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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

Suppression of PP2A potentiates the Wnt/β-catenin signaling. Panel A, OA enhanced the Lef/Tcf-sensitive transcription activity in response to Wnt3a. F9 cells expressing Rfz1 were treated with Wnt3a for 8 hr in the absence or presence of OA, added 60 min prior to stimulation with Wnt3a. Cells were lysed and the Lef/Tcf-sensitive gene transcription was assayed in samples of cell lysates using the M50 luciferase gene reporter. The results showed mean values ± S.E. that were obtained from five separate experiments. Statistical significance is noted (*, p < 0.001; **, p < 0.05). Panel B, OA enhanced the time-course of activation of Lef/Tcf-sensitive transcription in response to stimulation by Wnt3a. F9 cells expressing Rfz1 were treated without (open circle) or with (closed circle) OA and Wnt3a added for a 10 hr stimulation. Cells were harvested at indicated time points and disrupted. The Lef/Tcf-sensitive gene transcription was assayed. The results shown are mean values ± S.E. obtained from four separate experiments. Statistical significance is denoted (*, for p < 0.005). Panel C, effects of OA on the cellular abundance of Wnt/β-catenin signaling elements in response to long-term (0–10 hr) stimulation by Wnt3a. The cellular content of β-catenin, Dvl2, GSK3β, and GAPDH (as a control) were established in F9 cells expressing Rfz1 and stimulated with Wnt3a, in the absence or presence of OA, for 0–10 hr. The results shown are mean values ± S.E. obtained from four separate experiments. Representative blots are displayed.
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Figure 1: Suppression of PP2A potentiates the Wnt/β-catenin signaling. Panel A, OA enhanced the Lef/Tcf-sensitive transcription activity in response to Wnt3a. F9 cells expressing Rfz1 were treated with Wnt3a for 8 hr in the absence or presence of OA, added 60 min prior to stimulation with Wnt3a. Cells were lysed and the Lef/Tcf-sensitive gene transcription was assayed in samples of cell lysates using the M50 luciferase gene reporter. The results showed mean values ± S.E. that were obtained from five separate experiments. Statistical significance is noted (*, p < 0.001; **, p < 0.05). Panel B, OA enhanced the time-course of activation of Lef/Tcf-sensitive transcription in response to stimulation by Wnt3a. F9 cells expressing Rfz1 were treated without (open circle) or with (closed circle) OA and Wnt3a added for a 10 hr stimulation. Cells were harvested at indicated time points and disrupted. The Lef/Tcf-sensitive gene transcription was assayed. The results shown are mean values ± S.E. obtained from four separate experiments. Statistical significance is denoted (*, for p < 0.005). Panel C, effects of OA on the cellular abundance of Wnt/β-catenin signaling elements in response to long-term (0–10 hr) stimulation by Wnt3a. The cellular content of β-catenin, Dvl2, GSK3β, and GAPDH (as a control) were established in F9 cells expressing Rfz1 and stimulated with Wnt3a, in the absence or presence of OA, for 0–10 hr. The results shown are mean values ± S.E. obtained from four separate experiments. Representative blots are displayed.

Mentions: The mouse F9 teratocarcinoma cells (F9 cell) were transfected with an expression vector harboring rat Frizzled-1 (Rfz1) [19]. In previous studies, we demonstrated that these cells display both cytoplasmic and nuclear accumulation of β-catenin, activation of Lef/Tcf-sensitive gene transcription, and primitive endoderm formation in response to Wnt3a [19-22], providing an ideal model for the current study. The ability of PP2A to dephosphorylate several members of the Axin-based degradation complex in vitro and the central role of phosphorylation in the canonical Wnt/β-catenin pathway provoked our interest in probing its role in the trafficking of these signaling molecules. We first made use of okadaic acid (OA), a chemical inhibitor of serine/threonine phosphoprotein phosphatases (PP1 and PP2A), and probed its effects on Lef/Tcf-sensitive gene transcription in response to Wnt3a (figs. 1A, B). Basal activity of Lef/Tcf-sensitive gene transcription was unaffected by OA. Cells were treated with OA (30 nM) for 1 hr and then stimulated with purified Wnt3a (fig. 1A). OA elevated the Lef/Tcf-sensitive transcriptional response to Wnt3a from 25-fold to 45-fold, although not altering significantly the basal transcriptional response (fig. 1A). The ability of Wnt3a to stimulate Lef/Tcf-sensitive gene transcription in the absence or presence of OA was probed over time (fig. 1B). OA enhanced temporally the activation of Lef/Tcf-sensitive transcription in response to Wnt3a, half-maximal activation at 4 hr post Wnt3a; the amplitude of the response was not altered (fig. 1B).


