Using a nanofluidic proteomic immunoassay (NIA), consisting of isoelectric focusing followed by sensitive chemiluminescence detection, we demonstrate that under basal and ligand-induced conditions that the pattern of phosphorylation events is markedly different between AKT1 and AKT2. 
Nanofluidic proteomic immunoassay (NIA)
Coordinate phosphorylation of multiple residues on single AKT1 and AKT2 molecules.Aberrant AKT activation is prevalent across multiple human cancer lineages providing an important new target for therapy.  Twenty-two independent phosphorylation sites have been identified on specific AKT isoforms likely contributing to differential isoform regulation.  However, the mechanisms regulating phosphorylation of individual AKT isoform molecules have not been elucidated because of the lack of robust approaches able to assess phosphorylation of multiple sites on a single AKT molecule.  Using a nanofluidic proteomic immunoassay (NIA), consisting of isoelectric focusing followed by sensitive chemiluminescence detection, we demonstrate that under basal and ligand-induced conditions that the pattern of phosphorylation events is markedly different between AKT1 and AKT2.  Indeed, there are at least 12 AKT1 peaks and at least 5 AKT2 peaks consistent with complex combinations of phosphorylation of different sites on individual AKT molecules.  Following insulin stimulation, AKT1 was phosphorylated at Thr308 in the T-loop and Ser473 in the hydrophobic domain.  In contrast, AKT2 was only phosphorylated at the equivalent sites (Thr309 and Ser474) at low levels.  Further, Thr308 and Ser473 phosphorylation occurred predominantly on the same AKT1 molecules, whereas Thr309 and Ser474 were phosphorylated primarily on different AKT2 molecules.  PI3K inhibition decreased pThr451 on AKT2 but not pThr450 on AKT1.  Thus, NIA technology provides an ability to characterize coordinate phosphorylation of individual AKT molecules providing important information about AKT isoform-specific phosphorylation, which is required for optimal development and implementation of drugs targeting aberrant AKT activation.
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