Subcellular localisation of AMPK and effects on membrane trafficking

Abstract

AMPK is a heterotrimeric energy-sensing protein kinase comprised of a catalytic AMPKα subunit, a scaffolding AMPK-β subunit, and a regulatory AMPK-γ subunit. It appears to have originated in single-celled eukaryotes to promote survival during starvation and has evolved in multicellular organisms to integrate cellular metabolism with energy status and whole-body homeostasis by activating catabolic pathways and inhibiting anabolic pathways. AMPK is activated by: 1) increases in AMP:ATP and ADP:ATP ratios resulting from energy stress; 2) the AXIN-LKB1-LAMTOR1 pathway under low glucose conditions; 3) phosphorylation by CaMKK2, which occurs during increased concentrations of cytosolic Ca2+; and 4) binding of ligands to the ADaM site. Downstream targets of AMPK are localised within the cytosol in addition to most cellular compartments, suggesting that AMPK may interact with cellular membranes to phosphorylate these targets; it has been suggested that this is mediated by Nmyristoylation of the b subunits of AMPK.

This thesis provides evidence that AMPK is activated in the absence of glucose and in the presence of glutamine and pyruvate in U2OS cells, independently of changes in nucleotide ratios. However, this appears to be via a mechanism independent of the AXINLKB1-LAMTOR pathway. AMPK phosphorylates ER transmembrane enzyme, HMGCoA reductase, the membrane-associated proteins, Raptor and GBF1, and the cytosolic enzyme, ACC, independent of the mechanism by which it is activated and independent of N-myristoylation of AMPK-β subunit, with the exception of HMG-CoA reductase, which was not phosphorylated by non-myristoylatable recombinant AMPK. The absence of an N-myristoyl anchor increased the basal activity of AMPK that was associated with increased phosphorylation of ACC and AMPK-α Thr172, suggesting that N-myristoylation may affect either the phosphorylation or dephosphorylation of AMPK.

In U2OS cells, small pools of AMPK were located at the Golgi apparatus, the mitochondria network, and lysosomes. The activation of AMPK with C13, MK-8722, or A-23187 did not affect the association of AMPK with these compartments, but it did induce Golgi disassembly and re-positioning of the mitochondrial network. These effects were mimicked by an inhibitor (Golgicide A) of the ARF1 guanine nucleotide exchange factor, GBF1, and were associated with phosphorylation of the AMPK site on GBF1, Thr1337, and were abolished in AMPK-α1/-α2 knockout cells. Furthermore, AMPK was significantly increased in the Golgi pull-down fraction upon activation with either C13, MK-8722, or A-23187 compared to controls.

Date of Award	2023
Original language	English
Awarding Institution	University of Dundee
Supervisor	Grahame Hardie (Supervisor) & Hari Hundal (Supervisor)