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Abundance, complexation, and trafficking of Wnt/beta-catenin signaling elements in response to Wnt3a.

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

Bottom Line: Subcellular localization of Axin in the absence of Wnt3a is symmetric, found evenly distributed among plasma membrane-, cytosol-, and nuclear-enriched fractions.Dishevelled-2, in contrast, is found predominately in the cytosol, whereas beta-catenin is localized to the plasma membrane-enriched fraction.We quantify, for the first time, the Wnt-dependent regulation of cellular abundance and intracellular trafficking of these signaling molecules.

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 regulates a canonical signaling pathway in early development that controls the nuclear accumulation of beta-catenin and its activation of Lef/Tcf-sensitive transcription of developmentally important genes.

Results: Using totipotent mouse F9 teratocarcinoma cells expressing Frizzled-1 and biochemical analyses, we detail the influence of Wnt3a stimulation on the expression, complexation, and subcellular trafficking of key signaling elements of the canonical pathway, i.e., Dishevelled-2, Axin, glycogen synthase kinase-3beta, and beta-catenin. Cellular content of beta-catenin and Axin, and phospho-glycogen synthase kinase-3beta, but not Dishevelled-2, increases in response to Wnt3a. Subcellular localization of Axin in the absence of Wnt3a is symmetric, found evenly distributed among plasma membrane-, cytosol-, and nuclear-enriched fractions. Dishevelled-2, in contrast, is found predominately in the cytosol, whereas beta-catenin is localized to the plasma membrane-enriched fraction. Wnt3a stimulates trafficking of Dishevelled-2, Axin, and glycogen synthase kinase-3beta initially to the plasma membrane, later to the nucleus. Bioluminescence resonance energy transfer measurements reveal that complexes of Axin with Dishevelled-2, with glycogen synthase kinase-3beta, and with beta-catenin are demonstrable and they remain relatively stable in response to Wnt3a stimulation, although trafficking has occurred. Mammalian Dishevelled-1 and Dishevelled-2 display similar patterns of trafficking in response to Wnt3a, whereas that of Dishevelled-3 differs from the other two.

Conclusion: This study provides a detailed biochemical analysis of signaling elements key to Wnt3a regulation of the canonical pathway. We quantify, for the first time, the Wnt-dependent regulation of cellular abundance and intracellular trafficking of these signaling molecules. In contrast, we observe little effect of Wnt3a stimulation on the level of protein-protein interactions among these constituents of Axin-based complexes themselves.

No MeSH data available.


Related in: MedlinePlus

Wnt3a stimulates changes in cellular content of Wnt/β-catenin pathway signaling components. Panel A, formation of PE was assayed in F9 cells expressing Rfz1 and stimulated without or with purified Wnt3a for 3 days. PE formation was assayed by measuring of expression of PE-marker, cytokeratin endo A, using immunoblotting and staining with TROMA-1 antibody. The results shown are mean values ± S.E. from 3 independent experiments. A representative immunoblot is shown and the mean values of the quantification displayed in the graph. Panel B, activation of Lcf/Tcf-sensitive transcription was assayed in F9 cells co-transfected for one-day with Rfz1 and Super8xTOPFlash (M50) or Super8xFOPFlash (M51) and then stimulated without and with purified Wnt3a for 8 hr. The luciferase gene reporter was assayed and is displayed relative to the unstimulated cells (set to 1). The results shown are mean values ± S.E. from 5 independent experiments. Panel C, cellular abundance of Axin, Dvl2, GSK3β, p-GSK3β, PP2A C, and β-catenin was assayed in F9 cells expressing Rfz1 and stimulated with Wnt3a for 0 to 90 min. Cells were harvested and lysed in a lysis buffer [50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA, 2 mM Na3VO4, 50 mM NaF, 1 % Triton X-100, 1 mM phenymethysulfonyfluoride (PMSF), 10 μg/ml leupeptin, and 10 μg/ml aprotinin]. Protein expression was established by SDS-PAGE, immunoblotting, and densitometric scans of the immune complexes. A representative blot is shown and the quantification of results is shown as mean values ± S.E. from 4 independent experiments.
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Figure 1: Wnt3a stimulates changes in cellular content of Wnt/β-catenin pathway signaling components. Panel A, formation of PE was assayed in F9 cells expressing Rfz1 and stimulated without or with purified Wnt3a for 3 days. PE formation was assayed by measuring of expression of PE-marker, cytokeratin endo A, using immunoblotting and staining with TROMA-1 antibody. The results shown are mean values ± S.E. from 3 independent experiments. A representative immunoblot is shown and the mean values of the quantification displayed in the graph. Panel B, activation of Lcf/Tcf-sensitive transcription was assayed in F9 cells co-transfected for one-day with Rfz1 and Super8xTOPFlash (M50) or Super8xFOPFlash (M51) and then stimulated without and with purified Wnt3a for 8 hr. The luciferase gene reporter was assayed and is displayed relative to the unstimulated cells (set to 1). The results shown are mean values ± S.E. from 5 independent experiments. Panel C, cellular abundance of Axin, Dvl2, GSK3β, p-GSK3β, PP2A C, and β-catenin was assayed in F9 cells expressing Rfz1 and stimulated with Wnt3a for 0 to 90 min. Cells were harvested and lysed in a lysis buffer [50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA, 2 mM Na3VO4, 50 mM NaF, 1 % Triton X-100, 1 mM phenymethysulfonyfluoride (PMSF), 10 μg/ml leupeptin, and 10 μg/ml aprotinin]. Protein expression was established by SDS-PAGE, immunoblotting, and densitometric scans of the immune complexes. A representative blot is shown and the quantification of results is shown as mean values ± S.E. from 4 independent experiments.

