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 the A-Myb expression plasmid or control plasmid, with or without increasing amounts of the R-Fz1, R-Fz2, HIPK2, or TAK1/TAB1 expression plasmids. Luciferase activity was then measured. The relative trans-activation capacity of A-Myb is indicated. The degree of activation by A-Myb was 8.0 ± 0.6-fold when no component of Wnt-TAK1 signaling was coexpressed. Averages of three experiments ± SD are shown. (E) Wnt1-TAK1 signaling suppresses A-Myb activity via HIPK2. CV-1 cells were transfected with a mixture of 6MBS-I-luc reporter, A-Myb expression plasmid or control plasmid, with or without the R-Fz1 or TAK1+TAB1 expression plasmids. In some cases, the plasmid that expresses the kinase-negative form of HIPK2 was added. Luciferase activity was then measured, and the relative trans-activation capacity of A-Myb is indicated as described above. (F) Coexpression of Wnt-NLK pathway components do not affect the A-Myb levels. CV-1 cells were transfected with the FLAG-A-Myb expression plasmid and the internal control plasmid pact-β-gal together with a plasmid expressing the protein indicated above each lane. The total cell lysates were prepared and analyzed by Western blotting using anti-FLAG antibody.