Figure 2.

Opposing effects of anionic phospholipids regulate the formation of active PDK1 homodimers. All experiments were performed under resting conditions (−), stimulation of the PI3K pathway with PDGF (+) or treated with the PI3K inhibitor (LY294002) prior to stimulation. NIH3T3 cells were cotransfected with GFP-PDK1 and PDK1-mCherry. (A) Representative intensity and FRET-FLIM f_D images. (B) Relative distribution of PDK1 at the PM and the cytoplasm of NIH3T3 cells. The dashed line indicates the reference level for a control cytoplasmic protein (Table 1). (C) Quantification of the homodimerisation efficiency of PDK1 wild type and mutants. E_D calculation is based on FRET-FLIM measurements on a single-cell level, as illustrated in (A). (D) Localisation of the PDK1 homodimers in NIH3T3 cells. The graph shows the ratio of PDK1 dimers at the PM and the cytoplasm based on the segmentation of the f_D images shown in (A). (E) Phosphorylation of Akt/PKB at T308 in NIH3T3 cells. The upper panel shows a representative western blot of phosphorylated Akt/PKB on T308 and of total Akt/PKB (pan AKT). The bars show the ratio of T308 phosphorylation over total protein. The data were normalised to the condition basal wild type PDK1. Scale bar: 20 µm. (B) N > 30. Error bars: SEM. (C,D) N > 30. Box: 2xSEM (95.4% confidence); Whiskers: 80% population. Mann-Whitney test *p < 0.05. (B-E) Three independent experiments.