Mentions: Treating the F9 cells with purified Wnt3a stimulates the formation of primitive endoderm (fig. 1A). Primitive endoderm formation was observed within 3–6 days of treatment with Wnt3a, established by immunoblotting of the PE-specific marker, cytokeratin Endo-A. Staining of blots of whole-cell lysates of Wnt3a-stimulated F9 cells with the TROMA-1 monoclonal antibody to Endo-A reveals increased expression of Endo-A. Wnt3a provokes a 4-fold increase in the expression of the PE-marker. Activation of Lef/Tcf-sensitive transcription by β-catenin provides an earlier and robust read-out of the Wnt-stimulated canonical pathway [10]. We analyzed the activity of a Lef/Tcf-sensitive reporter gene, the M50 construct, which harbors multiple β-catenin binding sites in its promoter (fig. 1B). At 8 hr post stimulation with Wnt3a, a robust activation (~80 fold) of the M50 reporter gene was obvious. The activity of the control gene reporter M51, which lacks the β-catenin binding sites [20], was unaffected by treatment with Wnt3a. Activation of Lef/Tcf-sensitive transcription of F9 cells in response to Wnt3a could be detected as early as 4 hr; Wnt5a, in contrast, did not activate Lef/Tcf-sensitive transcription (results not shown).


Abundance, complexation, and trafficking of Wnt/beta-catenin signaling elements in response to Wnt3a.

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

Wnt3a stimulates changes in cellular content of Wnt/β-catenin pathway signaling components. Panel A, formation of PE was assayed in F9 cells expressing Rfz1 and stimulated without or with purified Wnt3a for 3 days. PE formation was assayed by measuring of expression of PE-marker, cytokeratin endo A, using immunoblotting and staining with TROMA-1 antibody. The results shown are mean values ± S.E. from 3 independent experiments. A representative immunoblot is shown and the mean values of the quantification displayed in the graph. Panel B, activation of Lcf/Tcf-sensitive transcription was assayed in F9 cells co-transfected for one-day with Rfz1 and Super8xTOPFlash (M50) or Super8xFOPFlash (M51) and then stimulated without and with purified Wnt3a for 8 hr. The luciferase gene reporter was assayed and is displayed relative to the unstimulated cells (set to 1). The results shown are mean values ± S.E. from 5 independent experiments. Panel C, cellular abundance of Axin, Dvl2, GSK3β, p-GSK3β, PP2A C, and β-catenin was assayed in F9 cells expressing Rfz1 and stimulated with Wnt3a for 0 to 90 min. Cells were harvested and lysed in a lysis buffer [50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA, 2 mM Na3VO4, 50 mM NaF, 1 % Triton X-100, 1 mM phenymethysulfonyfluoride (PMSF), 10 μg/ml leupeptin, and 10 μg/ml aprotinin]. Protein expression was established by SDS-PAGE, immunoblotting, and densitometric scans of the immune complexes. A representative blot is shown and the quantification of results is shown as mean values ± S.E. from 4 independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2211465&req=5

