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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

Effects of inhibition of PP2A on cellular abundance of Wnt/β-catenin signaling elements. Graphs display the relative cellular abundance of Wnt/β-catenin signaling elements in cells following stimulation with Wnt3a. F9 cells expressing Rfz1 were untreated or treated with Wnt3a in the presence or absence of OA for 0 to 90 min. For OA treatment, F9 cells expressing Rfz1 were treated with OA for 1 hr prior to Wnt3a stimulation. For small t antigen experiments, F9 cells were transiently co-transfected with Rfz1 and small t antigen, and then stimulated with Wnt3a for the indicated times. Cells were collected and lysed. Lysates (60–100 μg protein) were subjected to SDS-PAGE and analyzed by immunoblotting, blots stained with antibodies targeting the signaling molecules indicated. Bands were quantified by densitometry, as described in Experimental Procedure and values are displayed as "fold", with time = 0 set as "1". The results are shown as mean values ± S.E. from 6–8 independent experiments. Representative blots are displayed, as is the quantitative analysis of cellular content extracted from 6–8 separate experiments (graphs).
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Figure 4: Effects of inhibition of PP2A on cellular abundance of Wnt/β-catenin signaling elements. Graphs display the relative cellular abundance of Wnt/β-catenin signaling elements in cells following stimulation with Wnt3a. F9 cells expressing Rfz1 were untreated or treated with Wnt3a in the presence or absence of OA for 0 to 90 min. For OA treatment, F9 cells expressing Rfz1 were treated with OA for 1 hr prior to Wnt3a stimulation. For small t antigen experiments, F9 cells were transiently co-transfected with Rfz1 and small t antigen, and then stimulated with Wnt3a for the indicated times. Cells were collected and lysed. Lysates (60–100 μg protein) were subjected to SDS-PAGE and analyzed by immunoblotting, blots stained with antibodies targeting the signaling molecules indicated. Bands were quantified by densitometry, as described in Experimental Procedure and values are displayed as "fold", with time = 0 set as "1". The results are shown as mean values ± S.E. from 6–8 independent experiments. Representative blots are displayed, as is the quantitative analysis of cellular content extracted from 6–8 separate experiments (graphs).

Mentions: We investigated if PP2A also regulated Wnt3a-induced changes in cellular abundance of Dvl2, Axin, GSK3β, phospho-GSK3β, β-catenin, and GAPDH (as a control, fig. 4). Although having little influence on the abundance of Dvl2, GSK3β, PP2A, and the control GAPDH, Wnt3a stimulated a progressive increase in the cellular content of β-catenin (2-fold), phospho-GSK3β (2–3-fold), and Axin (3-fold) over 90 min. OA treatment as well as expression of small t antigen mimicked the effects of Wnt3a, increasing the expression of Axin (3- to 6-fold), phospho-GSK3β (4- to 5-fold) and β-catenin (2- to 3-fold). Thus, suppression of PP2A by any one of the strategies alone appears to mimic Wnt3a action, stabilizing β-catenin and phospho-GSK3β levels, while transiently increasing, then decreasing the cellular content of Axin and Dvl2 (fig. 4). PP2A levels increase modestly in response to either Wnt3a or suppression of PP2A. These data show that cellular content of Axin, phospho-GSK3β and β-catenin each reflect an inhibitory control of PP2A which can be reversed by suppression of PP2A action, thereby mimicking aspects of Wnt3a action.


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

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

Effects of inhibition of PP2A on cellular abundance of Wnt/β-catenin signaling elements. Graphs display the relative cellular abundance of Wnt/β-catenin signaling elements in cells following stimulation with Wnt3a. F9 cells expressing Rfz1 were untreated or treated with Wnt3a in the presence or absence of OA for 0 to 90 min. For OA treatment, F9 cells expressing Rfz1 were treated with OA for 1 hr prior to Wnt3a stimulation. For small t antigen experiments, F9 cells were transiently co-transfected with Rfz1 and small t antigen, and then stimulated with Wnt3a for the indicated times. Cells were collected and lysed. Lysates (60–100 μg protein) were subjected to SDS-PAGE and analyzed by immunoblotting, blots stained with antibodies targeting the signaling molecules indicated. Bands were quantified by densitometry, as described in Experimental Procedure and values are displayed as "fold", with time = 0 set as "1". The results are shown as mean values ± S.E. from 6–8 independent experiments. Representative blots are displayed, as is the quantitative analysis of cellular content extracted from 6–8 separate experiments (graphs).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 4: Effects of inhibition of PP2A on cellular abundance of Wnt/β-catenin signaling elements. Graphs display the relative cellular abundance of Wnt/β-catenin signaling elements in cells following stimulation with Wnt3a. F9 cells expressing Rfz1 were untreated or treated with Wnt3a in the presence or absence of OA for 0 to 90 min. For OA treatment, F9 cells expressing Rfz1 were treated with OA for 1 hr prior to Wnt3a stimulation. For small t antigen experiments, F9 cells were transiently co-transfected with Rfz1 and small t antigen, and then stimulated with Wnt3a for the indicated times. Cells were collected and lysed. Lysates (60–100 μg protein) were subjected to SDS-PAGE and analyzed by immunoblotting, blots stained with antibodies targeting the signaling molecules indicated. Bands were quantified by densitometry, as described in Experimental Procedure and values are displayed as "fold", with time = 0 set as "1". The results are shown as mean values ± S.E. from 6–8 independent experiments. Representative blots are displayed, as is the quantitative analysis of cellular content extracted from 6–8 separate experiments (graphs).
Mentions: We investigated if PP2A also regulated Wnt3a-induced changes in cellular abundance of Dvl2, Axin, GSK3β, phospho-GSK3β, β-catenin, and GAPDH (as a control, fig. 4). Although having little influence on the abundance of Dvl2, GSK3β, PP2A, and the control GAPDH, Wnt3a stimulated a progressive increase in the cellular content of β-catenin (2-fold), phospho-GSK3β (2–3-fold), and Axin (3-fold) over 90 min. OA treatment as well as expression of small t antigen mimicked the effects of Wnt3a, increasing the expression of Axin (3- to 6-fold), phospho-GSK3β (4- to 5-fold) and β-catenin (2- to 3-fold). Thus, suppression of PP2A by any one of the strategies alone appears to mimic Wnt3a action, stabilizing β-catenin and phospho-GSK3β levels, while transiently increasing, then decreasing the cellular content of Axin and Dvl2 (fig. 4). PP2A levels increase modestly in response to either Wnt3a or suppression of PP2A. These data show that cellular content of Axin, phospho-GSK3β and β-catenin each reflect an inhibitory control of PP2A which can be reversed by suppression of PP2A action, thereby mimicking aspects of Wnt3a action.

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