NLK positively regulates Wnt/β-catenin signalling by phosphorylating LEF1 in neural progenitor cells - PubMed (original) (raw)

NLK positively regulates Wnt/β-catenin signalling by phosphorylating LEF1 in neural progenitor cells

Satoshi Ota et al. EMBO J. 2012.

Abstract

Nemo-like kinase (NLK/Nlk) is an evolutionarily conserved protein kinase involved in Wnt/β-catenin signalling. However, the roles of NLK in Wnt/β-catenin signalling in vertebrates remain unclear. Here, we show that inhibition of Nlk2 function in zebrafish results in decreased Lymphoid enhancer factor-1 (Lef1)-mediated gene expression and cell proliferation in the presumptive midbrain, resulting in a reduction of midbrain tectum size. These defects are related to phosphorylation of Lef1 by Nlk2. Thus, Nlk2 is essential for the phosphorylation and activation of Lef1 transcriptional activity in neural progenitor cells (NPCs). In NPC-like mammalian cells, NLK is also required for the phosphorylation and activation of LEF1 transcriptional activity. Phosphorylation of LEF1 induces its dissociation from histone deacetylase, thereby allowing transcription activation. Furthermore, we demonstrate that NLK functions downstream of Dishevelled (Dvl) in the Wnt/β-catenin signalling pathway. Our findings reveal a novel role of NLK in the activation of the Wnt/β-catenin signalling pathway.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Figure 1

Figure 1

Nlk2 and Lef1 are required for the activation of Wnt/β-catenin signalling in zebrafish developing midbrain. (A) Whole mount in-situ hybridization staining for nlk2 in zebrafish embryos at the indicated stage. Scale bar: 250 μm. The expression of nlk2 in midbrain is indicated with arrows. (B, E) TOPdGFP-transgenic zebrafish embryos injected with lef1 spl MO, nlk2 MO, nlk2 spl MO, or p53 MO with or without mouse NLK (mNLK) mRNA, as indicated. Panels show the left side head views of 30 h.p.f. embryos with the anterior to the left. Cells expressing dGFP were visualized by fluorescence microscopy (right panels). Bright-field (BF) images are shown in left panels. Scale bar, 50 μm. Note that mNLK partially rescued nlk2 MO-induced reduction of TOPdGFP activity (_n_=28, 50%). (C, D) In-situ hybridization staining for TOPdGFP (C), lef1 (D), and nlk2 (D) in the transverse section at the level of midbrain in the indicated stage zebrafish embryos. Scale bar: 50 μm. The lateral dorsal region is indicated with arrows.

Figure 2

Figure 2

Nlk2 and Lef1 are essential for the midbrain tectum development in zebrafish. (A) HuC:kaede-transgenic zebrafish embryos injected with nlk2 MO, nlk2 spl MO, lef1 spl MO, or p53 MO as indicated. Panels show the dorsal head views of 80 h.p.f. embryos with the anterior to the left. Neurons expressing Kaede were visualized by fluorescence microscopy (right panels). Rectangles indicate the tectum. Bright-field (BF) images are shown in left panels. Broken lines indicate the tectum or presumptive tectal region. Scale bar, 50 μm. (B) Knockdown of either nlk2 or lef1 decreases the number of proliferating cells in midbrain. Anti-phospho-histone H3 immunostaining of 30 h.p.f. zebrafish embryos injected with nlk2 MO, nlk2 spl MO, or lef1 spl MO, as indicated. Top panels show the left side head views of embryos with the anterior to the left. The other panels show the left side midbrain view of embryos with the anterior to the left. Broken lines indicate the presumptive tectal region. Scale bar, 250 μm. (C) Knockdown of either nlk2 or lef1 reduces expression of zic2a in midbrain. Panels show whole mount in-situ hybridization for zic2a in 30 h.p.f. embryos. Embryos were injected with nlk2 MO, nlk2 spl MO, or lef1 spl MO as indicated. Panels show the left side head views of embryos with the anterior to the left. Expression of zic2a in midbrain is indicated with arrows. Scale bar, 250 μm.

