AKT1 and AKT2 Induce Distinct Phosphorylation Patterns in HL‑1 Cardiac Myocytes

The protein kinase AKT is a central
kinase in the heart and has
a major impact on growth/hypertrophy, survival/apoptosis, and metabolism.
To gain more insight into AKT isoform-specific signaling at the molecular
level, we investigated the phosphoproteome of HL-1 cardiomyocytes
carrying AKT1 or AKT2 isoform-specific knock down, respectively. We
combined stable isotope labeling with high resolution mass spectrometry
and identified 377 regulated phosphopeptides. Although AKT1 is expressed
at 4-fold higher levels, insulin stimulation mainly activated AKT2,
which might in part rely on a preferred interaction of AKT2 with the
mammalian target of rapamycin complex 2. In line with this result,
the highest number of regulated phosphopeptides was identified in
the AKT2 knock down cells. Isoform-specific regulation of AKT targets
not previously described could be observed, and specific regulation
of indirect target sites allows a deeper insight into affected biological
processes. In the myocardial context, we identified many phosphosites
supporting a connection of AKT to excitation–contraction coupling.
Phosphoproteins identified included L-type calcium channel, ryanodine
receptor, junctophilin, histidine-rich calcium binding protein, phospholamban,
heat shock protein beta-6, and Ca2+/calmodulin-dependent
kinase II. In conclusion, AKT isoform-specific knock down combined
with quantitative phosphoproteomics provided a powerful strategy to
unravel AKT isoform-specific signaling.