In Vitro Binding Assays
GST pulldown assays using GST-NLK and GST-HIPK2C were performed, as described previously (Kanei-Ishii et al., 2004a
). To increase the solubility of GST fusion proteins expressed in bacteria, the thioredoxin coexpression system (Yasukawa et al., 1995
) was used. The binding buffer used for most experiments consists of 20 mM HEPES, pH 7.5, 1 mM dithiothreitol (DTT), 0.1% NP-40, and 100 mM NaCl (for interactions between A-Myb and NLK or HIPK2) or 150 mM NaCl (for interactions between A-Myb and deletion mutants of CBP). GST pulldown assays using GST-CBP-KIX was performed, as described previously (Kanei-Ishii et al., 2004b
To investigate the in vivo interaction between A-Myb and NLK, CV-1 cells (5 × 105 cells per 100-mm dish) were transfected by LipofectAMINE Plus (Invitrogen, Carlsbad, CA) with the plasmids to express FLAG-A-Myb (2 μg) or HA-NLK (1 μg). Total plasmid amounts were adjusted to 8.0 μg by adding empty plasmid. The FLAG-A-Myb expression vector contains four tandem repeats of the FLAG tag at the N-terminus of A-Myb and the chicken cytoplasmic β-actin promoter. Transfectants were incubated for 24–36 h and lysed in TNE buffer (10 mM Tris-HCl, pH 7.8, 1 mM EDTA, 1% NP-40, protease inhibitor cocktail, 50 mM NaF, 25 mM β-glycerophosphate) containing 500 mM NaCl. Anti-HA (12CA5, Roche Diagnostics, Indianapolis, IN) or normal mouse IgG (Santa Cruz Biotechnology, Santa Cruz, CA) were used for immunoprecipitation. The immunocomplexes were washed three times with TNE buffer containing 1 M NaCl. To examine the effect of NLK on the A-Myb-CBP or B-Myb-CBP interaction, CV-1 cells were transfected with the plasmid encoding HA-tagged CBP (4 μg), A-Myb or B-Myb (2 μg), NLK (1 μg) or the control plasmid, and the internal control pCMV-luc (0.01 μg; total of 8.01 μg DNA) using LipofectAMINE Plus. In the case of B-Myb, transfected cells were treated with MG132 (50 μM) for 7 h before preparation of lysates. The immunoprecipitation was performed as described above. For immunoblotting, immunoprecipitates or whole cell lysates were resolved on SDS-PAGE and transferred to Hybond-P membranes (Amersham, Piscataway, NJ). The membranes were immunoblotted with various antibodies and the bound antibodies were visualized by horseradish peroxidase-conjugated antibodies against rabbit or mouse IgG using ECL (Amersham).
To examine the effect of NLK on the A-Myb levels, CV-1 cells were transfected with a mixture of FLAG-A-Myb expression plasmid (4 μg), NLK expression plasmid (2 μg), and the internal control plasmid pact-β-gal (0.3 μg). Total plasmid amounts were adjusted to 8.3 μg by adding empty plasmid. To investigate the effect of NLK on the B-Myb levels, CV-1 cells were transfected with a mixture of FLAG-B-Myb expression plasmid pact-4xFLAG-B-Myb (5 μg), NLK expression plasmid (2 μg), and the internal control plasmid pact-β-gal (0.3 μg). In some cases, the transfected cells were treated with MG132 (50 μM) for 7 h before preparation of lysates. To examine the effect of various components of the Wnt-NLK pathway on the A-Myb levels, CV-1 cells were transfected with a mixture of FLAG-A-Myb expression plasmid (2 μg), the plasmid to express each component (1 μg), and the internal control pCMV-luc (0.01 μg; total 8.01 μg). Cells were cultured for 40 h after transfection and then lysed in SDS sample buffer with mild sonication and subjected to SDS-PAGE, Western blotting with an anti-FLAG monoclonal antibody (Sigma, St. Louis, MO), and ECL detection. Aliquots of the cells were used to determine the transfection efficiency by measuring β-galactosidase or luciferase activity and the amounts of lysates used for Western blotting were normalized based on the β-galactosidase or luciferase activity.
For phosphorylation of A-Myb by NLK (), 293 cells were transfected with the FLAG-A-Myb expression plasmid (pact-FLAG-A-Myb) or the FLAG-NLK expression plasmid. The lysates were prepared from the transfected cells using NET buffer (20 mM Tris-HCl, pH 8.0, 1 mM EDTA, 0.5% NP-40, protease inhibitor cocktail) containing 150 mM NaCl and immunoprecipitated with anti-FLAG antibody (M2, Sigma). The immunocomplexes were sequentially washed with washing buffer (20 mM Tris-HCl, pH 8.0, 1 mM EDTA, 1 M NaCl, 2% NP-40, and 10% glycerol) and then NLK buffer (10 mM HEPES, pH 7.4, 1 mM DTT, and 5 mM MgCl2). An aliquot of the A-Myb immunocomplexes was used for Western blotting. FLAG-NLK proteins were eluted from the immunocomplexes with excess FLAG peptide. To phosphorylate A-Myb proteins, the A-Myb immunocomplexes were mixed with the eluted FLAG-NLK proteins and [γ-32P]ATP and incubated at 25°C for 2 h. The amount of A-Myb protein used in the kinase reaction was adjusted based on the Western blotting data. Phosphorylated proteins were analyzed by SDS-PAGE and autoradiography.
