AMPK is an energy-sensing protein kinase that has over 100 validated downstream targets located within different cellular compartments.1 While investigating the localisation of AMPK to different subcellular compartments using immunofluorescence microscopy, we observed that a subset of AMPK co-localises with Golgi membranes in U2OS cells. Using pulldowns from cells stably expressing the HA-tagged Golgi membrane protein, Tmem115, we showed that endogenous AMPK-α1/2, -β1/2, and -γ1 are enriched in Golgi membrane fractions.
GBF1, a GEF for ARF1, which regulates retrograde trafficking between the cis-Golgi and the ER, was suggested to be a target for AMPK involved in Golgi function.2 We have demonstrated that GBF1 is phosphorylated at Thr1337 by AMPK upon its activation with either C13 or MK-8722, causing disassembly of the Golgi apparatus and regulating trafficking of proteins through the secretory pathway. These effects were abolished in AMPK-α1/α2 double knockout and GBF1 T1337A phospho-mutant U2OS cells, however, remained in the absence of an N-myristoyl group on AMPK-β subunit.
Additionally, GBF1 was suggested to control mitochondrial positioning by regulating a pool of ARF1 localised to the outer mitochondrial membrane that interacts with Miro1/2.3 Using spinning disk microscopy, we demonstrated that AMPK regulates mitochondrial positioning, although this was only partially attributable to phosphorylation of GBF1 at Thr1337. We have created GBF1 T1337E phospho-mimetic knock-in U2OS cells to further investigate the role of phosphorylation of this site by AMPK, and to study effects on Golgi and mitochondrial morphology. Our findings contribute to the role of AMPK in regulating Golgi and mitochondrial function.