A key property of complex biological systems is the presence of interaction networks crucial for all levels of cellular function, including the regulation of cellular energy. Two enzymes take center stage in the regulation of cellular and whole body energy metabolism. Creatine kinase (CK) functions as an intracellular energy storage and transport system playing a crucial role in the acute response to increasing energy demands. AMP-activated protein kinase (AMPK) regulates cellular and whole body energy delivery and consumption. We first applied an original cytosolic Y2H screen to identify new protein-protein interactions of cytosolic brain type CK (BCK) and AMPK in human brain. Various interaction candidates were identified, among them vesicle associated membrane proteins (VAMPs) interacting with both kinases. AMPK-VAMP interaction was confirmed by co-immunoprecipitation from synaptic vesicles, but did not lead to VAMP phosphorylation, suggesting that VAMP rather recruits AMPK for a regulation of exo- and endocytotic processes. A second strategy combining a biophysical interaction screen (Surface Plasmon Resonance, SPR) with an in vitro phosphorylation assay revealed rather AMPK isoform-specific targets. One is fumarate hydratase, which is preferentially phosphorylated by AMPK221, leading to an increase in enzyme activity in vitro. Finally, one class of interaction candidates, the glutathione S-transferases GSTM1 and -P1, were characterized in detail by a panel of interactomics methods (Y2H, SPR, co-immunoprecipitation) as reliable, high affinity interactors and identified as new AMPK substrates. In case of GSTP1, this leads to an increase in its enzymatic activity, suggesting a direct role of AMPK signaling in oxidative stress defense.
