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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

N-terminal structure of NLK expression vectors.Schematic diagram of NLK constructs used in this study. NLK-L is the full-length protein without N-terminal tag sequences. NLK-S is the shorter NLK isoform. The rectangles indicate the open reading frame while the protein kinase domains are shown as black boxes. The tapered end on the left side of NLK-S indicates the leader sequence for the potential initiation codon (see Materials and methods). The thick line at the N-terminal of NLK-L indicates 5’ untranslated leader sequences of mouse NLK. The histidine rich regions are shown as hatched rectangles. The arrowheads indicate the methionine codons at the N-terminal regions. The mutation site of the kinase negative NLK is indicated with a hatched triangle above Flag-NLK.
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f1-83_016: N-terminal structure of NLK expression vectors.Schematic diagram of NLK constructs used in this study. NLK-L is the full-length protein without N-terminal tag sequences. NLK-S is the shorter NLK isoform. The rectangles indicate the open reading frame while the protein kinase domains are shown as black boxes. The tapered end on the left side of NLK-S indicates the leader sequence for the potential initiation codon (see Materials and methods). The thick line at the N-terminal of NLK-L indicates 5’ untranslated leader sequences of mouse NLK. The histidine rich regions are shown as hatched rectangles. The arrowheads indicate the methionine codons at the N-terminal regions. The mutation site of the kinase negative NLK is indicated with a hatched triangle above Flag-NLK.

Mentions: It has been previously suggested that mouse NLK may have a shorter isoform, translated from an alternative initiation codon at the 72nd amino acid from the N-terminal end.33) We generated mammalian NLK expression vectors to examine whether the shorter NLK isoform can have different function from full-length protein. Figure 1 shows a schematic diagram of the expression vectors used in this study. We designed to express Flag-NLK, the full-length NLK without a tag sequence (named NLK-L), and the shorter NLK isoform (named NLK-S). The native NLK mRNA sequences flanking each potential initiation codon were used as leader sequences for translation in mammalian cells (see Materials and methods).


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)

N-terminal structure of NLK expression vectors.Schematic diagram of NLK constructs used in this study. NLK-L is the full-length protein without N-terminal tag sequences. NLK-S is the shorter NLK isoform. The rectangles indicate the open reading frame while the protein kinase domains are shown as black boxes. The tapered end on the left side of NLK-S indicates the leader sequence for the potential initiation codon (see Materials and methods). The thick line at the N-terminal of NLK-L indicates 5’ untranslated leader sequences of mouse NLK. The histidine rich regions are shown as hatched rectangles. The arrowheads indicate the methionine codons at the N-terminal regions. The mutation site of the kinase negative NLK is indicated with a hatched triangle above Flag-NLK.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f1-83_016: N-terminal structure of NLK expression vectors.Schematic diagram of NLK constructs used in this study. NLK-L is the full-length protein without N-terminal tag sequences. NLK-S is the shorter NLK isoform. The rectangles indicate the open reading frame while the protein kinase domains are shown as black boxes. The tapered end on the left side of NLK-S indicates the leader sequence for the potential initiation codon (see Materials and methods). The thick line at the N-terminal of NLK-L indicates 5’ untranslated leader sequences of mouse NLK. The histidine rich regions are shown as hatched rectangles. The arrowheads indicate the methionine codons at the N-terminal regions. The mutation site of the kinase negative NLK is indicated with a hatched triangle above Flag-NLK.
Mentions: It has been previously suggested that mouse NLK may have a shorter isoform, translated from an alternative initiation codon at the 72nd amino acid from the N-terminal end.33) We generated mammalian NLK expression vectors to examine whether the shorter NLK isoform can have different function from full-length protein. Figure 1 shows a schematic diagram of the expression vectors used in this study. We designed to express Flag-NLK, the full-length NLK without a tag sequence (named NLK-L), and the shorter NLK isoform (named NLK-S). The native NLK mRNA sequences flanking each potential initiation codon were used as leader sequences for translation in mammalian cells (see Materials and methods).

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