified (fig. 5). Most notable, OA alone mimics with high fidelity the effects of Wnt3a on the trafficking of Axin, Dvl2, and β-catenin. In response to Wnt3a, Axin initially traffics to PM, CY and NU fractions (fueled by its increase in cellular abundance) and OA alone mimics each of these effects of Wnt3a. OA treatment stimulated an initial rise in the amount of Dvl2 localized to PM and NU subcellular fractions. β-catenin levels increase markedly in CY and NU subcellular fractions in responseβ to OA.

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