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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

BRET2 analysis of protein-protein interactions in the Wnt/β-catenin signaling pathway. Panel A, Lef/Tcf-sensitive gene activation in HEK 293 cells in response to Wnt3a. HEK 293 cells were co-transfected with either Rfz1 and M50 or Rfz1-Rluc and M50. HEK 293 cells were stimulated with Wnt3a (20 ng/ml) for 8 hr. Lef/Tcf-sensitive transcription activity was determined. Panel B, HEK 293 cells stably expressing Rfz1 were transiently co-transfected with Axin-Rluc and/or Dvl2-GFP2, Dvl2-Rluc, GSK3-GFP2, β-cat-GFP2, β-cat-Rluc as indicated for 48 h and then treated with Wnt3a (20 ng/ml) for 15 min. BRET ratios were measured by addition of DeepBlue C (5 μM) in cells co-expressing Rluc and GFP2 fusion proteins. Results are expressed as the mean ± S.E. of at least three independent experiments. Panel C,the β-adrenergic agonist isoproterenol (Iso) stimulates the interaction between GPCR and β-arrestin2. HEK293 cells were transiently transfected with β2AR-Rluc and β-arrestin2-GFP2 for 48 h, and then treated with Iso (10 μM) or Iso plus the β-adrenergic antagonist propranolol (Pro, 10 μM) for 0 to 60 min. Panel D, Iso stimulates the interaction between GPCR and the heterotrimeric G protein subunit Gβ1. HEK293 cells were transiently transfected with β2AR-Rluc, Gβ1-GFP2, Gαs, and Gγ2 for 48 h, and then treated with agonist Iso (10 μM) or Iso plus antagonist Pro (10 μM) for 0 to 30 min. BRET ratios (GFP2/Rluc activity) were measured by addition of DeepBlue C (5 μM) in cells coexpressing Rluc and GFP2 fusion proteins. Results are expressed as the mean ± S.E. of three independent experiments. Note that isoproterenol stimulates GPCR interaction with G-protein first (peaks within 5 min of agonist) and later leads to association of GPCR with β-arrestin2 (peaks at 30 min). Both sets of protein-protein interactions are blocked by simultaneous addition of propranolol with agonist.
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Figure 4: BRET2 analysis of protein-protein interactions in the Wnt/β-catenin signaling pathway. Panel A, Lef/Tcf-sensitive gene activation in HEK 293 cells in response to Wnt3a. HEK 293 cells were co-transfected with either Rfz1 and M50 or Rfz1-Rluc and M50. HEK 293 cells were stimulated with Wnt3a (20 ng/ml) for 8 hr. Lef/Tcf-sensitive transcription activity was determined. Panel B, HEK 293 cells stably expressing Rfz1 were transiently co-transfected with Axin-Rluc and/or Dvl2-GFP2, Dvl2-Rluc, GSK3-GFP2, β-cat-GFP2, β-cat-Rluc as indicated for 48 h and then treated with Wnt3a (20 ng/ml) for 15 min. BRET ratios were measured by addition of DeepBlue C (5 μM) in cells co-expressing Rluc and GFP2 fusion proteins. Results are expressed as the mean ± S.E. of at least three independent experiments. Panel C,the β-adrenergic agonist isoproterenol (Iso) stimulates the interaction between GPCR and β-arrestin2. HEK293 cells were transiently transfected with β2AR-Rluc and β-arrestin2-GFP2 for 48 h, and then treated with Iso (10 μM) or Iso plus the β-adrenergic antagonist propranolol (Pro, 10 μM) for 0 to 60 min. Panel D, Iso stimulates the interaction between GPCR and the heterotrimeric G protein subunit Gβ1. HEK293 cells were transiently transfected with β2AR-Rluc, Gβ1-GFP2, Gαs, and Gγ2 for 48 h, and then treated with agonist Iso (10 μM) or Iso plus antagonist Pro (10 μM) for 0 to 30 min. BRET ratios (GFP2/Rluc activity) were measured by addition of DeepBlue C (5 μM) in cells coexpressing Rluc and GFP2 fusion proteins. Results are expressed as the mean ± S.E. of three independent experiments. Note that isoproterenol stimulates GPCR interaction with G-protein first (peaks within 5 min of agonist) and later leads to association of GPCR with β-arrestin2 (peaks at 30 min). Both sets of protein-protein interactions are blocked by simultaneous addition of propranolol with agonist.