Figure 2. NLK phosphorylates A-Myb and inhibits A-Myb-dependent trans-activation without A-Myb degradation. (A) In vivo phosphorylation of A-Myb. CV-1 cells were transfected with the FLAG-A-Myb expression vector, with or without the expression vector for FLAG-tagged (more ...)
Luciferase Reporter Assays
Using LipofectAMINE PLUS, CV-1 cells (1 × 105 cells per 60-mm dish) were transfected with the 6MBS-I-SV40-luc reporter (0.5 μg), the A-Myb or B-Myb expression plasmid (0.2 μg), the NLK (0.03, 0.1 or 0.3 μg), or HIPK2 (0.1, 0.3, or 1.0 μg) expression plasmid, and the internal control plasmid pCMV-luc (0.1 μg), followed by luciferase assays. The chicken β-actin promoter was used to express A-Myb and B-Myb. In experiments to determine the effect of Wnt-NLK pathway components on A-Myb activity (), the pGL3-R2.2–6MBS-I-TK-luc reporter, which was made using the Rapid Response Reporter Vectors (Promega, Madison, WI) was used. CV-1 cells (2 × 105 cells per 60-mm dish) were transfected with the pGL3-R2.2–6MBS-I-TK-luc reporter (0.5 μg), and plasmids to express A-Myb (0.2 μg), R-Fz1 (0.1, 0.3, or 1.0 μg), R-Fz2 (0.1, 0.3, or 1.0 μg), TAK1/TAB1 (0.03, 0.1 or 0.3 μg), or the dominant-negative form of HIPK2 (1.0 μg), and the internal control plasmid pact-β-gal (0.05 μg). Luciferase assays were performed 24 h after transfection. The total amount of plasmid DNA was adjusted to 2.05 μg by adding empty plasmid.
Figure 3. Wnt signaling negatively regulates A-Myb-dependent trans-activation. (A–D) Effect of R-Fz1 (A), R-Fz2 (B), TAK1+TAB1 (C), or HIPK2 (D) on the trans-activating capacity of A-Myb. CV-1 cells were transfected with a mixture of 6MBS-I-luc and either (more ...)
In Vivo Acetylation Assay
To investigate the in vivo acetylation of A-Myb by CBP, CV-1 cells were transfected with the CBP expression plasmid pcDNA3-CBP-FLAG (4 μg) together with pact-FLAG-A-Myb (1 μg) and pCMV-FLAG-NLK or a control plasmid (1 μg). Cells were culture for 2 d after transfection, lysed in RIPA buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 10 mM sodium butylate) and centrifuged at 15,000 rpm. The supernatants were subjected to SDS-PAGE and Western blotting. The acetylated forms of A-Myb were detected with an anti-acetylated lysine polyclonal antibody (Cell Signaling, Beverly, MA), and a mixture of acetylated and nonacetylated forms of A-Myb were detected with the anti-FLAG M2 antibody.
Chromatin Immunoprecipitation Assays
Chromatin immunoprecipitation (ChIP) assays were carried out essentially as described (Tanikawa et al., 2004
) and in the Upstate Biotechnology protocol (Lake Placid, NY), with minor modifications. In brief, 1.5 × 105
CV-1 cells were transfected with FLAG-A-Myb expression plasmid or control plasmid (0.5 μg), CBP-HA expression plasmid (1.0 μg), NLK expression plasmid or control plasmid (0.5 μg), pGL3–6MBS-I-TK-Luc reporter plasmid (0.1 μg), and the internal control plasmid pCMV-luc (0.01 μg). The cytomegalovirus promoter was used in all expression plasmids. Cells were cultured for 40 h after transfection and then cross-linked and processed according to the manufacturer's protocol. Immunoprecipitation was performed overnight at 4°C with 6 μg of anti-FLAG antibody (M2, Sigma) or normal mouse IgG (Santa Cruz) as a negative control. The amounts of lysates used for immunoprecipitation was normalized based on the transfection efficiency (luciferase activity of pCMV-luc). In the ChIP assays to detect methylated histone H3, immunoprecipitation was performed using 2 μg of K9-H3m3 antibody (Abcam, Cambridge, UK). The immunocomplexes were washed and incubated at 65°C in 100 μl of IP elution buffer (1% SDS, 0.1 M NaHCO3
, 250 mM NaCl, 0.2 μg/μl proteinase K, 10 mM DTT) to release the proteins. In immunocomplexes containing FLAG-A-Myb, the complexes were incubated in IP elution buffer in the presence of the FLAG peptide (300 μg/ml) to enhance the elution efficiency. The de-cross-linked chromatin DNA was further purified using QIAquick PCR Purification Kit (QIAGEN, Santa Clarita, CA) and eluted in 40 μl sterile water. The eluted DNA samples (5 μl) were used for real-time PCR (7500 Real Time PCR System, Applied Biosystems, Foster City, CA). The primers and TaqMan probe (QIAGEN) used for the amplification of the 6MBS-I tk promoter were as follows: forward (5′-AATTCGAACACGCAGATGCA-3′), reverse (5′-GCCACACGCGTCACCTTAAT-3′), TaqMan probe (5′-CGCGGTCCCAGGTCCACTTCG-3′).