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

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

Suppression of PP2A potentiates the Wnt/β-catenin signaling. Panel A, OA enhanced the Lef/Tcf-sensitive transcription activity in response to Wnt3a. F9 cells expressing Rfz1 were treated with Wnt3a for 8 hr in the absence or presence of OA, added 60 min prior to stimulation with Wnt3a. Cells were lysed and the Lef/Tcf-sensitive gene transcription was assayed in samples of cell lysates using the M50 luciferase gene reporter. The results showed mean values ± S.E. that were obtained from five separate experiments. Statistical significance is noted (*, p < 0.001; **, p < 0.05). Panel B, OA enhanced the time-course of activation of Lef/Tcf-sensitive transcription in response to stimulation by Wnt3a. F9 cells expressing Rfz1 were treated without (open circle) or with (closed circle) OA and Wnt3a added for a 10 hr stimulation. Cells were harvested at indicated time points and disrupted. The Lef/Tcf-sensitive gene transcription was assayed. The results shown are mean values ± S.E. obtained from four separate experiments. Statistical significance is denoted (*, for p < 0.005). Panel C, effects of OA on the cellular abundance of Wnt/β-catenin signaling elements in response to long-term (0–10 hr) stimulation by Wnt3a. The cellular content of β-catenin, Dvl2, GSK3β, and GAPDH (as a control) were established in F9 cells expressing Rfz1 and stimulated with Wnt3a, in the absence or presence of OA, for 0–10 hr. The results shown are mean values ± S.E. obtained from four separate experiments. Representative blots are displayed.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Suppression of PP2A potentiates the Wnt/β-catenin signaling. Panel A, OA enhanced the Lef/Tcf-sensitive transcription activity in response to Wnt3a. F9 cells expressing Rfz1 were treated with Wnt3a for 8 hr in the absence or presence of OA, added 60 min prior to stimulation with Wnt3a. Cells were lysed and the Lef/Tcf-sensitive gene transcription was assayed in samples of cell lysates using the M50 luciferase gene reporter. The results showed mean values ± S.E. that were obtained from five separate experiments. Statistical significance is noted (*, p < 0.001; **, p < 0.05). Panel B, OA enhanced the time-course of activation of Lef/Tcf-sensitive transcription in response to stimulation by Wnt3a. F9 cells expressing Rfz1 were treated without (open circle) or with (closed circle) OA and Wnt3a added for a 10 hr stimulation. Cells were harvested at indicated time points and disrupted. The Lef/Tcf-sensitive gene transcription was assayed. The results shown are mean values ± S.E. obtained from four separate experiments. Statistical significance is denoted (*, for p < 0.005). Panel C, effects of OA on the cellular abundance of Wnt/β-catenin signaling elements in response to long-term (0–10 hr) stimulation by Wnt3a. The cellular content of β-catenin, Dvl2, GSK3β, and GAPDH (as a control) were established in F9 cells expressing Rfz1 and stimulated with Wnt3a, in the absence or presence of OA, for 0–10 hr. The results shown are mean values ± S.E. obtained from four separate experiments. Representative blots are displayed.
Mentions: The mouse F9 teratocarcinoma cells (F9 cell) were transfected with an expression vector harboring rat Frizzled-1 (Rfz1) [19]. In previous studies, we demonstrated that these cells display both cytoplasmic and nuclear accumulation of β-catenin, activation of Lef/Tcf-sensitive gene transcription, and primitive endoderm formation in response to Wnt3a [19-22], providing an ideal model for the current study. The ability of PP2A to dephosphorylate several members of the Axin-based degradation complex in vitro and the central role of phosphorylation in the canonical Wnt/β-catenin pathway provoked our interest in probing its role in the trafficking of these signaling molecules. We first made use of okadaic acid (OA), a chemical inhibitor of serine/threonine phosphoprotein phosphatases (PP1 and PP2A), and probed its effects on Lef/Tcf-sensitive gene transcription in response to Wnt3a (figs. 1A, B). Basal activity of Lef/Tcf-sensitive gene transcription was unaffected by OA. Cells were treated with OA (30 nM) for 1 hr and then stimulated with purified Wnt3a (fig. 1A). OA elevated the Lef/Tcf-sensitive transcriptional response to Wnt3a from 25-fold to 45-fold, although not altering significantly the basal transcriptional response (fig. 1A). The ability of Wnt3a to stimulate Lef/Tcf-sensitive gene transcription in the absence or presence of OA was probed over time (fig. 1B). OA enhanced temporally the activation of Lef/Tcf-sensitive transcription in response to Wnt3a, half-maximal activation at 4 hr post Wnt3a; the amplitude of the response was not altered (fig. 1B).

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