Figure 5.

Reduced, but active, signaling from Akt T308S. A, serum-starved COS-7 cells expressing BKAR with or without minimally detectable levels of Cherry-tagged Akt were imaged during stimulation with EGF followed by treatment with the Akt inhibitor GDC 0068. Normalized average FRET ratios for WT (blue), T308S (red), and T308S/S473A (green) compared with BKAR alone (gray) are shown. Data were analyzed from cells expressing equal low levels of Cherry-Akt. Multiple cells were included from at least two independent experiments. FRET ratios from each cell were normalized, and their average was plotted over time. Error bars, S.E. B, following a 10-min treatment with EGF, lysates from serum-starved COS-7 cells expressing the indicated Cherry-tagged Akt were analyzed by Western blotting for Akt activation using phospho-specific antibodies toward the activation loop (α-p308) and hydrophobic motif (α-p473). *, migration of Cherry-Akt; −, endogenous Akt.