Evaluation of the BH3-only protein Puma as a direct Bak activator.
J Biol Chem
; 289(1): 89-99, 2014 Jan 03.
Article
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| MEDLINE
| ID: mdl-24265320
ABSTRACT
Interactions among Bcl-2 family proteins play critical roles in cellular life and death decisions. Previous studies have established the BH3-only proteins Bim, tBid, and Noxa as "direct activators" that are able to directly initiate the oligomerization and activation of Bak and/or Bax. Earlier studies of Puma have yielded equivocal results, with some concluding that it also acts as a direct activator and other studies suggesting that it acts solely as a sensitizer BH3-only protein. In the present study we examined the interaction of Puma BH3 domain or full-length protein with Bak by surface plasmon resonance, assessed Bak oligomerization status by cross-linking followed by immunoblotting, evaluated the ability of the Puma BH3 domain to induce Bak-mediated permeabilization of liposomes and mitochondria, and determined the effect of wild type and mutant Puma on cell viability in a variety of cellular contexts. Results of this analysis demonstrate high affinity (KD = 26 ± 5 nM) binding of the Puma BH3 domain to purified Bak ex vivo, leading to Bak homo-oligomerization and membrane permeabilization. Mutations in Puma that inhibit (L141E/M144E/L148E) or enhance (M144I/A145G) Puma BH3 binding to Bak also produce corresponding alterations in Bak oligomerization, Bak-mediated membrane permeabilization and, in a cellular context, Bak-mediated killing. Collectively, these results provide strong evidence that Puma, like Bim, Noxa, and tBid, is able to act as a direct Bak activator.
Palabras clave
Texto completo:
1
Bases de datos:
MEDLINE
Asunto principal:
Proteínas Proto-Oncogénicas
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Proteínas Supresoras de Tumor
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Proteínas Reguladoras de la Apoptosis
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Proteína Destructora del Antagonista Homólogo bcl-2
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Multimerización de Proteína
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Mitocondrias
Tipo de estudio:
Prognostic_studies
Límite:
Animals
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Humans
Idioma:
En
Revista:
J Biol Chem
Año:
2014
Tipo del documento:
Article