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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

PP2A associates directly with Dvl2 and dephosphorylates Dvl2. Panel A, probing interaction of Dvl2 and PP2A in vivo. Whole-cell extracts (2 mg) prepared from F9 cells expressing Rfz1 were incubated with immobilized GST gel alone or with GST-PP2A C-subunit-immobilized gel. Bound proteins were released and resolved by SDS-PAGE. The resolved proteins were subjected to blotting and stained with either with anti-PP2A C subunit antibodies (top panel) or anti-Dvl2 antibodies (bottom panel). Panel B, pull-downs of Dvl2 from F9 cells reveal associated PP2A. F9 cells lysates were subjected to analysis by pull-downs of either Dvl2 or of mouse IgG (as a control). The immune precipitates were subjected to SDS-PAGE, immunoblotting, and staining with either anti-Dvl2 antibodies (top panel) or anti-PP2A C-subunit antibodies (bottom panel). Panel C, Dvl2 domains structure. Panel D, direct association of Dvl2 and PP2A in vitro. Purified mouse PP2A enzyme (PP2A, AC subunit dimers) was incubated with one of four domains of Dvl2 engineered as a fusion protein with GST and then immobilized: immobilized GST-PDZ, GST-DIX, GST-DEP, GST-SH3 and GST itself (as a control) at 4°C for 1 hr. The bound proteins were separated by SDS-PAGE, blotted, and stained with either anti-PP2A C-subunit antibodies or anti-GST antibodies. Panel E, PP2A dephosphorylates phospho-rDvl2. 6-Histidinyl-tagged phosphorylated Dvl2 was expressed in Sf9 cell and purified by Ni-NTA column chromatography as described in Methods. Purified phospho-rDvl2 protein was incubated with either purified calf alkaline phosphatase or with purified PP2A for 1.5 hr. The incubation was terminated by addition of SDS-PAGE sample buffer. The samples were subjected to SDS-PAGE, blotted, and stained with either anti-phospho-Ser antibodies (top of panel) or with anti-Dvl2 antibodies (bottom of panel). The immune complexes were made visible by use of an alkaline phosphatase-conjugated, second antibody and BCIP/NBT as substrates. The results shown are from a single experiment, duplicated with essentially similar results.
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Figure 8: PP2A associates directly with Dvl2 and dephosphorylates Dvl2. Panel A, probing interaction of Dvl2 and PP2A in vivo. Whole-cell extracts (2 mg) prepared from F9 cells expressing Rfz1 were incubated with immobilized GST gel alone or with GST-PP2A C-subunit-immobilized gel. Bound proteins were released and resolved by SDS-PAGE. The resolved proteins were subjected to blotting and stained with either with anti-PP2A C subunit antibodies (top panel) or anti-Dvl2 antibodies (bottom panel). Panel B, pull-downs of Dvl2 from F9 cells reveal associated PP2A. F9 cells lysates were subjected to analysis by pull-downs of either Dvl2 or of mouse IgG (as a control). The immune precipitates were subjected to SDS-PAGE, immunoblotting, and staining with either anti-Dvl2 antibodies (top panel) or anti-PP2A C-subunit antibodies (bottom panel). Panel C, Dvl2 domains structure. Panel D, direct association of Dvl2 and PP2A in vitro. Purified mouse PP2A enzyme (PP2A, AC subunit dimers) was incubated with one of four domains of Dvl2 engineered as a fusion protein with GST and then immobilized: immobilized GST-PDZ, GST-DIX, GST-DEP, GST-SH3 and GST itself (as a control) at 4°C for 1 hr. The bound proteins were separated by SDS-PAGE, blotted, and stained with either anti-PP2A C-subunit antibodies or anti-GST antibodies. Panel E, PP2A dephosphorylates phospho-rDvl2. 6-Histidinyl-tagged phosphorylated Dvl2 was expressed in Sf9 cell and purified by Ni-NTA column chromatography as described in Methods. Purified phospho-rDvl2 protein was incubated with either purified calf alkaline phosphatase or with purified PP2A for 1.5 hr. The incubation was terminated by addition of SDS-PAGE sample buffer. The samples were subjected to SDS-PAGE, blotted, and stained with either anti-phospho-Ser antibodies (top of panel) or with anti-Dvl2 antibodies (bottom of panel). The immune complexes were made visible by use of an alkaline phosphatase-conjugated, second antibody and BCIP/NBT as substrates. The results shown are from a single experiment, duplicated with essentially similar results.

Mentions: The ability of PP2A inhibition to influence the Wnt/β-catenin pathway and the trafficking of PP2A in response to Wnt3a stimulated us to hypothesize that this phosphoprotein phosphatase may interact directly with Dvl2. PP2A could well associate with Dvl2 directly or indirectly via Axin, APC, or some other unidentified protein. To test the hypothesis that PP2A binds directly to Dvl2, whole-cell lysates were subjected to pull-downs with either a fusion protein of PP2A C-subunit with glutathione-S-transferase (GST) immobilized to glutathione gel beads or GST itself immobilized to gel beads, as a control (fig. 8A). Pull-downs with PP2A C-subunit-GST beads, but not GST beads alone, reveal bound Dvl2, as evidenced by immunoblotting of the pull-down complexes. Pull-downs targeting the Dvl2 rather than PP2A were conducted and the immunoblotting confirmed the presence of PP2A in the Dvl2-based pull-downs (fig. 8B).


