RESUMEN
The S184 residue of Bax is the target of several protein kinases regulating cell fate, including AKT. It is well-established that, in cellulo, the substitution of S184 by a non-phosphorylatable residue stimulates both the mitochondrial localization of Bax, cytochrome c release, and apoptosis. However, in in vitro experiments, substituted mutants did not exhibit any increase in their binding capacity to isolated mitochondria or liposomes. Despite exhibiting a significant increase of the 6A7 epitope exposure, substituted mutants remain limited in their ability to form large oligomers, suggesting that they high capacity to promote apoptosis in cells was more related to a high content than to an increased ability to form large pores in the outer mitochondrial membranes.
RESUMEN
Bax is a major player in the apoptotic process, being at the core of the mitochondria permeabilization events. In spite of the major recent advances in the knowledge of Bax organization within the membrane, the precise behavior of the C-terminal helix α9 remains elusive, since it was absent from the resolved structure of active Bax. The Proline 168 (P168) residue, located in the short loop between α8 and α9, has been the target of site-directed mutagenesis experiments, with conflicting results. We have produced and purified a recombinant mutant Bax-P168A, and we have compared its behavior with that of wild-type Bax in a series of tests on Large Unilamellar Vesicles (LUVs) and isolated mitochondria. We conclude that Bax-P168A had a greater ability to oligomerize and bind to membranes. Bax-P168A was not more efficient than wild-type Bax to permeabilize liposomes to small molecules but was more prone to release cytochrome c from mitochondria.
Asunto(s)
Alanina/química , Mitocondrias/metabolismo , Prolina/química , Liposomas Unilamelares/metabolismo , Proteína X Asociada a bcl-2/química , Alanina/metabolismo , Sustitución de Aminoácidos , Clonación Molecular , Citocromos c/metabolismo , Expresión Génica , Células HCT116 , Humanos , Liposomas/química , Liposomas/metabolismo , Mitocondrias/química , Mutación , Permeabilidad , Prolina/metabolismo , Unión Proteica , Multimerización de Proteína , Estructura Secundaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Liposomas Unilamelares/química , Proteína X Asociada a bcl-2/genética , Proteína X Asociada a bcl-2/metabolismoRESUMEN
Bax-dependent mitochondrial permeabilization during apoptosis is controlled by multiple factors, including the phosphorylation by the protein kinase AKT. We used the heterologous co-expression of human Bax and AKT1 in yeast to investigate how the kinase modulates the different steps underlying Bax activation. We found that AKT activated Bax and increased its cellular content. Both effects were dependent on Ser184, but a phosphorylation of this residue did not fully explain the effects of AKT. Additional experiments with mutants substituted on Ser184 suggested that the regulation of Bax dynamic equilibrium between the cytosol and mitochondria might be more tightly regulated by Bcl-xL when Bax is phosphorylated.