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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 activity by siRNA or small t antigen enhances Lef/Tcf-sensitive transcription. Panel A, Rfz1-expressing F9 cells were treated with OA for 1 hr or siRNA targeting PP2A C subunit for 48 hr, or co-expression of small t antigen for 48 hr. Cell lysates were applied to a small Sephadex-G50 column and PP2A activity assay was carried out using pNPP as a substrate. The results are shown as mean values ± S.E. from 8–10 independent experiments. Abundance of PP2A C subunit was determined by Western immunoblotting with anti-PP2A C subunit antibody. Panel B, cells were treated with siRNAs targeting the PP2A C-subunit for one day before co-transfection of the cells with Rfz1 and Super8xpTOPFlash plasmids. Cells were stimulated with or without Wnt 3a for 8 hr. The luciferase gene reporter was assayed and is displayed relative to the unstimulated cells (set to "1"). The results showed mean values ± S.E., obtained from five separate experiments. Statistical significance is indicated (*, p < 0.001; ***, p < 0.005). Cell extracts also were analyzed for abundance of PP2A C-subunit by immunoblotting. Immunoblots were stained with anti-GAPDH antibodies to establish loading equivalence.Panel C, activation of Lcf/Tcf-sensitive transcription was assayed in F9 cells co-transfected for one-day with Rfz1, Super8xTOPFlash (M50) and small t antigen then stimulated without and with purified Wnt3a for 8 hr. The luciferase gene reporter was assayed and the transcriptional response displayed relative to the unstimulated cells (set to "1"). Cell lysates were analyzed by immunoblotting, blots stained with anti-PP2A C-subunit, anti-GAPDH, or anti-small t antigen antibodies. The results shown are mean values ± S.E. from 5 independent experiments.
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Figure 2: Suppression of PP2A activity by siRNA or small t antigen enhances Lef/Tcf-sensitive transcription. Panel A, Rfz1-expressing F9 cells were treated with OA for 1 hr or siRNA targeting PP2A C subunit for 48 hr, or co-expression of small t antigen for 48 hr. Cell lysates were applied to a small Sephadex-G50 column and PP2A activity assay was carried out using pNPP as a substrate. The results are shown as mean values ± S.E. from 8–10 independent experiments. Abundance of PP2A C subunit was determined by Western immunoblotting with anti-PP2A C subunit antibody. Panel B, cells were treated with siRNAs targeting the PP2A C-subunit for one day before co-transfection of the cells with Rfz1 and Super8xpTOPFlash plasmids. Cells were stimulated with or without Wnt 3a for 8 hr. The luciferase gene reporter was assayed and is displayed relative to the unstimulated cells (set to "1"). The results showed mean values ± S.E., obtained from five separate experiments. Statistical significance is indicated (*, p < 0.001; ***, p < 0.005). Cell extracts also were analyzed for abundance of PP2A C-subunit by immunoblotting. Immunoblots were stained with anti-GAPDH antibodies to establish loading equivalence.Panel C, activation of Lcf/Tcf-sensitive transcription was assayed in F9 cells co-transfected for one-day with Rfz1, Super8xTOPFlash (M50) and small t antigen then stimulated without and with purified Wnt3a for 8 hr. The luciferase gene reporter was assayed and the transcriptional response displayed relative to the unstimulated cells (set to "1"). Cell lysates were analyzed by immunoblotting, blots stained with anti-PP2A C-subunit, anti-GAPDH, or anti-small t antigen antibodies. The results shown are mean values ± S.E. from 5 independent experiments.

Mentions: Two strategies in addition to OA treatment were tested to probe further the role of PP2A in Wnt3a stimulation of the Lef/Tcf-sensitive transcription. We compared the effects siRNA targeting the expression of the conserved catalytic or "C-"subunit of PP2A, the expression of SV40 small t antigen, inhibitor of PP2A, and the effects of OA (40 nM) treatment on PP2A activity (fig. 2A). OA reduces PP2A activity in this assay by >70%. Expression of small t antigen has been shown to inhibit PP2A activity [23,24] and transfection of F9 cells with an expression vector harboring the small t antigen reduced PP2A activity by ~70%. Treating the cells with siRNAs targeting the expression of PP2A C-subunit effectively suppresses C-subunit expression, reducing PP2A activity by ~90%.


