Conserved α-Helix Acts as Autoinhibitory Sequence in AMP-activated Protein Kinase α Subunits

Abstract

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 ␣ subunit and two regulatory subunits, ␤ and ␥. It had been reported that the mammalian AMPK ␣ subunit contained an autoinhibitory domain (␣1: residues 313-392) and had little kinase activity. We have found that a conserved short segment of the ␣ subunit (␣1-(313-335)), which includes a predicted ␣-helix, is responsible for ␣ 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 ␣1 autoinhibition. An autoinhibitory structural model of human AMPK ␣1-(1-335) was constructed and revealed that Val-298 interacts with Leu-328 through hydrophobic bonding at a distance of about 4 Å 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 ␣1-(1-394) with deletion of residues 313-335 (⌬␣394) and a V298G or L328Q mutation, and the glucose uptake was also significantly enhanced in HepG2 cells transiently transfected with ⌬␣394, V298G, or L328Q mutants, which indicated that these AMPK ␣1 mutants are constitutively active in mammalian cells and that interaction between Leu-328 and Val-298 plays an important role in AMPK ␣ autoinhibitory function.The AMP-activated protein kinase (AMPK) 2 is a sensor of cellular energy state, being activated by a large variety of cellular stresses that increase cellular AMP and decrease ATP levels, such as glucose deprivation, hypoxia, oxidative stress, heat shock, and ischemia (1-3). AMPK is also activated by physiological stimuli, such as exercise, muscle contraction, hormones like leptin and adiponectin, pharmacological agents like thiazolidinediones and metformin, and a widely used AMPK activator, 5-aminoimidazole-4-carboxamide-1-␤-D-ribofuranoside (4 -10), modulating multiple metabolic pathways (11). Therefore, AMPK has been investigated for the treatment of type II diabetes, obesity, and even cancer (12).AMPK is a heterotrimeric serine/threonine protein kinase consisting of a catalytic ␣ subunit and two regulatory subunits, ␤ and ␥ (13). In mammals, each AMPK subunit has multiple isoforms, ␣1, ␣2, ␤1, ␤2, ␥1, ␥2, and ␥3 (14), suggesting that multiple heterotrimeric complexes may exist in different tissues and play different roles. Optimal kinase activity requires the formation of a heterotrimeric complex, involving AMP allosteric activation, and phosphorylation on Thr 172 within the activation loop of the catalytic ␣ subunit by an upstream kinase, AMPK kinase (AMPKK), identified as LKB1 or calcium/calmodulin-dependent protein kinase kinase ␤ (CAMKK␤) (13,(15)(16)(17)(18)(19)(20...

AMP-activated protein kinase: an emerging drug target to regulate imbalances in lipid and carbohydrate metabolism to treat cardio-metabolic diseases

“…Phosphorylation of Thr-172 within this loop is critical for enzyme activity ( 18 ). In addition to regulation through phosphorylation, AMPK ␣ -subunit activity has been shown to be self-regulated by a region identifi ed C-terminal to the kinase domain ( 19,20 ). This autoinhibitory sequence (AIS) is similar to ubiquitinassociated domains, which are highly conserved sequences phosphorylating and reducing DNA binding of the glucose-sensitive ChREBP ( 59 ), resulting in effects on hepatic lipogenic gene targets, which signifi cantly overlap with effects observed on SREBP1 ( Fig.…”

“…Studies performed using AIS deletion constructs show a greater than 10-fold increase in kinase activity as compared with constructs containing the AIS ( 19,20 ).…”

Adenosine Monophosphate-Activated Protein Kinase: A Central Regulator of Metabolism with Roles in Diabetes, Cancer, and Viral Infection

“…1) by the "autoinhibitory domain," so called because truncated constructs containing this domain plus the kinase domain are much less active that those containing the kinase domain alone (Goransson et al 2007;Pang et al 2007). Figure 1.…”

Down-regulation of adenosine monophosphate–activated protein kinase activity: A driver of cancer

“…Another study suggests that the 313-392 sequence may inhibit AMPK function by affecting the accessibility of substrates to the catalytic cleft of AMPK kinase. 17 Through structural modeling following truncation, deletion, and site-directed mutagenesis of the human AMPK α1 catalytic subunit, Pang et al 18 found that the fragment of 313-335 residues with the α-helix plays an important role in the inhibition of α1 catalytic subunit activity. Further study found that leucine 328 is the key residue for α1 subunit activity.…”

“…46 AMPK activation may also increase the expression of ubiquitin E3 ligase or MAFBx/Atrogin-1 and MuRF1 ubiquitin enzymes, which in turn mediate a negative feedback regulation. 47 Residues 313-548 of AMPK α1 subunit are the potential targets of ubiquitination, 18 as deletion of these amino acids increases AMPK α subunit protein halflife by fourfold. 16 PEST domain between the α1 subunit residues 504 and 526 may also contribute to the rapid degradation of AMPK α1 subunit by ubiquitin-26S proteasome pathway.…”