Biochemical Regulation of Mammalian AMP-activated Protein Kinase Activity by NAD and NADH

• Published in: The Journal of biological chemistry Vol. 279; no. 51; pp. 52934 - 52939
• Main Authors: Rafaeloff-Phail, Ronit, Ding, Liyun, Conner, Laura, Yeh, Wu-Kuang, McClure, Don, Guo, Haihong, Emerson, Kimberlee, Brooks, Harold
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 17-12-2004; American Society for Biochemistry and Molecular Biology
• Subjects: Adenine - chemistry; Adenosine Triphosphate - chemistry; AMP-Activated Protein Kinases; Binding, Competitive; Cell Line; Dose-Response Relationship, Drug; Humans; Inhibitory Concentration 50; Kinetics; Models, Chemical; Multienzyme Complexes - chemistry; Muscles - metabolism; NAD - chemistry; Oxidation-Reduction; Peptides - chemistry; Phosphorylation; Protein Binding; Protein-Serine-Threonine Kinases - chemistry; Recombinant Proteins - chemistry; Transfection
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M409574200

AMP-activated protein kinase (AMPK) serves as an energy-sensing protein kinase that is activated by a variety of metabolic stresses that lower cellular energy levels. When activated, AMPK modulates a network of metabolic pathways that result in net increased substrate oxidation, generation of reduced nucleotide cofactors, and production of ATP. AMPK is activated by a high AMP:ATP ratio and phosphorylation on threonine 172 by an upstream kinase. Recent studies suggest that mechanisms that do not involve changes in adenine nucleotide levels can activate AMPK. Another sensor of the metabolic state of the cell is the NAD/NADH redox potential. To test whether the redox state might have an effect on AMPK activity, we examined the effect of β-NAD and NADH on this enzyme. The recombinant T172D-AMPK, which was mutated to mimic the phosphorylated state, was activated by β-NAD in a dose-dependent manner, whereas NADH inhibited its activity. We explored the effect of NADH on AMPK by systematically varying the concentrations of ATP, NADH, peptide substrate, and AMP. Based on our findings and established activation of AMPK by AMP, we proposed a model for the regulation by NADH. Key features of this model are as follows. (a) NADH has an apparent competitive behavior with respect to ATP and uncompetitive behavior with respect to AMP resulting in improved binding constant in the presence of AMP, and (b) the binding of the peptide is not significantly altered by NADH. In the absence of AMP, the binding constant of NADH becomes higher than physiologically relevant. We conclude that AMPK senses both components of cellular energy status, redox potential, and phosphorylation potential.