The AMPK alpha-beta-gamma heterotrimer is a highly conserved serine/threonine protein kinase that acts as a metabolic fuel sensor and is crucial for maintaining cellular energy homeostasis. It forms complexes in a 1:1:1 ratio made of unique subunit isoform variations (alpha1, alpha2, beta1, beta2, gamma1, gamma2, gamma3) and is subject to modifications such as phosphorylation (on all subunits) and NH2-terminal myristoylation of the beta-subunit. Each isoform depicts its own distinct tissue-expression signature, where gamma3 shows the highest selectivity as it is exclusively expressed in skeletal muscle. AMPK is capable of stimulating glucose uptake independently of insulin, hence, the gamma3 isoform is recognised as a promising therapeutic target for metabolic disorders such as type II diabetes. However, this isoform is yet to undergo structural characterisation and has only been looked at briefly in a functional perspective. The gamma2 and gamma3 subunits contain large NH2-terminal extensions that don’t exist on gamma1, and despite high throughput phosphoproteomic studies identifying >50 phosphorylation sites on the gamma2 extension, only two sites have been identified on the gamma3 isoform. We believe this is due to an underrepresentation of gamma3 in these studies and that more sites are yet to be discovered.
In the present study, we are using a bacterial tricistronic expression construct that expresses AMPK gamma 3 complexes which will be used in cryo-EM experiments to elucidate the structure of gamma3. We have utilised HEK293T/17-expressed AMPK to characterise AMPK allosteric activation in response to different gamma-binding activators, which corroborate with previous findings that subunit composition is an important determinant of AMPK activation characteristics and that gamma3 heterotrimeric complexes are rather recalcitrant to allosteric stimulation. To further examine the function of the gamma3 NH2-terminal extension, mass spectrometry will be employed to identify novel phosphorylation sites, which will then be characterised using a range of biochemical techniques.