FIGURE 2.

c-Jun enhances PDK1 transactivation through a Sp1 site. A, structure of the proximal region of the human PDK1 promoter. The region from −45 to +82 contains 3 boxes highly conserved between human and mouse (box 1 from −14 to +2, box 2 from +4 to +15, and box 3 from +26 to +37 of the human promoter). In addition to the WT sequence, fragments containing mutations in box 1 and deletion of box 2 as well as mutation in box 3 were cloned in pGL2 vector for reporter assays. B, c-Jun enhances PDK1 transactivation through a Sp1 site in box 3. Reporter plasmids described in panel A were transfected into MeWo cells together with empty vector or pcDNA-c-Jun. Results are shown as the mean ± S.D. C, c-Jun siRNA no longer affect transcription driven by a mutant Sp1 site on box 3. Reporter plasmids described in panel A were transfected into Lu1205 cells together with scramble (sc) or c-Jun siRNA. D, mutation on the Sp1 site on box 3 abrogates responsiveness to c-Jun of the PDK1 promoter. A large region of the PDK1 promoter (−875 to +82) was subjected to mutagenesis of the Sp1 site on box 3 and the activity of both the WT and mutant promoter was assessed using reporter assays. Luciferase data are shown as the mean ± S.D.