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GSK3beta is involved in JNK2-mediated beta-catenin inhibition.

Hu D, Bi X, Fang W, Han A, Yang W - PLoS ONE (2009)

Bottom Line: Additionally, physical interaction and co-localization among JNK2, beta-catenin and GSK3beta were observed by immunoprecipitation, mammalian two-hybridization assay and confocal microscopy, respectively.In general, our data suggested that JNK2, like JNK1, interacts with and suppresses beta-catenin signaling in vitro and in vivo, in which GSK3beta plays a key role, although previous studies have shown distinct functions of JNK1 and JNK2.Our study also provides a novel insight into the crosstalk between Wnt/beta-catenin and MAPK JNKs signaling.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, University of Illinois at Chicago, Chicago, Illinois, United States of America.

ABSTRACT

Background: We have recently reported that mitogen-activated protein kinase (MAPK) JNK1 downregulates beta-catenin signaling and plays a critical role in regulating intestinal homeostasis and in suppressing tumor formation. This study was designed to determine whether JNK2, another MAPK, has similar and/or different functions in the regulation of beta-catenin signaling.

Methodology and principal findings: We used an in vitro system with manipulation of JNK2 and beta-catenin expression and found that activated JNK2 increased GSK3beta activity and inhibited beta-catenin expression and transcriptional activity. However, JNK2-mediated downregulation of beta-catenin was blocked by the proteasome inhibitor MG132 and GSK3beta inhibitor lithium chloride. Moreover, targeted mutations at GSK3beta phosphorylation sites (Ser33 and Ser37) of beta-catenin abrogated JNK2-mediated suppression of beta-catenin. In vivo studies further revealed that JNK2 deficiency led to upregulation of beta-catenin and increase of GSK3-beta phosphorylation in JNK2-/- mouse intestinal epithelial cells. Additionally, physical interaction and co-localization among JNK2, beta-catenin and GSK3beta were observed by immunoprecipitation, mammalian two-hybridization assay and confocal microscopy, respectively.

Conclusion and significance: In general, our data suggested that JNK2, like JNK1, interacts with and suppresses beta-catenin signaling in vitro and in vivo, in which GSK3beta plays a key role, although previous studies have shown distinct functions of JNK1 and JNK2. Our study also provides a novel insight into the crosstalk between Wnt/beta-catenin and MAPK JNKs signaling.

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Active JNK2 downregulated β-catenin expression, inhibited its transcriptional activity and reduced GSK3β phosphorylation.(A) Active JNK2 suppressed β-catenin expression and GSK3β phosphorylation in HEK293T cells. HEK293T cells were transfected with pcDNA3-HA-β-catenin together with pcDNA3-Flag-MKK7-JNK1 or pcDNA3-Flag-MKK7-JNK2. Forty-eight hours after transfection, cells were harvested for immunoblotting analysis to detect the alterations of HA-β-catenin, p-JNK, p-c-Jun, phospho-Ser9 GSK3β, and GSK3β. β-actin served as loading control. (B) Active JNK2 reduced GSK3β phosphorylation and downregulated β-catenin expression in human lung cancer cell line A549. A549 cells were co-transfected with pcDNA3-HA-β-catenin and pcDNA3-Flag-MKK7-JNK2. Forty-eight hours after transfection, cells were harvested for immunoblotting analysis to detect the alterations of β-catenin, p-JNK, and phospho-Ser9 GSK3β. β-actin served as loading control. (C) Active JNK inhibited β-catenin-mediated transcriptional activity of TCF. HEK293T cells were co-transfected with pcDNA3-Flag-MKK7-JNK1 or pcDNA3-Flag-MKK7-JNK2, pcDNA3-HA-β-catenin, TOPFLASH (TOP) or FOPFLASH (FOP), and Renilla. 48 h after transfection, cells were harvested for luciferase activity assay. Each bar represents the mean ± standard deviation (SD) for triplicated samples.
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pone-0006640-g001: Active JNK2 downregulated β-catenin expression, inhibited its transcriptional activity and reduced GSK3β phosphorylation.(A) Active JNK2 suppressed β-catenin expression and GSK3β phosphorylation in HEK293T cells. HEK293T cells were transfected with pcDNA3-HA-β-catenin together with pcDNA3-Flag-MKK7-JNK1 or pcDNA3-Flag-MKK7-JNK2. Forty-eight hours after transfection, cells were harvested for immunoblotting analysis to detect the alterations of HA-β-catenin, p-JNK, p-c-Jun, phospho-Ser9 GSK3β, and GSK3β. β-actin served as loading control. (B) Active JNK2 reduced GSK3β phosphorylation and downregulated β-catenin expression in human lung cancer cell line A549. A549 cells were co-transfected with pcDNA3-HA-β-catenin and pcDNA3-Flag-MKK7-JNK2. Forty-eight hours after transfection, cells were harvested for immunoblotting analysis to detect the alterations of β-catenin, p-JNK, and phospho-Ser9 GSK3β. β-actin served as loading control. (C) Active JNK inhibited β-catenin-mediated transcriptional activity of TCF. HEK293T cells were co-transfected with pcDNA3-Flag-MKK7-JNK1 or pcDNA3-Flag-MKK7-JNK2, pcDNA3-HA-β-catenin, TOPFLASH (TOP) or FOPFLASH (FOP), and Renilla. 48 h after transfection, cells were harvested for luciferase activity assay. Each bar represents the mean ± standard deviation (SD) for triplicated samples.

