ABSTRACT
Skeletal muscle contraction results in the phosphorylation and activation of the AMP-activated protein kinase (AMPK) by an upstream kinase (AMPKK). The LKB1-STE-related adaptor (STRAD)-mouse protein 25 (MO25) complex is the major AMPKK in skeletal muscle; however, LKB1-STRAD-MO25 activity is not increased by muscle contraction. This relationship suggests that phosphorylation of AMPK by LKB1-STRAD-MO25 during skeletal muscle contraction may be regulated by allosteric mechanisms. In this study, we tested an array of metabolites including, glucose 6-phosphate, fructose 6-phosphate, fructose 1,6-bisphosphate, 3-phosphoglycerate (3-PG), glucose 1-phosphate, glucose 1,6-bisphosphate, ADP, carnitine, acetylcarnitine, IMP, inosine, and ammonia for allosteric regulation. ADP inhibited both AMPK and LKB1-STRAD-MO25 actions, but probably is not important physiologically because of the low free ADP inside the muscle fiber. We found that 3-PG stimulated LKB1-STRAD-MO25 activity and allowed for increased AMPK phosphorylation. 3-PG did not stimulate LKB1-STRAD-MO25 activity toward the peptide substrate LKB1tide. These results have identified 3-PG as an AMPK-specific regulator of AMPK phosphorylation and activation by LKB1-STRAD-MO25.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Adaptor Proteins, Vesicular Transport/metabolism , Glyceric Acids/pharmacology , Multienzyme Complexes/metabolism , Protein Serine-Threonine Kinases/metabolism , AMP-Activated Protein Kinase Kinases , AMP-Activated Protein Kinases , Adenosine Diphosphate/metabolism , Animals , Calcium-Binding Proteins , Enzyme Activation/drug effects , Glyceric Acids/metabolism , Mice , Protein Binding , Protein Kinases/metabolismABSTRACT
Activation of the AMP-activated protein kinase (AMPK) results in acute changes in cellular metabolism and transcriptional events that make the cell more robust when encountering an energy challenge. AMPK is thought to be inhibited by glycogen, the major storage form of intracellular carbohydrate. We hypothesized that long-chain acyl-CoA esters (LCACEs) might also inhibit AMPK signaling. Cytosolic LCACEs are available for immediate transport and oxidation within the mitochondria and accordingly may be representative of the lipid energy charge of the cell. We found that LCACEs inhibited phosphorylation of AMPK by the recombinant AMPK kinase (AMPKK) LKB1/STRAD/MO25 in a concentration-dependent manner. Palmitoyl-CoA (PCoA) did not affect the activity of phosphothreonine-172 AMPK. PCoA potently inhibited AMPKK purified from liver. Conversely, PCoA stimulated the kinase activity of LKB1/STRAD/MO25 toward the peptide substrate LKB1tide. Octanoyl-CoA, palmitate, and palmitoylcarnitine did not inhibit AMPKK activity. Removal of AMP from the reaction mixture resulted in reduced AMPKK activity in the presence of PCoA. In conclusion, these results demonstrate that the AMPKK activity of LKB1/STRAD/MO25 is substrate specific and distinct from the kinase activity of LKB1/STRAD/MO25 toward the peptide substrate LKB1tide. They also demonstrate that LCACEs inhibit the AMPKK activity of LKB1/STRAD/MO25 in a specific manner with a dependence on both a long fatty chain and a CoA moiety. These results suggest that the AMPK signaling cascade may directly sense and respond to the lipid energy charge of the cell.
