Limits...
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

Suppression of PP2A activity alters cellular abundant of Wnt/β-catenin signaling elements. F9 cells expressing Rfz1 were either pretreated with OA for 1 hr or treated with siRNA targeting PP2A C- subunit for 48 hr, or transfected to express SV40 small t antigen for 48 hr. Cells were washed with PBS twice and lysed. Cell lysates were subjected to SDS-PAGE and analyzed by immunoblotting, blots stained with anti-Axin, anti-β-catenin, anti-Dvl2, anti-GSK3β, anti-p-Ser (9)-GSK3β, anti-PP2A C or anti-GAPDH antibody. The relative amounts of the proteins in each fraction were established by densitometry, as described in Methods. The relative abundance of each signaling molecule in the untreated cell, whole-cell extract was set to "1". The results are shown as mean values ± S.E. from 8–10 independent experiments. Right-handed panel displays the representative immunoblots, stained for Axin, β-catenin, GSK3β and p-Ser (9)-GSK3β.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2211464&req=5

Figure 3: Suppression of PP2A activity alters cellular abundant of Wnt/β-catenin signaling elements. F9 cells expressing Rfz1 were either pretreated with OA for 1 hr or treated with siRNA targeting PP2A C- subunit for 48 hr, or transfected to express SV40 small t antigen for 48 hr. Cells were washed with PBS twice and lysed. Cell lysates were subjected to SDS-PAGE and analyzed by immunoblotting, blots stained with anti-Axin, anti-β-catenin, anti-Dvl2, anti-GSK3β, anti-p-Ser (9)-GSK3β, anti-PP2A C or anti-GAPDH antibody. The relative amounts of the proteins in each fraction were established by densitometry, as described in Methods. The relative abundance of each signaling molecule in the untreated cell, whole-cell extract was set to "1". The results are shown as mean values ± S.E. from 8–10 independent experiments. Right-handed panel displays the representative immunoblots, stained for Axin, β-catenin, GSK3β and p-Ser (9)-GSK3β.

Mentions: Making use of the three independent strategies to suppress PP2A action, we probed further the linkage between PP2A and cellular content of key signaling elements in the Wnt3a-stimulated canonical pathway (fig. 3). OA, siRNA targeting PP2A C-subunit, or expression small t antigen were tested individually for their influence on the cellular content of β-catenin, Dvl2, Axin, GSK3β, phospho-GSK3β, PP2A C-subunit, and GAPDH (as a control). In order to facilitate comparisons, the cellular content of each molecule in the untreated cells was set as "1"; changes in cellular abundance in response to each of the three treatments were quantified and are reported as "fold-"changes. β-catenin levels, increasing 50% by OA treatment (fig. 1C), were similarly enhanced by either siRNA treatment or expression of small t antigen (fig. 3). The abundance of Axin as well as that of phospho-GSK3β increased 2- to 4-fold by suppression of PP2A action, the increases being somewhat greater in response to OA than to the other treatments. Cellular abundance of Dvl2 and GSK3β, like that of the control GAPDH, largely were unaffected by suppression of PP2A action. The abundance of PP2A was unaffected by either OA treatment or small t antigen expression. Treatment of siRNA resulted in ~70% suppression of PP2A C-subunit expression. Thus, the abundance of the scaffold Axin, of phospho-GSK3β, and of β-catenin itself are subject to regulation by PP2A, i.e., suppressing PP2A action increases their cellular content while enhancing Wnt3a stimulation of the canonical pathway (figs. 1, 3).


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

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

Suppression of PP2A activity alters cellular abundant of Wnt/β-catenin signaling elements. F9 cells expressing Rfz1 were either pretreated with OA for 1 hr or treated with siRNA targeting PP2A C- subunit for 48 hr, or transfected to express SV40 small t antigen for 48 hr. Cells were washed with PBS twice and lysed. Cell lysates were subjected to SDS-PAGE and analyzed by immunoblotting, blots stained with anti-Axin, anti-β-catenin, anti-Dvl2, anti-GSK3β, anti-p-Ser (9)-GSK3β, anti-PP2A C or anti-GAPDH antibody. The relative amounts of the proteins in each fraction were established by densitometry, as described in Methods. The relative abundance of each signaling molecule in the untreated cell, whole-cell extract was set to "1". The results are shown as mean values ± S.E. from 8–10 independent experiments. Right-handed panel displays the representative immunoblots, stained for Axin, β-catenin, GSK3β and p-Ser (9)-GSK3β.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Suppression of PP2A activity alters cellular abundant of Wnt/β-catenin signaling elements. F9 cells expressing Rfz1 were either pretreated with OA for 1 hr or treated with siRNA targeting PP2A C- subunit for 48 hr, or transfected to express SV40 small t antigen for 48 hr. Cells were washed with PBS twice and lysed. Cell lysates were subjected to SDS-PAGE and analyzed by immunoblotting, blots stained with anti-Axin, anti-β-catenin, anti-Dvl2, anti-GSK3β, anti-p-Ser (9)-GSK3β, anti-PP2A C or anti-GAPDH antibody. The relative amounts of the proteins in each fraction were established by densitometry, as described in Methods. The relative abundance of each signaling molecule in the untreated cell, whole-cell extract was set to "1". The results are shown as mean values ± S.E. from 8–10 independent experiments. Right-handed panel displays the representative immunoblots, stained for Axin, β-catenin, GSK3β and p-Ser (9)-GSK3β.
Mentions: Making use of the three independent strategies to suppress PP2A action, we probed further the linkage between PP2A and cellular content of key signaling elements in the Wnt3a-stimulated canonical pathway (fig. 3). OA, siRNA targeting PP2A C-subunit, or expression small t antigen were tested individually for their influence on the cellular content of β-catenin, Dvl2, Axin, GSK3β, phospho-GSK3β, PP2A C-subunit, and GAPDH (as a control). In order to facilitate comparisons, the cellular content of each molecule in the untreated cells was set as "1"; changes in cellular abundance in response to each of the three treatments were quantified and are reported as "fold-"changes. β-catenin levels, increasing 50% by OA treatment (fig. 1C), were similarly enhanced by either siRNA treatment or expression of small t antigen (fig. 3). The abundance of Axin as well as that of phospho-GSK3β increased 2- to 4-fold by suppression of PP2A action, the increases being somewhat greater in response to OA than to the other treatments. Cellular abundance of Dvl2 and GSK3β, like that of the control GAPDH, largely were unaffected by suppression of PP2A action. The abundance of PP2A was unaffected by either OA treatment or small t antigen expression. Treatment of siRNA resulted in ~70% suppression of PP2A C-subunit expression. Thus, the abundance of the scaffold Axin, of phospho-GSK3β, and of β-catenin itself are subject to regulation by PP2A, i.e., suppressing PP2A action increases their cellular content while enhancing Wnt3a stimulation of the canonical pathway (figs. 1, 3).

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