Mentions: The studies from us and others have demonstrated that JNK1 can antagonize the canonical Wnt/β-catenin signaling [2], [4]. To elucidate the potential role of JNK2 in the regulation of Wnt/β-catenin signaling, constitutively active JNK2 (MKK7-JNK2) was co-transfected with β-catenin into HEK293T cells. As shown in figure 1A (Lane 3 versus lane 1), β-catenin protein level was dramatically reduced in MKK7-JNK2-transfected HEK293T cells, even to a greater extent than that in MKK7-JNK1-transfected cells (Figure 1A, lane 3 versus 2), suggesting that both JNK1 and JNK2 activation downregulate β-catenin expression although to a different extend.


GSK3beta is involved in JNK2-mediated beta-catenin inhibition.

Hu D, Bi X, Fang W, Han A, Yang W - PLoS ONE (2009)

Active JNK2 downregulated β-catenin expression, inhibited its transcriptional activity and reduced GSK3β phosphorylation.(A) Active JNK2 suppressed β-catenin expression and GSK3β phosphorylation in HEK293T cells. HEK293T cells were transfected with pcDNA3-HA-β-catenin together with pcDNA3-Flag-MKK7-JNK1 or pcDNA3-Flag-MKK7-JNK2. Forty-eight hours after transfection, cells were harvested for immunoblotting analysis to detect the alterations of HA-β-catenin, p-JNK, p-c-Jun, phospho-Ser9 GSK3β, and GSK3β. β-actin served as loading control. (B) Active JNK2 reduced GSK3β phosphorylation and downregulated β-catenin expression in human lung cancer cell line A549. A549 cells were co-transfected with pcDNA3-HA-β-catenin and pcDNA3-Flag-MKK7-JNK2. Forty-eight hours after transfection, cells were harvested for immunoblotting analysis to detect the alterations of β-catenin, p-JNK, and phospho-Ser9 GSK3β. β-actin served as loading control. (C) Active JNK inhibited β-catenin-mediated transcriptional activity of TCF. HEK293T cells were co-transfected with pcDNA3-Flag-MKK7-JNK1 or pcDNA3-Flag-MKK7-JNK2, pcDNA3-HA-β-catenin, TOPFLASH (TOP) or FOPFLASH (FOP), and Renilla. 48 h after transfection, cells were harvested for luciferase activity assay. Each bar represents the mean ± standard deviation (SD) for triplicated samples.
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pone-0006640-g001: Active JNK2 downregulated β-catenin expression, inhibited its transcriptional activity and reduced GSK3β phosphorylation.(A) Active JNK2 suppressed β-catenin expression and GSK3β phosphorylation in HEK293T cells. HEK293T cells were transfected with pcDNA3-HA-β-catenin together with pcDNA3-Flag-MKK7-JNK1 or pcDNA3-Flag-MKK7-JNK2. Forty-eight hours after transfection, cells were harvested for immunoblotting analysis to detect the alterations of HA-β-catenin, p-JNK, p-c-Jun, phospho-Ser9 GSK3β, and GSK3β. β-actin served as loading control. (B) Active JNK2 reduced GSK3β phosphorylation and downregulated β-catenin expression in human lung cancer cell line A549. A549 cells were co-transfected with pcDNA3-HA-β-catenin and pcDNA3-Flag-MKK7-JNK2. Forty-eight hours after transfection, cells were harvested for immunoblotting analysis to detect the alterations of β-catenin, p-JNK, and phospho-Ser9 GSK3β. β-actin served as loading control. (C) Active JNK inhibited β-catenin-mediated transcriptional activity of TCF. HEK293T cells were co-transfected with pcDNA3-Flag-MKK7-JNK1 or pcDNA3-Flag-MKK7-JNK2, pcDNA3-HA-β-catenin, TOPFLASH (TOP) or FOPFLASH (FOP), and Renilla. 48 h after transfection, cells were harvested for luciferase activity assay. Each bar represents the mean ± standard deviation (SD) for triplicated samples.
Mentions: The studies from us and others have demonstrated that JNK1 can antagonize the canonical Wnt/β-catenin signaling [2], [4]. To elucidate the potential role of JNK2 in the regulation of Wnt/β-catenin signaling, constitutively active JNK2 (MKK7-JNK2) was co-transfected with β-catenin into HEK293T cells. As shown in figure 1A (Lane 3 versus lane 1), β-catenin protein level was dramatically reduced in MKK7-JNK2-transfected HEK293T cells, even to a greater extent than that in MKK7-JNK1-transfected cells (Figure 1A, lane 3 versus 2), suggesting that both JNK1 and JNK2 activation downregulate β-catenin expression although to a different extend.