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

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

PP2A associates directly with Dvl2 and dephosphorylates Dvl2. Panel A, probing interaction of Dvl2 and PP2A in vivo. Whole-cell extracts (2 mg) prepared from F9 cells expressing Rfz1 were incubated with immobilized GST gel alone or with GST-PP2A C-subunit-immobilized gel. Bound proteins were released and resolved by SDS-PAGE. The resolved proteins were subjected to blotting and stained with either with anti-PP2A C subunit antibodies (top panel) or anti-Dvl2 antibodies (bottom panel). Panel B, pull-downs of Dvl2 from F9 cells reveal associated PP2A. F9 cells lysates were subjected to analysis by pull-downs of either Dvl2 or of mouse IgG (as a control). The immune precipitates were subjected to SDS-PAGE, immunoblotting, and staining with either anti-Dvl2 antibodies (top panel) or anti-PP2A C-subunit antibodies (bottom panel). Panel C, Dvl2 domains structure. Panel D, direct association of Dvl2 and PP2A in vitro. Purified mouse PP2A enzyme (PP2A, AC subunit dimers) was incubated with one of four domains of Dvl2 engineered as a fusion protein with GST and then immobilized: immobilized GST-PDZ, GST-DIX, GST-DEP, GST-SH3 and GST itself (as a control) at 4°C for 1 hr. The bound proteins were separated by SDS-PAGE, blotted, and stained with either anti-PP2A C-subunit antibodies or anti-GST antibodies. Panel E, PP2A dephosphorylates phospho-rDvl2. 6-Histidinyl-tagged phosphorylated Dvl2 was expressed in Sf9 cell and purified by Ni-NTA column chromatography as described in Methods. Purified phospho-rDvl2 protein was incubated with either purified calf alkaline phosphatase or with purified PP2A for 1.5 hr. The incubation was terminated by addition of SDS-PAGE sample buffer. The samples were subjected to SDS-PAGE, blotted, and stained with either anti-phospho-Ser antibodies (top of panel) or with anti-Dvl2 antibodies (bottom of panel). The immune complexes were made visible by use of an alkaline phosphatase-conjugated, second antibody and BCIP/NBT as substrates. The results shown are from a single experiment, duplicated with essentially similar results.
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Related In: Results  -  Collection

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Figure 8: PP2A associates directly with Dvl2 and dephosphorylates Dvl2. Panel A, probing interaction of Dvl2 and PP2A in vivo. Whole-cell extracts (2 mg) prepared from F9 cells expressing Rfz1 were incubated with immobilized GST gel alone or with GST-PP2A C-subunit-immobilized gel. Bound proteins were released and resolved by SDS-PAGE. The resolved proteins were subjected to blotting and stained with either with anti-PP2A C subunit antibodies (top panel) or anti-Dvl2 antibodies (bottom panel). Panel B, pull-downs of Dvl2 from F9 cells reveal associated PP2A. F9 cells lysates were subjected to analysis by pull-downs of either Dvl2 or of mouse IgG (as a control). The immune precipitates were subjected to SDS-PAGE, immunoblotting, and staining with either anti-Dvl2 antibodies (top panel) or anti-PP2A C-subunit antibodies (bottom panel). Panel C, Dvl2 domains structure. Panel D, direct association of Dvl2 and PP2A in vitro. Purified mouse PP2A enzyme (PP2A, AC subunit dimers) was incubated with one of four domains of Dvl2 engineered as a fusion protein with GST and then immobilized: immobilized GST-PDZ, GST-DIX, GST-DEP, GST-SH3 and GST itself (as a control) at 4°C for 1 hr. The bound proteins were separated by SDS-PAGE, blotted, and stained with either anti-PP2A C-subunit antibodies or anti-GST antibodies. Panel E, PP2A dephosphorylates phospho-rDvl2. 6-Histidinyl-tagged phosphorylated Dvl2 was expressed in Sf9 cell and purified by Ni-NTA column chromatography as described in Methods. Purified phospho-rDvl2 protein was incubated with either purified calf alkaline phosphatase or with purified PP2A for 1.5 hr. The incubation was terminated by addition of SDS-PAGE sample buffer. The samples were subjected to SDS-PAGE, blotted, and stained with either anti-phospho-Ser antibodies (top of panel) or with anti-Dvl2 antibodies (bottom of panel). The immune complexes were made visible by use of an alkaline phosphatase-conjugated, second antibody and BCIP/NBT as substrates. The results shown are from a single experiment, duplicated with essentially similar results.
Mentions: The ability of PP2A inhibition to influence the Wnt/β-catenin pathway and the trafficking of PP2A in response to Wnt3a stimulated us to hypothesize that this phosphoprotein phosphatase may interact directly with Dvl2. PP2A could well associate with Dvl2 directly or indirectly via Axin, APC, or some other unidentified protein. To test the hypothesis that PP2A binds directly to Dvl2, whole-cell lysates were subjected to pull-downs with either a fusion protein of PP2A C-subunit with glutathione-S-transferase (GST) immobilized to glutathione gel beads or GST itself immobilized to gel beads, as a control (fig. 8A). Pull-downs with PP2A C-subunit-GST beads, but not GST beads alone, reveal bound Dvl2, as evidenced by immunoblotting of the pull-down complexes. Pull-downs targeting the Dvl2 rather than PP2A were conducted and the immunoblotting confirmed the presence of PP2A in the Dvl2-based pull-downs (fig. 8B).

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