Study On The Regulatory Mechanism Of AMP-activated Protein Kinase (AMPK)

AMPK (AMP-activated protein kinase) is a conserved heterotrimeric protein kinase. Its catalytic activity can be activated by AMP, which enables it to maintain the balance between ATP production and consumption (energy homeostasis) in eukaryotic cells by sensing the cellular energy status. It also plays important roles in the regulation of cell growth and proliferation, in the establishment and maintenance of cell polarity, in regulation of animal life span and circadian clock. These important functions have rendered AMPK a major drug target for obesity, type 2 diabetes and certain cancer treatments. However, the regulation mechanism of AMPK activity by AMP-binding remains unsolved.Here we solved the crystal structures of a fragment of AMPKα-subunit (KD-AID) from fission yeast that contains the catalytic kinase domain and an autoinhibitory domain (AID). AID binds, from the"backside", to the hinge region of its KD, forming contacts with both amino-terminal and carboxy-terminal lobes. Structural analysis indicated that AID-binding might constrain the mobility of helixαC, hence resulting in an autoinhibited KD-AID with much lower kinase activity than that of the kinase domain alone. AMP activates AMPK both allosterically and by inhibiting dephosphorylation of T172 site onαsubunit. Our further in vitro kinetic studies demonstrated that disruption of the KD-AID interface reverses the autoinhibition and these AMPK heterotrimeric mutants no longer respond to the change of AMP concentration. The structural and biochemical data have revealed the primary mechanism of AMPK autoinhibition. Based on these data, we proposed a conformational switch model for AMPK activation by AMP.In addition, we questioned the position of glycogen binding domain (GBD) proposed by previous studies. And a new possible position of GBD has been proposed, along with the corresponding physiological significance. The crystallization conditions of AMPK holoenzyme have also been screened, which provides the basis for further structure study.Over all, these results have contributed greatly in understanding the mechanism of AMPK regulation.