Biochemical regulation of mammalian AMP-activated protein kinase activity by NAD and NADH.
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 beta-NAD and NADH on this enzyme. The recombinant T172D-AMPK, which was mutated to mimic the phosphorylated state, was activated by beta-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.
Endocrine Discovery, Lilly Research Laboratories, Lilly Corporate Center, Indianapolis, IN 46285-0424, USA. PHAIL_RONIT_R@Lilly.com
Ronit
R
Rafaeloff-Phail
Liyun
L
Ding
Laura
L
Conner
Wu-Kuang
WK
Yeh
Don
D
McClure
Haihong
H
Guo
Kimberlee
K
Emerson
Harold
H
Brooks
D055372:AMP-Activated Protein Kinases; D000225:Adenine / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000737:chemistry; D001667:Binding, Competitive; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D006801:Humans; D020128:Inhibitory Concentration 50; D007700:Kinetics; D008956:Models, Chemical; D009097:Multienzyme Complexes / Q000737:chemistry*; D009132:Muscles / Q000378:metabolism; D009243:NAD / Q000737:chemistry*; D010084:Oxidation-Reduction; D010455:Peptides / Q000737:chemistry; D010766:Phosphorylation; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry; D014162:Transfection
D009097:Multienzyme Complexes; D010455:Peptides; D011994:Recombinant Proteins; D009243:NAD; D000255:Adenosine Triphosphate; D017346:Protein Serine-Threonine Kinases; D055372:AMP-Activated Protein Kinases; D000225:Adenine