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Mammalian Nemo-like kinase enhances β-catenin-TCF transcription activity in human osteosarcoma and neuroblastoma cells.

Yasuda J, Ichikawa H - Proc. Jpn. Acad., Ser. B, Phys. Biol. Sci. (2007)

Bottom Line: The nuclear localization of Lymphoid enhancer factor 1 (LEF1) and β-catenin ΔN was not altered by NLK overexpression regardless of its effect on β-catenin-TCF complex activity.Mutations in the potential NLK phosphorylation sites in β-catenin did not change its transcription activity either.Our results suggest that NLK positively regulates Wnt/β-catenin signaling in a cell type dependent manner through an unidentified mechanism.

View Article: PubMed Central - PubMed

Affiliation: Cancer Transcriptome Project, National Cancer Center Research Institute, Tokyo, Japan .

ABSTRACT
Nemo-like kinase (NLK) is an evolutionarily conserved serine/threonine kinase and has been considered to be a suppressor of Wnt signaling in mammalian cells. Our study, however, has raised the possibility that NLK also functions as a Wnt signaling activator. In human osteosarcoma and neuroblastoma cell lines, NLK specifically enhanced β-catenin-TCF complex transcription activity. The effect required kinase activity of NLK and co-expression of the β-catenin ΔN (constitutive active mutant of β-catenin). The nuclear localization of Lymphoid enhancer factor 1 (LEF1) and β-catenin ΔN was not altered by NLK overexpression regardless of its effect on β-catenin-TCF complex activity. Reporter analysis using LEF1 mutants at known NLK target sites indicated that NLK may have different activation targets for β-catenin-TCF complex. Mutations in the potential NLK phosphorylation sites in β-catenin did not change its transcription activity either. Our results suggest that NLK positively regulates Wnt/β-catenin signaling in a cell type dependent manner through an unidentified mechanism.

No MeSH data available.


Related in: MedlinePlus

NLK activates the pTOPFLASH transcription in osteosarcoma and neuroblastoma cells.The vertical axis shows the relative values of normalized fire fly luciferase activity in the cell lysates. The luciferase activity of each cell lysate was further normalized with that of the lysate transfected with pcDNA3-MYC-β-catenin ΔN only as 1 otherwise specified. The error bars indicate the standard error of measurement (SEM). A. NLK can activate the pTOPFLASH transcription in SaOS-2 (osteosarcoma) cells but not in 293 (epithelial) cells. 0.3 μg/well and 0.1 μg/well of MYC-β-catenin ΔN and NLK expression vectors were used respectively. B. Specific enhancement of pTOPFLASH transcription by NLK depends on its kinase activity and co-expression of β-catenin ΔN. The amount of MYC-β-catenin ΔN and NLK expression vectors used was 0.1 μg/well. In the case of leftmost panel, which indicates the lack of enhancement of β-catenin-TCF activity by NLK without β-catenin ΔN, luciferase activity of each cell lysate was further normalized with that of the lysate transfected with pTOPFLASH only as 1. C. NLK can activate pTOPFLASH reporter with β-catenin ΔN in neuroblastoma cells. The amount of MYC-β-catenin ΔN and NLK expression vectors used was 0.3 μg/well.
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f2-83_016: NLK activates the pTOPFLASH transcription in osteosarcoma and neuroblastoma cells.The vertical axis shows the relative values of normalized fire fly luciferase activity in the cell lysates. The luciferase activity of each cell lysate was further normalized with that of the lysate transfected with pcDNA3-MYC-β-catenin ΔN only as 1 otherwise specified. The error bars indicate the standard error of measurement (SEM). A. NLK can activate the pTOPFLASH transcription in SaOS-2 (osteosarcoma) cells but not in 293 (epithelial) cells. 0.3 μg/well and 0.1 μg/well of MYC-β-catenin ΔN and NLK expression vectors were used respectively. B. Specific enhancement of pTOPFLASH transcription by NLK depends on its kinase activity and co-expression of β-catenin ΔN. The amount of MYC-β-catenin ΔN and NLK expression vectors used was 0.1 μg/well. In the case of leftmost panel, which indicates the lack of enhancement of β-catenin-TCF activity by NLK without β-catenin ΔN, luciferase activity of each cell lysate was further normalized with that of the lysate transfected with pTOPFLASH only as 1. C. NLK can activate pTOPFLASH reporter with β-catenin ΔN in neuroblastoma cells. The amount of MYC-β-catenin ΔN and NLK expression vectors used was 0.3 μg/well.

Mentions: We tested whether NLK isoforms can suppress the transcription activity of β-catenin ΔN (constitutive active mutant β-catenin31)) by the use of pTOPFLASH, a specific reporter for Wnt/β-catenin signaling, in 293 and SaOS-2 cells. In 293 cells, Flag-NLK suppressed reporter gene expression, a finding in agreement with previous reports.17), 34) NLK-S also suppressed the reporter gene expression but to a lesser degree (Fig. 2A). Unexpectedly, NLK showed apparent induction of the reporter gene expression in SaOS-2 cells, suggesting that NLK enhances β-catenin-TCF transcription (Fig. 2A).