Figure 3

Figure 3

Nlk2 is involved in Lef1 phosphorylation in zebrafish. (A) Amino-acid sequence alignment of the NLK phosphorylation regions within vertebrate LEF1/Lef1 proteins. The Thr and Ser residues, which are phosphorylated by NLK, are indicated with red and blue letters, respectively. The bar under the sequence alignment indicates the immunogen for anti-pLef1 antibody. (B) Nlk2 phosphorylates Lef1 at the conserved Thr residue. Neuro-2a cells were transfected with Flag–Nlk2 wild-type (WT), Flag–Nlk2 kinase-negative mutant (KN), Myc–Lef1 (WT) and Myc–Lef1(T151A) as indicated. Cell lysates were immunoblotted with anti-pLef1, anti-Myc, and anti-Flag antibodies. (C) Nlk2 is required for phosphorylation of Lef1 at the conserved Thr residue in zebrafish. Zebrafish embryos were not injected or injected with nlk2 MO, as indicated. Extracts were harvested from the embryos at 24 h.p.f., and immunoblotted with anti-pLef1, anti-Lef1, anti-NLK, and anti-β-tubulin antibodies. Arrow and arrowhead indicate Lef1 and phosphorylated Lef1 proteins, respectively. Anti-NLK antibody, which was generated in rabbit with a synthetic peptide corresponding to the carboxyl terminal conserved region of NLK, can recognize Nlk2, but not Nlk1. (D) The nlk2 MO-induced tectum size reduction phenotype is rescued by expression of mouse NLK (mNLK) and zebrafish Lef1(T151E). Zebrafish embryos were injected with nlk2 MO with or without transposase mRNA and Tol2-donor plasmid containing cDNA encoding mNLK-WT, mNLK-KN, Lef1-WT, or Lef1(TE), and then tectum size was determined. Embryos were classified into three groups based on the extent of tectum size reduction (normal, slightly reduced, and reduced). Upper panels show an example of each class. Broken lines indicate the tectum or presumptive tectal region. Lower graph shows the percentages of embryos exhibiting each class of tectum size reduction. The number shown in the right side of graph is the total number of embryos. Figure source data can be found in Supplementary data.

Figure 4

Figure 4

Wnt1 is involved in Lef1 phosphorylation in zebrafish. (A) Wnt1 is required for Lef1 phosphorylation at the conserved Thr residue in zebrafish. Zebrafish embryos were co-injected with p53 MO and control MO or wnt1 MO, as indicated. Extracts were harvested from the embryos at 24 h.p.f., and immunoblotted with anti-pLef1 and anti-Lef1 antibodies. (B) Wnt1 is required for activation of TOPdGFP in zebrafish midbrain. TOPdGFP-transgenic zebrafish embryos were co-injected with p53 MO and control MO or wnt1 MO, as indicated. Panels show the left side head views of 24 h.p.f. zebrafish embryos with the anterior to the left. Cells expressing dGFP were visualized by fluorescence microscopy (upper panels). Bright-field (BF) images are shown in lower panels. Scale bar, 250 μm. (C, D) Wnt1 is required for the formation of 80 h.p.f. zebrafish midbrain. Zebrafish embryos were injected with p53 MO and wnt1 MO with or without transposase mRNA and a Tol2-donor plasmid containing cDNA encoding mouse Wnt-1 (mWnt-1) or GFP, as indicated. Panels in (C) show a typical example. Broken lines indicate the tectum or presumptive tectal region. Embryos were classified into three groups based on the extent of tectum size reduction (normal, slightly reduced, and reduced). Graph in (D) shows the percentages of embryos exhibiting each class of tectum size reduction. The number shown in the right side of graph is the total number of embryos. Figure source data can be found in Supplementary data.

Figure 5

Figure 5

NLK promotes LEF1-mediated transcription in NPC-like mammalian cell lines. (A, B) The Wnt/β-catenin signalling reporter plasmids and expression plasmids encoding β-cateninΔN, human LEF1-WT, LEF1-2A, LEF1-2E, mouse NLK-WT, and NLK-KN were transfected as indicated and the luciferase activities were measured in neuro-2a (A) and PC12 (B) cells. (C) NLK phosphorylates LEF1 at Thr-155 and Ser-166 in neuro-2a and PC12 cells. Neuro-2a and PC12 cells were transfected with Flag-tagged mouse NLK (Flag–NLK-WT), Flag–NLK-KN, T7-tagged human LEF1 (T7–LEF1-WT) and T7–LEF1-2A mutant as indicated. Cell lysates were immunoblotted with anti-T7, anti-pLef1, and anti-Flag antibodies. Note that anti-pLef1 also recognized the phosphorylation of human LEF1. Figure source data can be found in Supplementary data.

