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Evaluation of the BH3-only Protein Puma as a Direct Bak Activator*

Haiming Dai,Y. Pang,M. