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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

Differential effects of Wnt3a stimulation on Dvl1, Dvl2, and Dvl3. Panel A, the comparison of the cellular abundance of mammalian Dvl isoforms (Dvl1, Dvl2, and Dvl3) in subcellular fractions. F9 cells expressing Rfz1 were used to prepare subcellular fractions. Plasma membrane-enriched (PM), cytosol-enriched (CY), and nuclear-enriched (NU) fractions were prepared as described in detail in Methods and are displayed. The results of multiple densitometric scans of immunoblots were quantified. The results are displayed based upon the distribution (%), the total protein content of the whole-cell homogenate and fractions, and quantified analysis of blots from SDS-PAGE for each molecule and various controls for sample loading and blotting. The results are shown as mean values ± S.E. from 6–8 independent experiments. Panel B, the comparison of the cellular abundance of Dvls in response to Wnt3a. F9 cells expressing Rfz1 were stimulated with purified Wnt3a for indicated time and then disrupted 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]. Abundance of each Dvl isoform was determined by staining of blots with specific antibodies against each Dvl. Fractions were also stained with anti-GAPDH antibody to establish loading equivalence. Values are displayed as ''fold'' of zero-time point (set to 1). A representative blot is shown and the quantification of results is shown as mean values ± S.E. from 6 independent experiments. Panel C, F9 cells expressing Rfz1 receptor were stimulated without or with Wnt3a. Cells were harvested at each time point indicated, cell lysates were fractionated into plasma membrane (PM), cytoplasm (CY) and nuclei (NU) fractions. Quantified immunoblotting was performed as described in Methods. Each fraction (100 μg) was subjected to SDS-PAGE and the resolved proteins analyzed by immunoblotting with anti-Dvl1-, anti-Dvl2- and anti-Dvl3-specific antibodies. The three panels displayed at the bottom of the immunoblot set show blots stained with antibodies to well known subcellular marker proteins: Na+-K+-ATPase (plasma membrane), GAPDH (cytoplasm) and fibrillarin (nuclei), respectively. Representative blots are shown (top panel). Summary of quantified analysis of blots from SDS-PAGE are shown (bottom panel). The results are shown as mean values ± S.E. from 4–6 independent experiments. Dvl1 (blue line), Dvl2 (pink line) and Dvl3 (green line) are displayed.
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Figure 5: Differential effects of Wnt3a stimulation on Dvl1, Dvl2, and Dvl3. Panel A, the comparison of the cellular abundance of mammalian Dvl isoforms (Dvl1, Dvl2, and Dvl3) in subcellular fractions. F9 cells expressing Rfz1 were used to prepare subcellular fractions. Plasma membrane-enriched (PM), cytosol-enriched (CY), and nuclear-enriched (NU) fractions were prepared as described in detail in Methods and are displayed. The results of multiple densitometric scans of immunoblots were quantified. The results are displayed based upon the distribution (%), the total protein content of the whole-cell homogenate and fractions, and quantified analysis of blots from SDS-PAGE for each molecule and various controls for sample loading and blotting. The results are shown as mean values ± S.E. from 6–8 independent experiments. Panel B, the comparison of the cellular abundance of Dvls in response to Wnt3a. F9 cells expressing Rfz1 were stimulated with purified Wnt3a for indicated time and then disrupted 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]. Abundance of each Dvl isoform was determined by staining of blots with specific antibodies against each Dvl. Fractions were also stained with anti-GAPDH antibody to establish loading equivalence. Values are displayed as ''fold'' of zero-time point (set to 1). A representative blot is shown and the quantification of results is shown as mean values ± S.E. from 6 independent experiments. Panel C, F9 cells expressing Rfz1 receptor were stimulated without or with Wnt3a. Cells were harvested at each time point indicated, cell lysates were fractionated into plasma membrane (PM), cytoplasm (CY) and nuclei (NU) fractions. Quantified immunoblotting was performed as described in Methods. Each fraction (100 μg) was subjected to SDS-PAGE and the resolved proteins analyzed by immunoblotting with anti-Dvl1-, anti-Dvl2- and anti-Dvl3-specific antibodies. The three panels displayed at the bottom of the immunoblot set show blots stained with antibodies to well known subcellular marker proteins: Na+-K+-ATPase (plasma membrane), GAPDH (cytoplasm) and fibrillarin (nuclei), respectively. Representative blots are shown (top panel). Summary of quantified analysis of blots from SDS-PAGE are shown (bottom panel). The results are shown as mean values ± S.E. from 4–6 independent experiments. Dvl1 (blue line), Dvl2 (pink line) and Dvl3 (green line) are displayed.

