AMP-activated protein kinase (AMPK) is a central energy sensor and regulator of cellular energy state, but the AMPK signaling network is still incompletely understood. Two earlier non-biased screens for AMPK interaction partners and substrates performed in the laboratory identified several candidate proteins, but functional and physiological roles remained unclear. Here we characterized the functional relationship of AMPK with four different protein interaction partners: gluthatione S-transferases (GSTP1 and GSTM1), fumarate hydratase (FH), an E3 ubiquitin-ligase (NRDP1), and vesicle-associated membrane proteins (VAMP2 and VAMP3). Each of these interaction partners seems to have a different function in AMPK signaling, either acting up- or down-stream of AMPK. GSTP1 and GSTM1 can contribute to AMPK activation by facilitating S-glutathionylation of AMPK under mildly oxidative conditions. This non-canonical regulation suggests AMPK as a sensor of cellular redox state. Mitochondrial FH was identified as the only clear AMPK downstream substrate, but surprisingly the phosphorylation site is present in the mitochondrial targeting prepeptide, possibly affecting mitochondrial import. NRDP1, whose expression as a full-length soluble protein was achieved here for the first time, is phosphorylated by AMPK only at low levels. The interaction does neither serve for AMPK ubiquitinylation, but rather affects NRDP1 turnover. Finally, interaction of VAMP2/3 with AMPK does not involve phosphorylation or activation events of one of the partners. Instead, we propose VAMP2/3 as scaffolding proteins that recruit AMPK to exocytotic vesicles which could favor phosphorylation of vesicular AMPK substrates for exocytosis. Collectively, our results add some new elements to the AMPK signaling network, suggesting that it is much more complex than anticipated. In addition to upstream kinases and downstream substrates, regulation of AMPK signaling occurs by secondary protein modifications other than phosphorylation, by effects on protein turnover, and probably also by specific subcellular recruitment of AMPK.
