RESUMO
The heterotrimeric AMP-activated protein kinase (AMPK) has a key role in regulating cellular energy metabolism; in response to a fall in intracellular ATP levels it activates energy-producing pathways and inhibits energy-consuming processes. AMPK has been implicated in a number of diseases related to energy metabolism including type 2 diabetes, obesity and, most recently, cancer. AMPK is converted from an inactive form to a catalytically competent form by phosphorylation of the activation loop within the kinase domain: AMP binding to the γ-regulatory domain promotes phosphorylation by the upstream kinase, protects the enzyme against dephosphorylation, as well as causing allosteric activation. Here we show that ADP binding to just one of the two exchangeable AXP (AMP/ADP/ATP) binding sites on the regulatory domain protects the enzyme from dephosphorylation, although it does not lead to allosteric activation. Our studies show that active mammalian AMPK displays significantly tighter binding to ADP than to Mg-ATP, explaining how the enzyme is regulated under physiological conditions where the concentration of Mg-ATP is higher than that of ADP and much higher than that of AMP. We have determined the crystal structure of an active AMPK complex. The structure shows how the activation loop of the kinase domain is stabilized by the regulatory domain and how the kinase linker region interacts with the regulatory nucleotide-binding site that mediates protection against dephosphorylation. From our biochemical and structural data we develop a model for how the energy status of a cell regulates AMPK activity.
Assuntos
Proteínas Quinases Ativadas por AMP/química , Proteínas Quinases Ativadas por AMP/metabolismo , Difosfato de Adenosina/metabolismo , Difosfato de Adenosina/farmacologia , Proteínas Quinases Ativadas por AMP/genética , Monofosfato de Adenosina/metabolismo , Monofosfato de Adenosina/farmacologia , Trifosfato de Adenosina/metabolismo , Trifosfato de Adenosina/farmacologia , Regulação Alostérica/efeitos dos fármacos , Regulação Alostérica/genética , Animais , Sítios de Ligação , Cristalografia por Raios X , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/genética , Cinética , Magnésio/metabolismo , Mamíferos , Modelos Moleculares , Fosforilação/efeitos dos fármacos , Fosforilação/genética , Ligação Proteica , Estrutura Terciária de Proteína/efeitos dos fármacos , Estrutura Terciária de Proteína/genética , TermodinâmicaRESUMO
Maintaining sufficient levels of ATP (the immediate source of cellular energy) is essential for the proper functioning of all living cells. As a consequence, cells require mechanisms to balance energy demand with supply. In eukaryotic cells the AMP-activated protein kinase (AMPK) cascade has an important role in this homeostasis. AMPK is activated by a fall in ATP (concomitant with a rise in ADP and AMP), which leads to the activation of catabolic pathways and the inhibition of anabolic pathways. Here we review the role of AMPK as an energy sensor and consider the recent finding that ADP, as well as AMP, causes activation of mammalian AMPK. We also review recent progress in structural studies on phosphorylated AMPK that provides a mechanism for the regulation of AMPK in which AMP and ADP protect it against dephosphorylation. Finally, we briefly survey some of the outstanding questions concerning the regulation of AMPK.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Nucleotídeos de Adenina/fisiologia , Metabolismo Energético/fisiologia , Regulação Enzimológica da Expressão Gênica/fisiologia , Proteínas Quinases Ativadas por AMP/genética , Transdução de Sinais/fisiologia , Especificidade da EspécieRESUMO
AMP-activated protein kinase (AMPK) plays a key role in integrating metabolic pathways in response to energy demand. We identified a mutation in the γ1 subunit (γ1D316A) that leads to activation of AMPK. We generated mice with this mutation to study the effect of chronic liver-specific activation of AMPK in vivo. Primary hepatocytes isolated from these mice have reduced gluconeogenesis and fatty acid synthesis, but there is no effect on fatty acid oxidation compared to cells from wild-type mice. Liver-specific activation of AMPK decreases lipogenesis in vivo and completely protects against hepatic steatosis when mice are fed a high-fructose diet. Our findings demonstrate that liver-specific activation of AMPK is sufficient to protect against hepatic triglyceride accumulation, a hallmark of non-alcoholic fatty liver disease (NAFLD). These results emphasize the clinical relevance of activating AMPK in the liver to combat NAFLD and potentially other associated complications (e.g., cirrhosis and hepatocellular carcinoma).
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Dieta , Fígado/enzimologia , Hepatopatia Gordurosa não Alcoólica/enzimologia , Hepatopatia Gordurosa não Alcoólica/prevenção & controle , Animais , Células COS , Chlorocebus aethiops , Açúcares da Dieta , Ativação Enzimática , Frutose , Hepatócitos/metabolismo , Metabolismo dos Lipídeos , Fígado/patologia , Camundongos , Mutação/genética , Hepatopatia Gordurosa não Alcoólica/patologia , Especificidade de ÓrgãosRESUMO
The SNF1 protein kinase complex plays an essential role in regulating gene expression in response to the level of extracellular glucose in budding yeast. SNF1 shares structural and functional similarities with mammalian AMP-activated protein kinase. Both kinases are activated by phosphorylation on a threonine residue within the activation loop segment of the catalytic subunit. Here we show that ADP is the long-sought metabolite that activates SNF1 in response to glucose limitation by protecting the enzyme against dephosphorylation by Glc7, its physiologically relevant protein phosphatase. We also show that the regulatory subunit of SNF1 has two ADP binding sites. The tighter site binds AMP, ADP, and ATP competitively with NADH, whereas the weaker site does not bind NADH, but is responsible for mediating the protective effect of ADP on dephosphorylation. Mutagenesis experiments suggest that the general mechanism by which ADP protects against dephosphorylation is strongly conserved between SNF1 and AMPK.