Relative Expression Levels Rather Than Specific Activity Plays the Major Role in Determining In Vivo AKT Isoform Substrate Specificity
Figure 1
AKT3 is more active than AKT1 towards both the RPRAATF peptide and rpS7 protein substrate. HA-AKT isoforms were expressed in HEK293 cells, stimulated with 10% serum, 1 μM insulin, or 0.1 μM pervanadate and harvested into RLB. (a) expression of HA-AKT isoforms was detected by immunoblotting with the anti-HA antibody. (b) HA-AKT isoforms were immunoprecipitated from cleared protein lysates (100 μg) and assayed towards the RPRAATF peptide for activity. Samples were assayed in duplicate. . Error bars: mean ± SD. GST-AKT1 and GST-AKT3 were expressed in HEK293 cells, stimulated with 0.1 μM pervanadate, and harvested into RLB, then purified by GST-pull down and (c and d) assayed against increasing concentrations of the RPRAATF peptide or (e) rpS7. Data points were fitted to the Michaelis-Menten equation using GraphPad Prism version 5.00, GraphPad Software, San Diego, Calif, USA, http://www.graphpad.com/. , where samples were assayed in duplicate. Graph shows mean of duplicates.
