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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

Cellular distribution of Wnt/β-catenin signaling elements. Large-scale cultures of mouse F9 cells expressing Rfz1 were harvested and the cells disrupted. Subcellular fractions were prepared from the cell masses and the fractions probed for enrichment in plasma membrane (PM), cytoplasm (CY), and nuclei (NU) as described in detail in the Methods. Samples of the whole-cell homogenate were subjected to subcellular fractionation and their complements of cellular proteins to SDS-PAGE. Panel A, the distribution of well-known marker proteins for subcellular fractions (i.e., Na+-K+-ATPase as a marker for plasma membrane, GAPDH as a marker for cytoplasm, and fibrillarin as a marker for nuclei) also was analyzed in each fraction to establish purity/enrichment of markers in these fractions. Panel B, resolved proteins were analyzed by immunoblotting, stained with one of the following antibodies: anti-Axin, anti-β-catenin, anti-Dvl2, anti-GSK 3β, anti-p-Ser (9)-GSK 3β, and anti-PP2A C-subunit. The relative amounts of the proteins distributed in each subcellular fraction was established densitometrically, based upon their distribution (%), the total protein content of the homogenate and fractions, and quantified analysis of blots from SDS-PAGE. The results are shown as mean values ± S.E. from 8-10 independent experiments.
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Figure 2: Cellular distribution of Wnt/β-catenin signaling elements. Large-scale cultures of mouse F9 cells expressing Rfz1 were harvested and the cells disrupted. Subcellular fractions were prepared from the cell masses and the fractions probed for enrichment in plasma membrane (PM), cytoplasm (CY), and nuclei (NU) as described in detail in the Methods. Samples of the whole-cell homogenate were subjected to subcellular fractionation and their complements of cellular proteins to SDS-PAGE. Panel A, the distribution of well-known marker proteins for subcellular fractions (i.e., Na+-K+-ATPase as a marker for plasma membrane, GAPDH as a marker for cytoplasm, and fibrillarin as a marker for nuclei) also was analyzed in each fraction to establish purity/enrichment of markers in these fractions. Panel B, resolved proteins were analyzed by immunoblotting, stained with one of the following antibodies: anti-Axin, anti-β-catenin, anti-Dvl2, anti-GSK 3β, anti-p-Ser (9)-GSK 3β, and anti-PP2A C-subunit. The relative amounts of the proteins distributed in each subcellular fraction was established densitometrically, based upon their distribution (%), the total protein content of the homogenate and fractions, and quantified analysis of blots from SDS-PAGE. The results are shown as mean values ± S.E. from 8-10 independent experiments.

Mentions: Results from the analysis of the cellular complement of the signaling elements by immunoblotting clearly demonstrates the need for subcellular fractionation and characterization of the abundance of each of the proteins, in advance of determinations of apparent "protein shuttling" in response to Wnt. Subcellular fractionation of F9 cells was performed by standard protocols and the fractions characterized biochemically for enrichment in marker proteins. Nuclei (NU), plasma membrane (PM), and cytosol (CY, see Methods) subcellular fractions were prepared and characterized for their enrichment by immunoblotting and by staining of samples of fractions with antibodies to marker proteins: Na+-K+-ATPase for plasma membrane; glyceraldehyde-3-phosphate dehydrogenase (GAPDH) for cytoplasm; and the nucleolar-specific protein fibrillarin for nuclei (fig. 2A). The results from the immunoblotting testify to the enriched character of each subcellular fraction prepared from these cells (fig. 2A, B). Each fraction was highly enriched in the appropriate marker for the subcellular source and relatively devoid of marker proteins for the other subcellular fractions.


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

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

Cellular distribution of Wnt/β-catenin signaling elements. Large-scale cultures of mouse F9 cells expressing Rfz1 were harvested and the cells disrupted. Subcellular fractions were prepared from the cell masses and the fractions probed for enrichment in plasma membrane (PM), cytoplasm (CY), and nuclei (NU) as described in detail in the Methods. Samples of the whole-cell homogenate were subjected to subcellular fractionation and their complements of cellular proteins to SDS-PAGE. Panel A, the distribution of well-known marker proteins for subcellular fractions (i.e., Na+-K+-ATPase as a marker for plasma membrane, GAPDH as a marker for cytoplasm, and fibrillarin as a marker for nuclei) also was analyzed in each fraction to establish purity/enrichment of markers in these fractions. Panel B, resolved proteins were analyzed by immunoblotting, stained with one of the following antibodies: anti-Axin, anti-β-catenin, anti-Dvl2, anti-GSK 3β, anti-p-Ser (9)-GSK 3β, and anti-PP2A C-subunit. The relative amounts of the proteins distributed in each subcellular fraction was established densitometrically, based upon their distribution (%), the total protein content of the homogenate and fractions, and quantified analysis of blots from SDS-PAGE. The results are shown as mean values ± S.E. from 8-10 independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Cellular distribution of Wnt/β-catenin signaling elements. Large-scale cultures of mouse F9 cells expressing Rfz1 were harvested and the cells disrupted. Subcellular fractions were prepared from the cell masses and the fractions probed for enrichment in plasma membrane (PM), cytoplasm (CY), and nuclei (NU) as described in detail in the Methods. Samples of the whole-cell homogenate were subjected to subcellular fractionation and their complements of cellular proteins to SDS-PAGE. Panel A, the distribution of well-known marker proteins for subcellular fractions (i.e., Na+-K+-ATPase as a marker for plasma membrane, GAPDH as a marker for cytoplasm, and fibrillarin as a marker for nuclei) also was analyzed in each fraction to establish purity/enrichment of markers in these fractions. Panel B, resolved proteins were analyzed by immunoblotting, stained with one of the following antibodies: anti-Axin, anti-β-catenin, anti-Dvl2, anti-GSK 3β, anti-p-Ser (9)-GSK 3β, and anti-PP2A C-subunit. The relative amounts of the proteins distributed in each subcellular fraction was established densitometrically, based upon their distribution (%), the total protein content of the homogenate and fractions, and quantified analysis of blots from SDS-PAGE. The results are shown as mean values ± S.E. from 8-10 independent experiments.
Mentions: Results from the analysis of the cellular complement of the signaling elements by immunoblotting clearly demonstrates the need for subcellular fractionation and characterization of the abundance of each of the proteins, in advance of determinations of apparent "protein shuttling" in response to Wnt. Subcellular fractionation of F9 cells was performed by standard protocols and the fractions characterized biochemically for enrichment in marker proteins. Nuclei (NU), plasma membrane (PM), and cytosol (CY, see Methods) subcellular fractions were prepared and characterized for their enrichment by immunoblotting and by staining of samples of fractions with antibodies to marker proteins: Na+-K+-ATPase for plasma membrane; glyceraldehyde-3-phosphate dehydrogenase (GAPDH) for cytoplasm; and the nucleolar-specific protein fibrillarin for nuclei (fig. 2A). The results from the immunoblotting testify to the enriched character of each subcellular fraction prepared from these cells (fig. 2A, B). Each fraction was highly enriched in the appropriate marker for the subcellular source and relatively devoid of marker proteins for the other subcellular fractions.

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