Figure 1: Wnt3a stimulates changes in cellular content of Wnt/β-catenin pathway signaling components. Panel A, formation of PE was assayed in F9 cells expressing Rfz1 and stimulated without or with purified Wnt3a for 3 days. PE formation was assayed by measuring of expression of PE-marker, cytokeratin endo A, using immunoblotting and staining with TROMA-1 antibody. The results shown are mean values ± S.E. from 3 independent experiments. A representative immunoblot is shown and the mean values of the quantification displayed in the graph. Panel B, activation of Lcf/Tcf-sensitive transcription was assayed in F9 cells co-transfected for one-day with Rfz1 and Super8xTOPFlash (M50) or Super8xFOPFlash (M51) and then stimulated without and with purified Wnt3a for 8 hr. The luciferase gene reporter was assayed and is displayed relative to the unstimulated cells (set to 1). The results shown are mean values ± S.E. from 5 independent experiments. Panel C, cellular abundance of Axin, Dvl2, GSK3β, p-GSK3β, PP2A C, and β-catenin was assayed in F9 cells expressing Rfz1 and stimulated with Wnt3a for 0 to 90 min. Cells were harvested and lysed in a lysis buffer [50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA, 2 mM Na3VO4, 50 mM NaF, 1 % Triton X-100, 1 mM phenymethysulfonyfluoride (PMSF), 10 μg/ml leupeptin, and 10 μg/ml aprotinin]. Protein expression was established by SDS-PAGE, immunoblotting, and densitometric scans of the immune complexes. A representative blot is shown and the quantification of results is shown as mean values ± S.E. from 4 independent experiments.
Mentions: Treating the F9 cells with purified Wnt3a stimulates the formation of primitive endoderm (fig. 1A). Primitive endoderm formation was observed within 3–6 days of treatment with Wnt3a, established by immunoblotting of the PE-specific marker, cytokeratin Endo-A. Staining of blots of whole-cell lysates of Wnt3a-stimulated F9 cells with the TROMA-1 monoclonal antibody to Endo-A reveals increased expression of Endo-A. Wnt3a provokes a 4-fold increase in the expression of the PE-marker. Activation of Lef/Tcf-sensitive transcription by β-catenin provides an earlier and robust read-out of the Wnt-stimulated canonical pathway [10]. We analyzed the activity of a Lef/Tcf-sensitive reporter gene, the M50 construct, which harbors multiple β-catenin binding sites in its promoter (fig. 1B). At 8 hr post stimulation with Wnt3a, a robust activation (~80 fold) of the M50 reporter gene was obvious. The activity of the control gene reporter M51, which lacks the β-catenin binding sites [20], was unaffected by treatment with Wnt3a. Activation of Lef/Tcf-sensitive transcription of F9 cells in response to Wnt3a could be detected as early as 4 hr; Wnt5a, in contrast, did not activate Lef/Tcf-sensitive transcription (results not shown).

Bottom Line: Subcellular localization of Axin in the absence of Wnt3a is symmetric, found evenly distributed among plasma membrane-, cytosol-, and nuclear-enriched fractions.Dishevelled-2, in contrast, is found predominately in the cytosol, whereas beta-catenin is localized to the plasma membrane-enriched fraction.We quantify, for the first time, the Wnt-dependent regulation of cellular abundance and intracellular trafficking of these signaling molecules.

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 regulates a canonical signaling pathway in early development that controls the nuclear accumulation of beta-catenin and its activation of Lef/Tcf-sensitive transcription of developmentally important genes.

Results: Using totipotent mouse F9 teratocarcinoma cells expressing Frizzled-1 and biochemical analyses, we detail the influence of Wnt3a stimulation on the expression, complexation, and subcellular trafficking of key signaling elements of the canonical pathway, i.e., Dishevelled-2, Axin, glycogen synthase kinase-3beta, and beta-catenin. Cellular content of beta-catenin and Axin, and phospho-glycogen synthase kinase-3beta, but not Dishevelled-2, increases in response to Wnt3a. Subcellular localization of Axin in the absence of Wnt3a is symmetric, found evenly distributed among plasma membrane-, cytosol-, and nuclear-enriched fractions. Dishevelled-2, in contrast, is found predominately in the cytosol, whereas beta-catenin is localized to the plasma membrane-enriched fraction. Wnt3a stimulates trafficking of Dishevelled-2, Axin, and glycogen synthase kinase-3beta initially to the plasma membrane, later to the nucleus. Bioluminescence resonance energy transfer measurements reveal that complexes of Axin with Dishevelled-2, with glycogen synthase kinase-3beta, and with beta-catenin are demonstrable and they remain relatively stable in response to Wnt3a stimulation, although trafficking has occurred. Mammalian Dishevelled-1 and Dishevelled-2 display similar patterns of trafficking in response to Wnt3a, whereas that of Dishevelled-3 differs from the other two.

Conclusion: This study provides a detailed biochemical analysis of signaling elements key to Wnt3a regulation of the canonical pathway. We quantify, for the first time, the Wnt-dependent regulation of cellular abundance and intracellular trafficking of these signaling molecules. In contrast, we observe little effect of Wnt3a stimulation on the level of protein-protein interactions among these constituents of Axin-based complexes themselves.

No MeSH data available.


Related in: MedlinePlus