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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 a rapid shuttling of Wnt/β-catenin signaling elements. Panel A, quantified immunoblot analysis of subcellular fractions obtained from control cells (time = 0) and cells stimulated with purified Wnt3a. F9 cells expressing Rfz1 receptor were stimulated with Wnt3a for the indicated times. Cell cultures were collected, disrupted, and fractionated to the plasma membrane, cytoplasm and nuclei fractions, as described in Methods. Each fraction (100 μg) was subjected to SDS-PAGE and analyzed by immunoblotting with specific antibodies targeting the signaling molecules indicated. 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. The result shown is representative of 10 independent experiments. Panel B, summary of the quantified trafficking of signal elements in response to Wnt3a. Bands were quantified by densitometry as described in Methods and values are displayed as fold of zero time point. The content of key signaling molecules in the plasma membrane- (PM, blue line), cytoplasmic- (CY, pink line) and nuclear- (NU, green line) enriched subcellular fractions are displayed. The results are shown as mean values ± S.E. from 6–10 independent experiments.
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Figure 3: Wnt3a stimulates a rapid shuttling of Wnt/β-catenin signaling elements. Panel A, quantified immunoblot analysis of subcellular fractions obtained from control cells (time = 0) and cells stimulated with purified Wnt3a. F9 cells expressing Rfz1 receptor were stimulated with Wnt3a for the indicated times. Cell cultures were collected, disrupted, and fractionated to the plasma membrane, cytoplasm and nuclei fractions, as described in Methods. Each fraction (100 μg) was subjected to SDS-PAGE and analyzed by immunoblotting with specific antibodies targeting the signaling molecules indicated. 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. The result shown is representative of 10 independent experiments. Panel B, summary of the quantified trafficking of signal elements in response to Wnt3a. Bands were quantified by densitometry as described in Methods and values are displayed as fold of zero time point. The content of key signaling molecules in the plasma membrane- (PM, blue line), cytoplasmic- (CY, pink line) and nuclear- (NU, green line) enriched subcellular fractions are displayed. The results are shown as mean values ± S.E. from 6–10 independent experiments.

Mentions: Our overarching goal was to quantify the trafficking of key signaling molecules of the Wnt/β-catenin pathway in response to Wnt3a stimulation. Within 90 min of stimulation with Wnt3a, F9 cells display a substantial increase in the abundance of Axin, of β-catenin, and to a lesser extent of the protein kinase GSK3β (fig. 1C). We incorporated the changes in cellular abundance, referencing the subcellular localization established for each molecule in the absence of Wnt (i.e., the starting time, t = 0) and quantified the cellular distribution of the Axin, β-catenin, Dvl2 and GSK3β in F9 cells treated with Wnt3a (fig. 3). The cells were stimulated with purified Wnt3a for periods up to two hours. Immunoblots of subcellular fractions prepared from large-scale growths of F9 cells treated with Wnt3a at various times from a representative experiment are displayed for each signaling molecule (fig. 3A). The relative changes in distribution of the signaling molecules among the subcellular fractions obtained from 6–10 individual, separate experiments were compiled, analyzed statistically, and displayed (fig. 3B).


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 a rapid shuttling of Wnt/β-catenin signaling elements. Panel A, quantified immunoblot analysis of subcellular fractions obtained from control cells (time = 0) and cells stimulated with purified Wnt3a. F9 cells expressing Rfz1 receptor were stimulated with Wnt3a for the indicated times. Cell cultures were collected, disrupted, and fractionated to the plasma membrane, cytoplasm and nuclei fractions, as described in Methods. Each fraction (100 μg) was subjected to SDS-PAGE and analyzed by immunoblotting with specific antibodies targeting the signaling molecules indicated. 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. The result shown is representative of 10 independent experiments. Panel B, summary of the quantified trafficking of signal elements in response to Wnt3a. Bands were quantified by densitometry as described in Methods and values are displayed as fold of zero time point. The content of key signaling molecules in the plasma membrane- (PM, blue line), cytoplasmic- (CY, pink line) and nuclear- (NU, green line) enriched subcellular fractions are displayed. The results are shown as mean values ± S.E. from 6–10 independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
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Figure 3: Wnt3a stimulates a rapid shuttling of Wnt/β-catenin signaling elements. Panel A, quantified immunoblot analysis of subcellular fractions obtained from control cells (time = 0) and cells stimulated with purified Wnt3a. F9 cells expressing Rfz1 receptor were stimulated with Wnt3a for the indicated times. Cell cultures were collected, disrupted, and fractionated to the plasma membrane, cytoplasm and nuclei fractions, as described in Methods. Each fraction (100 μg) was subjected to SDS-PAGE and analyzed by immunoblotting with specific antibodies targeting the signaling molecules indicated. 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. The result shown is representative of 10 independent experiments. Panel B, summary of the quantified trafficking of signal elements in response to Wnt3a. Bands were quantified by densitometry as described in Methods and values are displayed as fold of zero time point. The content of key signaling molecules in the plasma membrane- (PM, blue line), cytoplasmic- (CY, pink line) and nuclear- (NU, green line) enriched subcellular fractions are displayed. The results are shown as mean values ± S.E. from 6–10 independent experiments.
Mentions: Our overarching goal was to quantify the trafficking of key signaling molecules of the Wnt/β-catenin pathway in response to Wnt3a stimulation. Within 90 min of stimulation with Wnt3a, F9 cells display a substantial increase in the abundance of Axin, of β-catenin, and to a lesser extent of the protein kinase GSK3β (fig. 1C). We incorporated the changes in cellular abundance, referencing the subcellular localization established for each molecule in the absence of Wnt (i.e., the starting time, t = 0) and quantified the cellular distribution of the Axin, β-catenin, Dvl2 and GSK3β in F9 cells treated with Wnt3a (fig. 3). The cells were stimulated with purified Wnt3a for periods up to two hours. Immunoblots of subcellular fractions prepared from large-scale growths of F9 cells treated with Wnt3a at various times from a representative experiment are displayed for each signaling molecule (fig. 3A). The relative changes in distribution of the signaling molecules among the subcellular fractions obtained from 6–10 individual, separate experiments were compiled, analyzed statistically, and displayed (fig. 3B).

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