AKT2 is the predominant AKT isoform expressed in human skeletal muscle

Skeletal muscle physiology and metabolism are regulated by complex networks of intracellular signaling pathways. Among many of these pathways, the protein kinase AKT plays a prominent role. While three AKT isoforms have been identified (AKT1, AKT2, and AKT3), surprisingly little is known regarding isoform-specific expression of AKT in human skeletal muscle. To address this, we examined the expressions of each AKT isoform in muscle biopsy samples collected from the vastus lateralis of healthy male adults at rest. In muscle, AKT2 was the most highly expressed AKT transcript, exhibiting a 15.4-fold increase over AKT1 and AKT3 transcripts. Next, the abundance of AKT protein isoforms was determined using antibody immunoprecipitation followed by Liquid Chromatography-Parallel Reaction Monitoring/Mass Spectrometry. Immunoprecipitation was performed using either mouse or rabbit pan AKT antibodies that were immunoreactive with all three AKT isoforms. We found that AKT2 was the most abundant AKT isoform in human skeletal muscle (4.2-fold greater than AKT1 using the rabbit antibody and 1.6-fold greater than AKT1 using the mouse antibody). AKT3 was virtually undetectable. Next, cultured primary human myoblasts were virally-transduced with cDNAs encoding either wild-type (WT) or kinase-inactive AKT1 (AKT1-K179M) or AKT2 (AKT2-K181M) and allowed to terminally differentiate. Myotubes expressing WT-AKT1 or WT-AKT2 showed enhanced fusion compared to control myotubes, while myotubes expressing AKT1-K179M showed a 14% reduction in fusion. Myotubes expressing AKT2-K181M displayed 63% decreased fusion compared to control. Together, these data identify AKT2 as the most highly-expressed AKT isoform in human skeletal muscle and as the principal AKT isoform regulating human myoblast differentiation.