FIGURE 1.

c-Jun regulates PDK1 transcription. A, c-Jun regulates PDK1 expression. Protein extracts (40 μg) from SW1 cells stably transfected with FLAG-TAM67 and control SW1 cells were blotted with PDK1 and FLAG antibodies. B, PDK1 levels are regulated by c-Jun expression. Protein extracts (40 μg) from c-Jun^+/+ fibroblasts and c-Jun^−/− fibroblasts were blotted with the indicated antibodies. C, c-Jun siRNA decreases PDK1 protein levels in melanoma. Protein extracts (40 μg) from Lu1205 cells transfected with scrambled (sc) oligonucleotides or a siRNA specific for c-Jun were analyzed by Western blots using the indicated antibodies. D, PDK1 activity is reduced in c-Jun^−/− fibroblasts. Protein extracts from c-Jun^+/+ or c-Jun^−/− fibroblasts were immunoprecipitated with PDK1 antibody, and PDK1 kinase activity was measured by an in vitro kinase assay as indicated under “Experimental Procedures.” Results shown are mean ± S.D. of three independent experiments. E, activation of the JNK/c-Jun pathway increases PDK1 expression. Human embryonic kidney 293T cells were transfected with 0.2 μg of ΔMEKK1 or empty pEF plasmid. Protein extracts were obtained 48 h post-transfection and blotted with the indicated antibodies. The arrow indicates bands corresponding to PDK1 and ΔMEKK1. F, inhibition of c-Jun decreases PDK1 mRNA levels. Relative levels of PDK1 mRNA were determined by real time PCR in c-Jun^−/− and control fibroblasts and in control SW1 and SW1-TAM67 cells. Results shown are mean ± S.D. of the respective relative concentrations. A representative experiment (of three performed) is shown.