SGK-1 inhibits FOXO3 transcriptional activity and subsequently LKB1 promoter activation.

<p>(<b>A</b>) Illustration of the LKB1 promoter region, extending from nucleotide position -2537 to +727 relative to the transcription start site. Positions of the transcription start site (TSS) and binding sites of transcription factors involved in LKB1 regulation (FOXO3, NF-Y and Sp1) as well as their positions relative to the transcription start site are indicated in brackets. (<b>B</b>) LKB1 reporter activity in HEK293 cells after ectopic expression of SGK-1 (dark grey) is compared to the activity obtained from co-transfections with the empty vector (Ctrl, light grey). Reporter activity is expressed as fold of luciferase activity (relative light units normalized to renilla luciferase activity) obtained from co-transfection of the plasmid containing the LKB1 promoter (nucleotides −2537 to +727) together with the empty expression vector (pcDNA3). Instead of the empty vector the same amount of either a FOXO3 wild-type expression plasmid (FOXO3 WT) or a SGK-1 phosphorylation site deficient triple mutant (T32A/S253A/S315A) of FOXO3 (FOXO3 A3) has been co-transfected. Each bar represents the means ± standard deviation of three measurements. (<b>C</b>) Western blot analysis of the same lysates used in the LKB1 reporter assay described in (B) using antibodies against phospho-FOXO3 (T32), total FOXO3 and actin. The ratios of p-FOXO3/FOXO3 for each experimental condition are indicated. The shown blot is a representative of three independent experiments.