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

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

Suppression of PP2A activity by siRNA or small t antigen enhances Lef/Tcf-sensitive transcription. Panel A, Rfz1-expressing F9 cells were treated with OA for 1 hr or siRNA targeting PP2A C subunit for 48 hr, or co-expression of small t antigen for 48 hr. Cell lysates were applied to a small Sephadex-G50 column and PP2A activity assay was carried out using pNPP as a substrate. The results are shown as mean values ± S.E. from 8–10 independent experiments. Abundance of PP2A C subunit was determined by Western immunoblotting with anti-PP2A C subunit antibody. Panel B, cells were treated with siRNAs targeting the PP2A C-subunit for one day before co-transfection of the cells with Rfz1 and Super8xpTOPFlash plasmids. Cells were stimulated with or without Wnt 3a for 8 hr. The luciferase gene reporter was assayed and is displayed relative to the unstimulated cells (set to "1"). The results showed mean values ± S.E., obtained from five separate experiments. Statistical significance is indicated (*, p < 0.001; ***, p < 0.005). Cell extracts also were analyzed for abundance of PP2A C-subunit by immunoblotting. Immunoblots were stained with anti-GAPDH antibodies to establish loading equivalence.Panel C, activation of Lcf/Tcf-sensitive transcription was assayed in F9 cells co-transfected for one-day with Rfz1, Super8xTOPFlash (M50) and small t antigen then stimulated without and with purified Wnt3a for 8 hr. The luciferase gene reporter was assayed and the transcriptional response displayed relative to the unstimulated cells (set to "1"). Cell lysates were analyzed by immunoblotting, blots stained with anti-PP2A C-subunit, anti-GAPDH, or anti-small t antigen antibodies. The results shown are mean values ± S.E. from 5 independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Suppression of PP2A activity by siRNA or small t antigen enhances Lef/Tcf-sensitive transcription. Panel A, Rfz1-expressing F9 cells were treated with OA for 1 hr or siRNA targeting PP2A C subunit for 48 hr, or co-expression of small t antigen for 48 hr. Cell lysates were applied to a small Sephadex-G50 column and PP2A activity assay was carried out using pNPP as a substrate. The results are shown as mean values ± S.E. from 8–10 independent experiments. Abundance of PP2A C subunit was determined by Western immunoblotting with anti-PP2A C subunit antibody. Panel B, cells were treated with siRNAs targeting the PP2A C-subunit for one day before co-transfection of the cells with Rfz1 and Super8xpTOPFlash plasmids. Cells were stimulated with or without Wnt 3a for 8 hr. The luciferase gene reporter was assayed and is displayed relative to the unstimulated cells (set to "1"). The results showed mean values ± S.E., obtained from five separate experiments. Statistical significance is indicated (*, p < 0.001; ***, p < 0.005). Cell extracts also were analyzed for abundance of PP2A C-subunit by immunoblotting. Immunoblots were stained with anti-GAPDH antibodies to establish loading equivalence.Panel C, activation of Lcf/Tcf-sensitive transcription was assayed in F9 cells co-transfected for one-day with Rfz1, Super8xTOPFlash (M50) and small t antigen then stimulated without and with purified Wnt3a for 8 hr. The luciferase gene reporter was assayed and the transcriptional response displayed relative to the unstimulated cells (set to "1"). Cell lysates were analyzed by immunoblotting, blots stained with anti-PP2A C-subunit, anti-GAPDH, or anti-small t antigen antibodies. The results shown are mean values ± S.E. from 5 independent experiments.
Mentions: Two strategies in addition to OA treatment were tested to probe further the role of PP2A in Wnt3a stimulation of the Lef/Tcf-sensitive transcription. We compared the effects siRNA targeting the expression of the conserved catalytic or "C-"subunit of PP2A, the expression of SV40 small t antigen, inhibitor of PP2A, and the effects of OA (40 nM) treatment on PP2A activity (fig. 2A). OA reduces PP2A activity in this assay by >70%. Expression of small t antigen has been shown to inhibit PP2A activity [23,24] and transfection of F9 cells with an expression vector harboring the small t antigen reduced PP2A activity by ~70%. Treating the cells with siRNAs targeting the expression of PP2A C-subunit effectively suppresses C-subunit expression, reducing PP2A activity by ~90%.

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