Conserved α-helix acts as autoinhibitory sequence in AMP-activated protein kinase α subunits

AMP-activated protein kinase (AMPK) acts as an energy sensor, being activated by metabolic stresses and regulating cellular metabolism. AMPK is a heterotrimer consisting of a catalytic a subunit and two regulatory subunits, beta and gamma. It had been reported that the mammalian AMPK a subunit contained an autoinhibitory domain (alpha 1: residues 313-392) and had little kinase activity. We have found that a conserved short segment of the a subunit (alpha 1-(313-335)), which includes a predicted a-helix, is responsible for a subunit autoinhibition. The role of the residues in this segment for autoinhibition was further investigated by systematic site-directed mutation. Several hydrophobic and charged residues, in particular Leu-328, were found to be critical for alpha 1 autoinhibition. An autoinhibitory structural model of human AMPK alpha 1-(1-335) was constructed and revealed that Val-298 interacts with Leu-328 through hydrophobic bonding at a distance of about 4 A and may stabilize the autoinhibitory conformation. Further mutation analysis showed that V298G mutation significantly activated the kinase activity. Moreover, the phosphorylation level of acetyl-CoA carboxylase, the AMPK downstream substrate, was significantly increased in COS7 cells overexpressing AMPK alpha 1-(1-394) with deletion of residues 313-335 (Delta alpha 394) and a V298G or L328Q mutation, and the glucose uptake was also significantly enhanced in HepG2 cells transiently transfected with Delta alpha 394, V298G, or L328Q mutants, which indicated that these AMPK al mutants are constitutively active in mammalian cells and that interaction between Leu-328 and Val-298 plays an important role in AMPK alpha autoinhibitor-gamma function.