Mentions: Our data suggest a close, perhaps "coupled" interaction of Axin with Dvl2 and β-catenin during activation of the canonical pathway by Wnt3a in mouse F9 cells (figs. 2, 3). As early as 15 min following stimulation of the cells with Wnt3a, we observe strikingly similar patterns of trafficking of all three signaling elements to the plasma membrane- and to the nuclear-enriched subcellular fractions (fig. 3B). Not so for the results obtained by analysis of the content of these molecules in the cytosol-enriched subcellular fractions, likely reflecting the differential effects of Wnt3a on the cellular abundance, rather than shuttling for both Axin and β-catenin. To probe this question more directly and in situ in live cells, we made use of bioluminescence response energy transfer (BRET) in which biomolecular interactions can be probed in live cells [38]. The BRET partners of R-luciferase-fusion proteins and eGFP2-fusion proteins of Axin, Dvl2, and β-catenin were engineered and expressed in various combinations in cells co-expressing Rfz1. Despite repeated efforts, we were not able to express the BRET fusion proteins in F9 cells at the levels that are necessary to provide unambiguous results of protein-protein interaction (results not shown). As an alternative, we employed HEK 293 cells that display a robust synthetic and chaperone capacity for exogenously introduced expression vectors [39]. Expression of the necessary fusion protein partners was successful in the HEK 293 cells, permitting BRET2 analysis of protein-protein interactions. BRET2 assay is performed with improved fusion moieties (e.g., GFP2) in combination with a more stable substrate for the Renilla luciferase (Rluc) [38,40]. Wnt3a stimulation of HEK 293 cells expressing Rfz1 resulted in robust activation of Lef/Tcf-sensitive luciferase gene reporter (fig. 4A), similar to that observed for F9 cells (fig. 1B). We tested the function of the fusion proteins, exemplified by the analysis of Rfz1-Rluc, which was shown to be fully functional (fig. 4A). The BRET ratio was measured in live cells at 15 min following Wnt3a stimulation (fig. 4B).


