Question:

The protein kinase AKT acts as a signaling node in the cell and has widespread functions in cell metabolism, cell growth and apoptosis. To investigate AKT-mediated signal transduction we examined protein interactions of AKT2.

Methods:

Screening for AKT2- interaction partners was performed by a combination of Tandem affinity purification (TAP) and quantitative mass spectrometry using HEK 293 cells stably expressing TAP-tagged AKT2. For the verification of possible candidates the Proximity Ligation Assay (PLA) in HEK293 cells and neonatal rat cardiomyocytes was used. Further functional analysis of confirmed binding partners was performed by measuring enzymatic activity.

Results:

As AKT2-interacting proteins we discovered Heat Shock Protein 90 (HSP 90), Heat shock protein 70 (HSP 70) Glucose regulated protein 78 (GRP 78), Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), Elongation factor 2 (EF2) and Enolase 1 (ENO1). Complex stability was analyzed via SILAC and revealed, that AKT2 is stably bound only to the HSP90/cdc37-complex whereas other proteins like GRP78 or Tubulin showed a low affinity-binding to AKT2. Proximity ligation assay (PLA) verified EF2 and ENO1 as new AKT2-specific protein interaction partners. Moreover, in embryonic rat cardiomyocytes, activation of AKT in response to insulin and angiotensin II induced a dissociation of the AKT-EF2 complex, whereas inhibition of the PI3-kinase pathway (LY294002) stabilized this complex. Despite complex formation, ENO1 enzymatic activity after AKT-inhibition and -stimulation did not reveal a relevant influence of AKT on ENO1 enzymatic activity.

Conclusion:

Our data show that transient protein interactions play an important role in AKT2-signaling. With EF2 and ENO1 we discovered new AKT2-binding proteins, which point to new AKT2-mediated effects in glycolysis (ENO1) and protein synthesis (EF2).