Mammalian Nemo-like kinase enhances β-catenin-TCF transcription activity in human osteosarcoma and neuroblastoma cells.

Yasuda J, Ichikawa H - Proc. Jpn. Acad., Ser. B, Phys. Biol. Sci. (2007)

NLK activates the pTOPFLASH transcription in osteosarcoma and neuroblastoma cells.The vertical axis shows the relative values of normalized fire fly luciferase activity in the cell lysates. The luciferase activity of each cell lysate was further normalized with that of the lysate transfected with pcDNA3-MYC-β-catenin ΔN only as 1 otherwise specified. The error bars indicate the standard error of measurement (SEM). A. NLK can activate the pTOPFLASH transcription in SaOS-2 (osteosarcoma) cells but not in 293 (epithelial) cells. 0.3 μg/well and 0.1 μg/well of MYC-β-catenin ΔN and NLK expression vectors were used respectively. B. Specific enhancement of pTOPFLASH transcription by NLK depends on its kinase activity and co-expression of β-catenin ΔN. The amount of MYC-β-catenin ΔN and NLK expression vectors used was 0.1 μg/well. In the case of leftmost panel, which indicates the lack of enhancement of β-catenin-TCF activity by NLK without β-catenin ΔN, luciferase activity of each cell lysate was further normalized with that of the lysate transfected with pTOPFLASH only as 1. C. NLK can activate pTOPFLASH reporter with β-catenin ΔN in neuroblastoma cells. The amount of MYC-β-catenin ΔN and NLK expression vectors used was 0.3 μg/well.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3756733&req=5

f2-83_016: NLK activates the pTOPFLASH transcription in osteosarcoma and neuroblastoma cells.The vertical axis shows the relative values of normalized fire fly luciferase activity in the cell lysates. The luciferase activity of each cell lysate was further normalized with that of the lysate transfected with pcDNA3-MYC-β-catenin ΔN only as 1 otherwise specified. The error bars indicate the standard error of measurement (SEM). A. NLK can activate the pTOPFLASH transcription in SaOS-2 (osteosarcoma) cells but not in 293 (epithelial) cells. 0.3 μg/well and 0.1 μg/well of MYC-β-catenin ΔN and NLK expression vectors were used respectively. B. Specific enhancement of pTOPFLASH transcription by NLK depends on its kinase activity and co-expression of β-catenin ΔN. The amount of MYC-β-catenin ΔN and NLK expression vectors used was 0.1 μg/well. In the case of leftmost panel, which indicates the lack of enhancement of β-catenin-TCF activity by NLK without β-catenin ΔN, luciferase activity of each cell lysate was further normalized with that of the lysate transfected with pTOPFLASH only as 1. C. NLK can activate pTOPFLASH reporter with β-catenin ΔN in neuroblastoma cells. The amount of MYC-β-catenin ΔN and NLK expression vectors used was 0.3 μg/well.
Mentions: We tested whether NLK isoforms can suppress the transcription activity of β-catenin ΔN (constitutive active mutant β-catenin31)) by the use of pTOPFLASH, a specific reporter for Wnt/β-catenin signaling, in 293 and SaOS-2 cells. In 293 cells, Flag-NLK suppressed reporter gene expression, a finding in agreement with previous reports.17), 34) NLK-S also suppressed the reporter gene expression but to a lesser degree (Fig. 2A). Unexpectedly, NLK showed apparent induction of the reporter gene expression in SaOS-2 cells, suggesting that NLK enhances β-catenin-TCF transcription (Fig. 2A).

Bottom Line: The nuclear localization of Lymphoid enhancer factor 1 (LEF1) and β-catenin ΔN was not altered by NLK overexpression regardless of its effect on β-catenin-TCF complex activity.Mutations in the potential NLK phosphorylation sites in β-catenin did not change its transcription activity either.Our results suggest that NLK positively regulates Wnt/β-catenin signaling in a cell type dependent manner through an unidentified mechanism.

View Article: PubMed Central - PubMed

Affiliation: Cancer Transcriptome Project, National Cancer Center Research Institute, Tokyo, Japan .

ABSTRACT
Nemo-like kinase (NLK) is an evolutionarily conserved serine/threonine kinase and has been considered to be a suppressor of Wnt signaling in mammalian cells. Our study, however, has raised the possibility that NLK also functions as a Wnt signaling activator. In human osteosarcoma and neuroblastoma cell lines, NLK specifically enhanced β-catenin-TCF complex transcription activity. The effect required kinase activity of NLK and co-expression of the β-catenin ΔN (constitutive active mutant of β-catenin). The nuclear localization of Lymphoid enhancer factor 1 (LEF1) and β-catenin ΔN was not altered by NLK overexpression regardless of its effect on β-catenin-TCF complex activity. Reporter analysis using LEF1 mutants at known NLK target sites indicated that NLK may have different activation targets for β-catenin-TCF complex. Mutations in the potential NLK phosphorylation sites in β-catenin did not change its transcription activity either. Our results suggest that NLK positively regulates Wnt/β-catenin signaling in a cell type dependent manner through an unidentified mechanism.

No MeSH data available.


Related in: MedlinePlus