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

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

BRET2 analysis of protein-protein interactions in the Wnt/β-catenin signaling pathway. Panel A, Lef/Tcf-sensitive gene activation in HEK 293 cells in response to Wnt3a. HEK 293 cells were co-transfected with either Rfz1 and M50 or Rfz1-Rluc and M50. HEK 293 cells were stimulated with Wnt3a (20 ng/ml) for 8 hr. Lef/Tcf-sensitive transcription activity was determined. Panel B, HEK 293 cells stably expressing Rfz1 were transiently co-transfected with Axin-Rluc and/or Dvl2-GFP2, Dvl2-Rluc, GSK3-GFP2, β-cat-GFP2, β-cat-Rluc as indicated for 48 h and then treated with Wnt3a (20 ng/ml) for 15 min. BRET ratios were measured by addition of DeepBlue C (5 μM) in cells co-expressing Rluc and GFP2 fusion proteins. Results are expressed as the mean ± S.E. of at least three independent experiments. Panel C,the β-adrenergic agonist isoproterenol (Iso) stimulates the interaction between GPCR and β-arrestin2. HEK293 cells were transiently transfected with β2AR-Rluc and β-arrestin2-GFP2 for 48 h, and then treated with Iso (10 μM) or Iso plus the β-adrenergic antagonist propranolol (Pro, 10 μM) for 0 to 60 min. Panel D, Iso stimulates the interaction between GPCR and the heterotrimeric G protein subunit Gβ1. HEK293 cells were transiently transfected with β2AR-Rluc, Gβ1-GFP2, Gαs, and Gγ2 for 48 h, and then treated with agonist Iso (10 μM) or Iso plus antagonist Pro (10 μM) for 0 to 30 min. BRET ratios (GFP2/Rluc activity) were measured by addition of DeepBlue C (5 μM) in cells coexpressing Rluc and GFP2 fusion proteins. Results are expressed as the mean ± S.E. of three independent experiments. Note that isoproterenol stimulates GPCR interaction with G-protein first (peaks within 5 min of agonist) and later leads to association of GPCR with β-arrestin2 (peaks at 30 min). Both sets of protein-protein interactions are blocked by simultaneous addition of propranolol with agonist.
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Figure 4: BRET2 analysis of protein-protein interactions in the Wnt/β-catenin signaling pathway. Panel A, Lef/Tcf-sensitive gene activation in HEK 293 cells in response to Wnt3a. HEK 293 cells were co-transfected with either Rfz1 and M50 or Rfz1-Rluc and M50. HEK 293 cells were stimulated with Wnt3a (20 ng/ml) for 8 hr. Lef/Tcf-sensitive transcription activity was determined. Panel B, HEK 293 cells stably expressing Rfz1 were transiently co-transfected with Axin-Rluc and/or Dvl2-GFP2, Dvl2-Rluc, GSK3-GFP2, β-cat-GFP2, β-cat-Rluc as indicated for 48 h and then treated with Wnt3a (20 ng/ml) for 15 min. BRET ratios were measured by addition of DeepBlue C (5 μM) in cells co-expressing Rluc and GFP2 fusion proteins. Results are expressed as the mean ± S.E. of at least three independent experiments. Panel C,the β-adrenergic agonist isoproterenol (Iso) stimulates the interaction between GPCR and β-arrestin2. HEK293 cells were transiently transfected with β2AR-Rluc and β-arrestin2-GFP2 for 48 h, and then treated with Iso (10 μM) or Iso plus the β-adrenergic antagonist propranolol (Pro, 10 μM) for 0 to 60 min. Panel D, Iso stimulates the interaction between GPCR and the heterotrimeric G protein subunit Gβ1. HEK293 cells were transiently transfected with β2AR-Rluc, Gβ1-GFP2, Gαs, and Gγ2 for 48 h, and then treated with agonist Iso (10 μM) or Iso plus antagonist Pro (10 μM) for 0 to 30 min. BRET ratios (GFP2/Rluc activity) were measured by addition of DeepBlue C (5 μM) in cells coexpressing Rluc and GFP2 fusion proteins. Results are expressed as the mean ± S.E. of three independent experiments. Note that isoproterenol stimulates GPCR interaction with G-protein first (peaks within 5 min of agonist) and later leads to association of GPCR with β-arrestin2 (peaks at 30 min). Both sets of protein-protein interactions are blocked by simultaneous addition of propranolol with agonist.
Mentions: Our data suggest a close, perhaps "coupled" interaction of Axin with Dvl2 and β-catenin during activation of the canonical pathway by Wnt3a in mouse F9 cells (figs. 2, 3). As early as 15 min following stimulation of the cells with Wnt3a, we observe strikingly similar patterns of trafficking of all three signaling elements to the plasma membrane- and to the nuclear-enriched subcellular fractions (fig. 3B). Not so for the results obtained by analysis of the content of these molecules in the cytosol-enriched subcellular fractions, likely reflecting the differential effects of Wnt3a on the cellular abundance, rather than shuttling for both Axin and β-catenin. To probe this question more directly and in situ in live cells, we made use of bioluminescence response energy transfer (BRET) in which biomolecular interactions can be probed in live cells [38]. The BRET partners of R-luciferase-fusion proteins and eGFP2-fusion proteins of Axin, Dvl2, and β-catenin were engineered and expressed in various combinations in cells co-expressing Rfz1. Despite repeated efforts, we were not able to express the BRET fusion proteins in F9 cells at the levels that are necessary to provide unambiguous results of protein-protein interaction (results not shown). As an alternative, we employed HEK 293 cells that display a robust synthetic and chaperone capacity for exogenously introduced expression vectors [39]. Expression of the necessary fusion protein partners was successful in the HEK 293 cells, permitting BRET2 analysis of protein-protein interactions. BRET2 assay is performed with improved fusion moieties (e.g., GFP2) in combination with a more stable substrate for the Renilla luciferase (Rluc) [38,40]. Wnt3a stimulation of HEK 293 cells expressing Rfz1 resulted in robust activation of Lef/Tcf-sensitive luciferase gene reporter (fig. 4A), similar to that observed for F9 cells (fig. 1B). We tested the function of the fusion proteins, exemplified by the analysis of Rfz1-Rluc, which was shown to be fully functional (fig. 4A). The BRET ratio was measured in live cells at 15 min following Wnt3a stimulation (fig. 4B).

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