FIGURE 7.

Activation of MITF and USF1 by GSK3. A, proliferating T98G cells were treated for 5 to 30 min with LY294002. Protein extracts were then subjected to immunoblotting with pan-GSK3β and GSK3β phospho-Ser-9 antibodies. B, proliferating T98G cells were transfected for 48 h with a luciferase reporter containing a 400-bp promoter fragment of ATROGIN-1 with or without co-transfection with plasmids expressing MITF and GSK3β S9A. Firefly luciferase activity was normalized against Renilla luciferase activity, derived from a co-transfected reporter construct. Data represent the mean ± S.E. of 5 transfections, except for GSK3β S9A alone, which was performed in duplicate. Expression from the ATROGIN-1 promoter-driven reporter construct was significantly higher when MITF was co-expressed with GSK3β S9A compared with MITF alone (*, p < 0.05). Immunoblots of MITF, GSK3β, and β-actin are shown in the lower panel. C, proliferating T98G cells were transfected as above, but with a plasmid expressing USF1 instead of MITF. Luciferase activity was normalized against β-gal activity from a co-transfected plasmid. Data are the mean ± S.E. of 3 transfections, except for GSK3β S9A alone, which was performed in duplicate. Expression from the ATROGIN-1 promoter-driven reporter construct was significantly higher when USF1 was coexpressed with GSK3β S9A compared with USF1 alone (*, p < 0.05). Immunoblots of USF1, GSK3β, and β-actin are shown in the lower panel. D, proliferating T98G cells were transfected with either the wild-type ATROGIN-1 luciferase reporter or a mutated ATROGIN-1 luciferase reporter lacking the single E-box-binding site within its 400-bp promoter region. These cells were also co-transfected with or without GSK3β S9A. Luciferase activity was normalized against β-gal activity from a co-transfected plasmid and data are the mean ± S.E. of 4 transfections. Expression from the ATROGIN-1 promoter-driven reporter construct was significantly lower when the single E-box was removed (p < 0.05).