Figure 7.

Effects of mutation of PS-binding residues on Akt downstream signaling and apoptotic cell death induced by serum starvation. Neuro 2A cells were transfected with GFP-Akt WT or mutants for 36 h and subjected to serum starvation for 48 h. (a) GFP-Akt (R15A) or GFP-Akt (K20A) expressed in Neuro 2A cells showed significantly impaired phosphorylation. Phosphorylation of GSK-3β, FOXO1, and Bad, the downstream signaling of the Akt activation, was also reduced in cells expressing these mutants, with a concomitant increase in active caspase-3. (b) Cells expressing the mutants showed significantly more TUNEL-positive cells (green + red = orange–∼yellow) in comparison to cells expressing GFP-Akt WT. TUNEL-positive cells were stained red using Click-iT TUNEL assay kit with Alexa Fluor 594 Azide. Nuclei were stained with Hoechst 33342. Bar, 30 µm (c) The percentages of GFP- and TUNEL-positive cells were determined by counting the total of 500–1,000 GFP-Akt WT or GFP-Akt mutant expressing cells from six randomly selected fields. Inset, Western blot analysis indicating comparable expression of WT and mutant GFP-Akt. Results represent two independent experiments performed in duplicates. Serum-sufficient cells transfected with WT or mutants showed negligible cell death. Statistical analysis was performed by post-hoc Tukey honestly significant difference test at the significance level of P < 0.05. Different letters indicate statistically significant differences.