Bottom Line: Additionally, physical interaction and co-localization among JNK2, beta-catenin and GSK3beta were observed by immunoprecipitation, mammalian two-hybridization assay and confocal microscopy, respectively.In general, our data suggested that JNK2, like JNK1, interacts with and suppresses beta-catenin signaling in vitro and in vivo, in which GSK3beta plays a key role, although previous studies have shown distinct functions of JNK1 and JNK2.Our study also provides a novel insight into the crosstalk between Wnt/beta-catenin and MAPK JNKs signaling.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, University of Illinois at Chicago, Chicago, Illinois, United States of America.

ABSTRACT

Background: We have recently reported that mitogen-activated protein kinase (MAPK) JNK1 downregulates beta-catenin signaling and plays a critical role in regulating intestinal homeostasis and in suppressing tumor formation. This study was designed to determine whether JNK2, another MAPK, has similar and/or different functions in the regulation of beta-catenin signaling.

Methodology and principal findings: We used an in vitro system with manipulation of JNK2 and beta-catenin expression and found that activated JNK2 increased GSK3beta activity and inhibited beta-catenin expression and transcriptional activity. However, JNK2-mediated downregulation of beta-catenin was blocked by the proteasome inhibitor MG132 and GSK3beta inhibitor lithium chloride. Moreover, targeted mutations at GSK3beta phosphorylation sites (Ser33 and Ser37) of beta-catenin abrogated JNK2-mediated suppression of beta-catenin. In vivo studies further revealed that JNK2 deficiency led to upregulation of beta-catenin and increase of GSK3-beta phosphorylation in JNK2-/- mouse intestinal epithelial cells. Additionally, physical interaction and co-localization among JNK2, beta-catenin and GSK3beta were observed by immunoprecipitation, mammalian two-hybridization assay and confocal microscopy, respectively.

Conclusion and significance: In general, our data suggested that JNK2, like JNK1, interacts with and suppresses beta-catenin signaling in vitro and in vivo, in which GSK3beta plays a key role, although previous studies have shown distinct functions of JNK1 and JNK2. Our study also provides a novel insight into the crosstalk between Wnt/beta-catenin and MAPK JNKs signaling.

Show MeSH
Related in: MedlinePlus