Question: 

AKT-GSK3b -signalling is a central mediator of signal transduction in cell survival, cell growth and metabolism. AKT acts downstream of receptor tyrosin kinases and is activated by phosphorylation. To analyse specific signalling, complex formation of the AKT 1 and 2-isoforms and GSK3b was examined.

Methods: 

Tandem Affinity Purification (TAP) allows purification of proteins from cells under native conditions. To copurify interacting proteins, AKT1, AKT2 and GSK3b were fused with a TAP-Tag, stably expressed in HEK293-cells and purified via TAP. Interacting partners were identified via MS/MS. For quantification of interacting proteins, the proteom of the cells was labeled by stable isotopes in cell culture (SILAC).

Results: 

TAP was performed with AKT 1, AKT 2 and GSK3b. Whereas GSK3b formed stable and abundant complexes e.g. with PKA, TAPs of AKT 1 and 2 mainly identified HSP 90/cdc 37 as interacting partners. Other proteins were only found in low amounts. Among these, we identified for AKT1 CAD-protein, T-complex-protein and Methylosome protein 50 and for AKT 2 Elongation factor 1 and a-Enolase. Using SILAC, we analysed these low level interactions and found, that they appear to be due to weak interactions of AKT with its binding partners. First AKT 2-results show, that there is only a stable complex formation with HSP90/cdc37. Other proteins like GRP 78 or HSP 70 appear to interact more transiently with AKT 2.

Conclusion: 

Whereas GSK3 b-signalling involves the formation of highly stable protein complexes, AKT-isoforms appear to transport signals via specific but only transient interactions.