Relative Expression Levels Rather Than Specific Activity Plays the Major Role in Determining In Vivo AKT Isoform Substrate Specificity
Figure 8
Identification of eEF2 as a potential AKT substrate. HEK293 cells transfected with the pCDNA3 vector (control) or expressing similar levels of myrAKT1 or myrAKT3 were serum-starved for 24 hours prior to harvesting into RLB. Samples were processed in duplicate. Cy2 labelled protein samples (250 μg) were loaded onto 18 cm broad range IPG strips with a nonlinear pH range of 3–11, focused and resolved by SDS-PAGE. After 2DGE, gels were transferred onto Hybond-LFP membrane and then immunoblotted with the PAS antibody and Cy5-conjugated secondary antibody. Membranes were scanned using the Typhoon trio9100 for both Cy2 and Cy5 signals. Cy2 and Cy5 signals were overlayed using ImageQuant (GE Healthcare). Cy2 (total protein) signals are represented in red. Cy5 (PAS) signals are represented in green. Overlayed signals are represented in yellow. (a) control. (b–d) enlarged region of membrane containing control, myrAKT1 or myrAKT3 samples, respectively. Proteins more efficiently phosphorylated by myrAKT1 are circled in white, by myrAKT3 are circled in black, and with equal efficiencies for both are circled in blue. The four protein spots (spots 1–4) were excised from the myrAKT1 Coomassie R-250 stained gel (Supplementary Figure 2) and identified as eEF2 by mass spectrometry analysis (Supplementary Figure 3)..
