The Regulation of AMP-Activated Protein Kinase by H 2 O 2 Sang-Lim Choi,* Soo-Ja Kim,† Kyung-Tae Lee,‡ Joungmok Kim,* James Mu,§ Morris J. Birnbaum,§ Sung Soo Kim,* and Joohun Ha* ,1 *Department of Molecular Biology, East–West Medical Research Center, College of Medicine, †Department of Chemistry, and ‡Biochemistry Laboratory, College of Pharmacy, Kyung Hee University, Seoul 130-701, Korea; and §Howard Hughes Medical Institute, Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104 Received August 6, 2001 AMP-activated protein kinase (AMPK), a heterotri- meric serine/threonine kinase, is activated by condi- tions leading to an increase of the intracellular AMP: ATP ratio. However, how AMPK is regulated under the oxidative stress is completely unknown. In the present study, we examined effects of the oxidative agent H 2 O 2 on AMPK. AMPK was transiently and concentration- dependently activated by H 2 O 2 in NIH-3T3 cells. This activation was tightly associated with an increased AMP:ATP ratio, an electrophoretic mobility shift of AMPK 1 catalytic subunit, and an increased phos- phorylation level of AMPK 1 threonine 172, which is a major in vitro phosphorylation site by the upstream AMPK kinase. All of these events were significantly blocked by the pretreatment of 0.5% dimethyl sulfox- ide, a potent hydroxyl radical scavenger, indicating that AMPK cascades are highly sensitive to the oxida- tive stress. Interestingly, a specific tyrosine kinase in- hibitor, genistein, further stimulated the H 2 O 2 - induced AMPK activity by 70% without altering the AMP:ATP. Taken together, our results suggest that AMP:ATP ratio is the major parameter to which AMPK responds under the oxidative stress, but AMPK may be regulated in part by a tyrosine kinase-dependent path- way, which is independent of the cellular adenosine nucleotides level. © 2001 Academic Press Key Words: AMP-activated protein kinase; oxidative stress; H 2 O 2 ; ATP; AMP; tyrosine kinase. Mammalian AMP-activated protein kinase (AMPK), a heterotrimeric serine/threonine kinase consisting of a catalytic  subunit and two regulatory  and  sub- units, plays a key role in the regulation of energy homeostasis (reviewed in Refs. 1–3). Elevation of intra- cellular AMP activates AMPK, and this is mediated via several distinct mechanisms. First, AMP causes direct allosteric activation of AMPK (4). Second, binding of AMP to AMPK renders it a better substrate for the upstream AMPK kinase (AMPKK) and a worse sub- strate for protein phosphatases (4, 5). Third, the up- stream kinase AMPKK, which is also allosterically ac- tivated by AMP, activates AMPK by phosphorylation (4). Since some of these effects of AMP are antagonized by high concentration of ATP in vitro (6), it has been speculated that AMPK is sensitively regulated by the cellular AMP:ATP ratio. Indeed, AMPK is activated by stresses that deplete ATP; in mammalian cells, deple- tion of ATP always results in a greater increase of AMP level due to adenylate kinase, which maintains the reaction 2 ADP ATP + AMP close to equilibrium (reviewed in 1). Once activated under ATP-depleting conditions, AMPK limits further ATP utilization by inhibiting key enzymes involved in ATP-consuming an- abolic pathways such as fatty acid synthesis and cho- lesterol synthesis (7, 8). In addition, AMPK stimulates the ATP-generating pathways such as fatty acid oxida- tion (9, 10), glycolysis (11), and glucose uptake (12, 13). Hence, it has been proposed that AMPK play a key role in protecting the cell against ATP depletion (1–3). The pathological or physiological conditions that ac- tivate AMPK include heat shock (14), hypoxia/ ischemia in heart muscle (15), exercise in skeletal mus- cle (16), and metabolic toxicity (17). However, effects of the oxidative stress on AMPK cascades are completely unknown. Many studies have suggested that excess reactive oxygen species (ROS) can cause oxidative damages to macromolecules of host cell and thus play important roles in the etiology of many disease pro- cesses including cancer, atherosclerosis, ischemia/ reperfusion-induced cardiac abnormalities, diabetes, and general process of aging (18). In addition to their roles in pathological processes, increasing evidence shows that ROS can be generated in a variety of cells in response to cytokines (19), growth factors (20), and agonists of receptors with seven transmembrane spans 1 To whom correspondence should be addressed at Department of Molecular Biology, Kyung Hee University College of Medicine, Seoul 130-701, Korea. Fax: 82-2-959-8168. E-mail: hajh@khu.ac.kr. Biochemical and Biophysical Research Communications 287, 92–97 (2001) doi:10.1006/bbrc.2001.5544, available online at http://www.idealibrary.com on 92 0006-291X/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.