Mentions: Unlike the fly which expresses a single Dishevelled (i.e., Dsh), mammalian cells can express three highly homologous isoforms of Dishevelled, termed Dvl1, Dvl2 and Dvl3 [41]. Dvl2 is the most abundant Dvl in mouse F9 cells (results not shown). We explored the cellular distribution of each mammalian Dvl expressed in F9 cells (fig. 5). All three Dvls were expressed in mouse F9 cells, as determined by immunoblotting with isoform-specific antibodies (fig 5A). The cellular content of Dvl1 and Dvl2 among the three subcellular fractions was nearly identical. The bulk of Dvl1 and of Dvl2 (> 80%) is found in the cytosol-enriched fraction, the least amount of each of these isoforms (> 5%) is observed in the nuclear-enriched fractions. Notable differences for the subcellular content and distribution of Dvl3 as compare to either Dvl1 or to Dvl2 were observed. Dvl3 content in the cytosol-enriched fractions is markedly reduced (~30%) in comparison to the content of either Dvl1 or Dvl2. More importantly, the content of Dvl3 in the nuclear-enriched fraction of unstimulated F9 cells is several folds greater than that for either Dvl1 or Dvl2 (fig 5A).


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

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

Differential effects of Wnt3a stimulation on Dvl1, Dvl2, and Dvl3. Panel A, the comparison of the cellular abundance of mammalian Dvl isoforms (Dvl1, Dvl2, and Dvl3) in subcellular fractions. F9 cells expressing Rfz1 were used to prepare subcellular fractions. Plasma membrane-enriched (PM), cytosol-enriched (CY), and nuclear-enriched (NU) fractions were prepared as described in detail in Methods and are displayed. The results of multiple densitometric scans of immunoblots were quantified. The results are displayed based upon the distribution (%), the total protein content of the whole-cell homogenate and fractions, and quantified analysis of blots from SDS-PAGE for each molecule and various controls for sample loading and blotting. The results are shown as mean values ± S.E. from 6–8 independent experiments. Panel B, the comparison of the cellular abundance of Dvls in response to Wnt3a. F9 cells expressing Rfz1 were stimulated with purified Wnt3a for indicated time and then disrupted 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]. Abundance of each Dvl isoform was determined by staining of blots with specific antibodies against each Dvl. Fractions were also stained with anti-GAPDH antibody to establish loading equivalence. Values are displayed as ''fold'' of zero-time point (set to 1). A representative blot is shown and the quantification of results is shown as mean values ± S.E. from 6 independent experiments. Panel C, F9 cells expressing Rfz1 receptor were stimulated without or with Wnt3a. Cells were harvested at each time point indicated, cell lysates were fractionated into plasma membrane (PM), cytoplasm (CY) and nuclei (NU) fractions. Quantified immunoblotting was performed as described in Methods. Each fraction (100 μg) was subjected to SDS-PAGE and the resolved proteins analyzed by immunoblotting with anti-Dvl1-, anti-Dvl2- and anti-Dvl3-specific antibodies. The three panels displayed at the bottom of the immunoblot set show blots stained with antibodies to well known subcellular marker proteins: Na+-K+-ATPase (plasma membrane), GAPDH (cytoplasm) and fibrillarin (nuclei), respectively. Representative blots are shown (top panel). Summary of quantified analysis of blots from SDS-PAGE are shown (bottom panel). The results are shown as mean values ± S.E. from 4–6 independent experiments. Dvl1 (blue line), Dvl2 (pink line) and Dvl3 (green line) are displayed.
