AKT is a serine-threonine protein kinase, which plays a central role in cell metabolism, growth, survival and apoptosis. AKT is acting in response to receptor tyrosine kinase stimulation such as the insulin receptor and to G-Protein-coupled receptors, like the ATII-receptor. The activation of AKT occurs in a PI3-Kinase dependent manner via membrane-translocation. Activated AKT phosphorylates a high number of downstream targets, for example GSK3ß, mTor or caspase 9.

There are three isoforms of AKT (AKT1-3). AKT2 and 3 share a high sequence homology with AKT1 of approx. 80%. Nevertheless, there are hints for different functions of the different isoforms: whereas AKT1 seems to play a major role in cell growth, AKT2 is more involved in glucose metabolism and AKT3 affects postnatal brain growth.

The aim of this project is to investigate AKT-isoform-specific signalling via isolation of AKT associated protein complexes and the identification of the protein components. To achieve this goal we expressed N-and C-terminally tagged AKT2 in HEK 293 cells. For the isolation of complexes under native conditions we use a novel 8 kDa Tag consisting of HA-, StrepII- and Flag-Tag which allows tandem affinity purification (TAP).

In cells expressing high levels of the recombinant AKT its regulation by phosphorylation was impaired, because we found AKT2-Tag to be phosphorylated even under PI3-Kinase inhibition. In contrast low level expression by stably transfected cells resulted in normal phosphorylation. Immunhistochemistry also showed that AKT2-Tag behaved like endogenous AKT in response to IGF-1 stimulation and PI3-Kinase inhibition by Ly 294002. TAP of protein complexes resulted in purification of several proteins which copurify with N-terminally but not C-terminally tagged AKT2. ESI-MS-MS identified HSP90, ATP-synthase, subunit alpha and phosphoglycerat-dehydrogenase as first possible binding partners.

Our data show that AKT2 mediated signal transduction appears to involve the formation of multiple protein complexes.