RESUMO
SU6656, a Src kinase inhibitor, was reported to increase fat oxidation and reduce body weight in mice, with proposed mechanisms involving AMP-activated protein kinase (AMPK) activation via inhibition of phosphorylation of either LKB1 or AMPK by the Src kinase, Fyn. However, we report that AMPK activation by SU6656 is independent of Src kinases or tyrosine phosphorylation of LKB1 or AMPK and is not due to decreased cellular energy status or binding at the ADaM site on AMPK. SU6656 is a potent AMPK inhibitor, yet binding at the catalytic site paradoxically promotes phosphorylation of Thr172 by LKB1. This would enhance phosphorylation of downstream targets provided the lifetime of Thr172 phosphorylation was sufficient to allow dissociation of the inhibitor and subsequent catalysis prior to its dephosphorylation. By contrast, sorafenib, a kinase inhibitor in clinical use, activates AMPK indirectly by inhibiting mitochondrial metabolism and increasing cellular AMP:ADP and/or ADP:ATP ratios.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Indóis/farmacologia , Niacinamida/análogos & derivados , Compostos de Fenilureia/farmacologia , Sulfonamidas/farmacologia , Quinases Proteína-Quinases Ativadas por AMP , Proteínas Quinases Ativadas por AMP/antagonistas & inibidores , Proteínas Quinases Ativadas por AMP/genética , Regulação Alostérica , Sítios de Ligação , Domínio Catalítico , Sistema Livre de Células , Ativação Enzimática/efeitos dos fármacos , Células HEK293 , Células HeLa , Humanos , Indanos/farmacologia , Indóis/química , Indóis/metabolismo , Mutagênese Sítio-Dirigida , Niacinamida/química , Niacinamida/metabolismo , Niacinamida/farmacologia , Compostos de Fenilureia/química , Compostos de Fenilureia/metabolismo , Fosforilação/efeitos dos fármacos , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Pirimidinas/farmacologia , Sorafenibe , Sulfonamidas/química , Sulfonamidas/metabolismo , Quinases da Família src/antagonistas & inibidores , Quinases da Família src/metabolismoRESUMO
AMPK is known to be activated by oxidative stress. Addition of glucose oxidase to cells generates H2O2 at a constant rate that is opposed by enzymic degradation, providing a good model for physiological oxidative stress. AMPK activation by glucose oxidase correlated with increases in cellular AMP:ATP and was greatly reduced in cells expressing an AMP-insensitive AMPK mutant, although a small degree of activation remained. The effects of increased AMP were partly due to inhibition of Thr172 dephosphorylation. These results suggest that changes in adenine nucleotides, rather than direct oxidative modification, are the major drivers of AMPK activation during oxidative stress.