Conserved alpha-helix acts as autoinhibitory sequence in AMP-activated protein kinase alpha 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 alpha subunit and two regulatory subunits, beta and gamma. It had been reported that the mammalian AMPK alpha subunit contained an autoinhibitory domain (alpha1: residues 313-392) and had little kinase activity. We have found that a conserved short segment of the alpha subunit (alpha1-(313-335)), which includes a predicted alpha-helix, is responsible for alpha 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 alpha1 autoinhibition. An autoinhibitory structural model of human AMPK alpha1-(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 alpha1-(1-394) with deletion of residues 313-335 (Deltaalpha394) and a V298G or L328Q mutation, and the glucose uptake was also significantly enhanced in HepG2 cells transiently transfected with Deltaalpha394, V298G, or L328Q mutants, which indicated that these AMPK alpha1 mutants are constitutively active in mammalian cells and that interaction between Leu-328 and Val-298 plays an important role in AMPK alpha autoinhibitory function.
Pang
Tao
T
Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Graduate School of the Chinese Academy of Sciences, Shanghai 201203, China.
Xiong
Bing
B
Li
Jing-Ya
JY
Qiu
Bei-Ying
BY
Jin
Guo-Zhang
GZ
Shen
Jin-Kang
JK
Li
Jia
J
D055372:AMP-Activated Protein Kinases; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D002522:Chlorocebus aethiops; D004252:DNA Mutational Analysis; D004926:Escherichia coli / Q000378:metabolism; D017353:Gene Deletion; D006801:Humans; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000737:chemistry*; D011487:Protein Conformation; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry*; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid
D009097:Multienzyme Complexes; D017346:Protein Serine-Threonine Kinases; D055372:AMP-Activated Protein Kinases; C526180:PRKAA1 protein, human