Figure 6

Figure 6

HDAC1 interacts with unphosphorylated LEF1. (A) NLK does not affect the interaction of LEF1 with β-catenin in neuro-2a cells. Neuro-2a cells were transfected with T7-tagged human LEF1, β-cateninΔN, and Flag-tagged mouse NLK as indicated. Cell extracts were subjected to immunoprecipitation with anti-T7 antibody. Immunoprecipitated complexes were immunoblotted with anti-β-catenin and anti-T7 antibodies. The amounts of β-catenin, β-cateninΔN, and Flag–NLK were confirmed by immunoblotting with anti-β-catenin and anti-Flag antibodies. (B, C) NLK-mediated LEF1 phosphorylation inhibits the interaction of HDAC1 with LEF1 in neuro-2a cells. Neuro-2a cells were transfected with plasmids encoding T7-tagged human LEF1-WT, T7–LEF1-2A, T7–LEF1-2E, and Flag-tagged mouse NLK as indicated. Cell extracts were immunoprecipitated with control IgG or anti-HDAC1 antibody. Immunoprecipitated complexes were immunoblotted with anti-T7 and anti-HDAC1 antibodies. The amounts of T7–LEF1 and Flag–NLK proteins were confirmed by immunoblotting with anti-T7 and anti-Flag antibodies, respectively. (D) Trichostatin A (TSA) treatment activates β-catenin–LEF1 complex-mediated transcription. Neuro-2a cells were transfected with Wnt/β-catenin reporter plasmids and plasmids encoding β-cateninΔN and LEF1. At 24 h after transfection, cells were left untreated or treated with 50 ng/ml TSA for 24 h and luciferase activity was measured. Figure source data can be found in Supplementary data.

Figure 7

Figure 7

Nlk2 positively regulates Wnt/β-catenin signalling by blocking Hdac1-mediated inhibition in zebrafish midbrain. TOPdGFP-transgenic zebrafish embryos were uninjected or injected with hdac1 MO, nlk2 MO, lef1 spl MO or p53 MO at one-cell stage, as indicated. At 24 h.p.f., embryos were untreated (A) or left treated with DMSO or 1.2 μM TSA for 6 h (B, C). Panels show the left side head views of 30 h.p.f. zebrafish embryos with the anterior to the left. The cells expressing dGFP were visualized by fluorescence microscopy (upper panels). BF images are shown in lower panels. Scale bar, 250 μm.

Figure 8

Figure 8

NLK functions downstream of Dvl in the Wnt/β-catenin signalling pathway. (A) Wnt-3a signalling activates the kinase activity of endogenous NLK. PC12 cells were untreated or treated with Wnt-3a and/or R-spondin 3 (Rspo3) for 30 min and endogenous NLK was immunoprecipitated (IP) with anti-NLK antibody. Aliquots of purified Flag–LEF1 and IP NLK proteins were subjected to a non-RI in-vitro kinase assay, and immunoblotted with anti-pLef1 antibody. Flag–LEF1 and NLK were confirmed by immunoblotting with anti-Flag and anti-NLK antibodies, respectively. (B) Wnt-3a signalling induces LEF1 phosphorylation. PC12 cells were treated with either control or NLK siRNA and then treated with or without Wnt-3a and Rspo3 for 30 min. Cell extracts were immunoprecipitated with anti-LEF1 antibody. IP complexes were immunoblotted with anti-pLef1 and anti-LEF1 antibodies. The amounts of endogenous NLK proteins were confirmed by immunoblotting with anti-NLK antibody. β-Tubulin was used as a loading control. (C) Wnt-3a signalling reduces the interaction of LEF1 with HDAC1. PC12 cells were treated with or without Wnt-3a and Rspo3 for the indicated time and then cell extracts were immunoprecipitated with control IgG or anti-HDAC1 antibody. IP complexes were immunoblotted with anti-LEF1 and anti-HDAC1 antibodies. The amounts of endogenous LEF1 were confirmed by immunoblotting with anti-LEF1 antibody. (D) Dvl1 induces LEF1 phosphorylation in a manner dependent on NLK. Neuro-2a cells transfected with Myc–Dvl1 and T7–Lef1 were treated with either control or NLK siRNA. Cell extracts were immunoprecipitated with anti-T7 antibody. IP complexes were immunoblotted with anti-pLef1 and anti-T7 antibodies. The amounts of Myc–Dvl1 and endogenous NLK were confirmed by immunoblotting with anti-Myc and anti-NLK antibodies, respectively. ERK was used as a loading control. (E) NLK binds to Dvl in a manner dependent on Wnt-3a signalling. PC12 cells were treated with or without Wnt-3a and Rspo3 for the indicated times and then cell extracts were immunoprecipitated with either control IgG or anti-NLK antibodies. IP complexes were immunoblotted with anti-Dvl and anti-NLK antibodies. The amounts of endogenous Dvl proteins were confirmed by immunoblotting with anti-Dvl antibody. Figure source data can be found in Supplementary data.

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