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Figure 5: Differential effects of Wnt3a stimulation on Dvl1, Dvl2, and Dvl3. Panel A, the comparison of the cellular abundance of mammalian Dvl isoforms (Dvl1, Dvl2, and Dvl3) in subcellular fractions. F9 cells expressing Rfz1 were used to prepare subcellular fractions. Plasma membrane-enriched (PM), cytosol-enriched (CY), and nuclear-enriched (NU) fractions were prepared as described in detail in Methods and are displayed. The results of multiple densitometric scans of immunoblots were quantified. The results are displayed based upon the distribution (%), the total protein content of the whole-cell homogenate and fractions, and quantified analysis of blots from SDS-PAGE for each molecule and various controls for sample loading and blotting. The results are shown as mean values ± S.E. from 6–8 independent experiments. Panel B, the comparison of the cellular abundance of Dvls in response to Wnt3a. F9 cells expressing Rfz1 were stimulated with purified Wnt3a for indicated time and then disrupted 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]. Abundance of each Dvl isoform was determined by staining of blots with specific antibodies against each Dvl. Fractions were also stained with anti-GAPDH antibody to establish loading equivalence. Values are displayed as ''fold'' of zero-time point (set to 1). A representative blot is shown and the quantification of results is shown as mean values ± S.E. from 6 independent experiments. Panel C, F9 cells expressing Rfz1 receptor were stimulated without or with Wnt3a. Cells were harvested at each time point indicated, cell lysates were fractionated into plasma membrane (PM), cytoplasm (CY) and nuclei (NU) fractions. Quantified immunoblotting was performed as described in Methods. Each fraction (100 μg) was subjected to SDS-PAGE and the resolved proteins analyzed by immunoblotting with anti-Dvl1-, anti-Dvl2- and anti-Dvl3-specific antibodies. The three panels displayed at the bottom of the immunoblot set show blots stained with antibodies to well known subcellular marker proteins: Na+-K+-ATPase (plasma membrane), GAPDH (cytoplasm) and fibrillarin (nuclei), respectively. Representative blots are shown (top panel). Summary of quantified analysis of blots from SDS-PAGE are shown (bottom panel). The results are shown as mean values ± S.E. from 4–6 independent experiments. Dvl1 (blue line), Dvl2 (pink line) and Dvl3 (green line) are displayed.
Mentions: Unlike the fly which expresses a single Dishevelled (i.e., Dsh), mammalian cells can express three highly homologous isoforms of Dishevelled, termed Dvl1, Dvl2 and Dvl3 [41]. Dvl2 is the most abundant Dvl in mouse F9 cells (results not shown). We explored the cellular distribution of each mammalian Dvl expressed in F9 cells (fig. 5). All three Dvls were expressed in mouse F9 cells, as determined by immunoblotting with isoform-specific antibodies (fig 5A). The cellular content of Dvl1 and Dvl2 among the three subcellular fractions was nearly identical. The bulk of Dvl1 and of Dvl2 (> 80%) is found in the cytosol-enriched fraction, the least amount of each of these isoforms (> 5%) is observed in the nuclear-enriched fractions. Notable differences for the subcellular content and distribution of Dvl3 as compare to either Dvl1 or to Dvl2 were observed. Dvl3 content in the cytosol-enriched fractions is markedly reduced (~30%) in comparison to the content of either Dvl1 or Dvl2. More importantly, the content of Dvl3 in the nuclear-enriched fraction of unstimulated F9 cells is several folds greater than that for either Dvl1 or Dvl2 (fig 5A).

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