RESUMO
AMP-activated protein kinase (AMPK) is an energy sensor that is activated by increases in the cellular AMP/ATP and ADP/ATP ratios by three mechanisms: (1) allosteric activation, (2) promotion of phosphorylation at Thr172 on the α subunit by upstream kinases, and (3) inhibition of dephosphorylation of Thr172 by protein phosphatases. All of these effects are triggered by the binding of AMP or ADP at one or more of three sites on the γ subunit, where they displace ATP. AMPK is also activated by ligands that bind in the ADaM site, which is located between the α and ß subunits. In this chapter we describe cell-free assays that can be used to study these varied activation mechanisms.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Difosfato de Adenosina/metabolismo , Monofosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Ensaios Enzimáticos/métodos , Proteínas Quinases Ativadas por AMP/química , Difosfato de Adenosina/química , Monofosfato de Adenosina/química , Trifosfato de Adenosina/química , Regulação Alostérica , Sítios de Ligação , Sistema Livre de Células/metabolismo , Ativação Enzimática , Marcação por Isótopo/métodos , Ligantes , Radioisótopos de Fósforo/química , Fosforilação , Ligação Proteica , Subunidades Proteicas/químicaRESUMO
AMP-activated protein kinase (AMPK) is extremely sensitive to cellular stress, so that nonphysiological activation of the kinase can readily occur during harvesting of cells or tissues. In this chapter we describe methods to harvest cells and tissues, and for kinase assays, that preserve the physiological activation status of AMPK as far as possible. Note that similar care with methods of cell or tissue harvesting is required when AMPK function is monitored by Western blotting, rather than by kinase assays. We also describe methods to determine whether compounds that activate AMPK in intact cells do so indirectly by interfering with cellular ATP synthesis or directly by binding to AMPK and, if the latter, whether this occurs by binding at the AMP-binding sites on the γ subunit or at the ADaM site located between the α and ß subunits.