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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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PP2A shuttles to the plasma membrane and nuclear subcellular fractions in response to Wnt3a. Cells expressing Rfz1 receptor were harvested at indicated time point after Wnt3a stimulation. Cells were fractionated to the plasma membrane (PM), cytoplasm (CY) and nuclei (NU), as described in the Methods. Subcelluar fractions obtained from control (time = 0) and Wnt3a-stimulated cells were analyzed by immunoblotting with anti-PP2A C-subunit antibody. PM (blue line), CY (pink line) and NU (green line) fractions are displayed. Stained protein bands were quantified and the values are presented as "fold of zero time point". Bottom three panels of immunoblots are stained with antibodies against the following subcellular fraction marker proteins: Na+-K+-ATPase (plasma membrane), GAPDH (cytoplasm) and fibrillarin (nuclei), respectively. The results are shown as mean values ± S.E. from 5 or more independent experiments. Representative blots are displayed, as is the quantitative analysis of cellular content extracted from a compilation of data from the separate experiments (graphs).
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Figure 7: PP2A shuttles to the plasma membrane and nuclear subcellular fractions in response to Wnt3a. Cells expressing Rfz1 receptor were harvested at indicated time point after Wnt3a stimulation. Cells were fractionated to the plasma membrane (PM), cytoplasm (CY) and nuclei (NU), as described in the Methods. Subcelluar fractions obtained from control (time = 0) and Wnt3a-stimulated cells were analyzed by immunoblotting with anti-PP2A C-subunit antibody. PM (blue line), CY (pink line) and NU (green line) fractions are displayed. Stained protein bands were quantified and the values are presented as "fold of zero time point". Bottom three panels of immunoblots are stained with antibodies against the following subcellular fraction marker proteins: Na+-K+-ATPase (plasma membrane), GAPDH (cytoplasm) and fibrillarin (nuclei), respectively. The results are shown as mean values ± S.E. from 5 or more independent experiments. Representative blots are displayed, as is the quantitative analysis of cellular content extracted from a compilation of data from the separate experiments (graphs).

Mentions: We wondered if PP2A itself was subject to trafficking in response to Wnt3a. Cells were treated without and with Wnt3a and then subjected to lysis and subcellular fractionation. Wnt3a stimulated rapid trafficking of and accumulation of PP2A in the PM and to a lesser extent in CY subcellular fractions, at the expense of the NU fraction (fig. 7). At 60 min post stimulation with Wnt3a, PP2A reverses course and begins to accumulate in the nucleus [much like Axin, β-catenin, and Dvl2 (fig. 5) and see additional file: Supplementary fig. 3A and 3B)]. PP2A localizes predominantly (>70%) in the cytosol; ~20% residing in the plasma membrane-enriched fraction, and the remaining <8%in the nuclear fraction of unstimulated F9 cells [20].


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

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

PP2A shuttles to the plasma membrane and nuclear subcellular fractions in response to Wnt3a. Cells expressing Rfz1 receptor were harvested at indicated time point after Wnt3a stimulation. Cells were fractionated to the plasma membrane (PM), cytoplasm (CY) and nuclei (NU), as described in the Methods. Subcelluar fractions obtained from control (time = 0) and Wnt3a-stimulated cells were analyzed by immunoblotting with anti-PP2A C-subunit antibody. PM (blue line), CY (pink line) and NU (green line) fractions are displayed. Stained protein bands were quantified and the values are presented as "fold of zero time point". Bottom three panels of immunoblots are stained with antibodies against the following subcellular fraction marker proteins: Na+-K+-ATPase (plasma membrane), GAPDH (cytoplasm) and fibrillarin (nuclei), respectively. The results are shown as mean values ± S.E. from 5 or more independent experiments. Representative blots are displayed, as is the quantitative analysis of cellular content extracted from a compilation of data from the separate experiments (graphs).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 7: PP2A shuttles to the plasma membrane and nuclear subcellular fractions in response to Wnt3a. Cells expressing Rfz1 receptor were harvested at indicated time point after Wnt3a stimulation. Cells were fractionated to the plasma membrane (PM), cytoplasm (CY) and nuclei (NU), as described in the Methods. Subcelluar fractions obtained from control (time = 0) and Wnt3a-stimulated cells were analyzed by immunoblotting with anti-PP2A C-subunit antibody. PM (blue line), CY (pink line) and NU (green line) fractions are displayed. Stained protein bands were quantified and the values are presented as "fold of zero time point". Bottom three panels of immunoblots are stained with antibodies against the following subcellular fraction marker proteins: Na+-K+-ATPase (plasma membrane), GAPDH (cytoplasm) and fibrillarin (nuclei), respectively. The results are shown as mean values ± S.E. from 5 or more independent experiments. Representative blots are displayed, as is the quantitative analysis of cellular content extracted from a compilation of data from the separate experiments (graphs).
Mentions: We wondered if PP2A itself was subject to trafficking in response to Wnt3a. Cells were treated without and with Wnt3a and then subjected to lysis and subcellular fractionation. Wnt3a stimulated rapid trafficking of and accumulation of PP2A in the PM and to a lesser extent in CY subcellular fractions, at the expense of the NU fraction (fig. 7). At 60 min post stimulation with Wnt3a, PP2A reverses course and begins to accumulate in the nucleus [much like Axin, β-catenin, and Dvl2 (fig. 5) and see additional file: Supplementary fig. 3A and 3B)]. PP2A localizes predominantly (>70%) in the cytosol; ~20% residing in the plasma membrane-enriched fraction, and the remaining <8%in the nuclear fraction of unstimulated F9 cells [20].

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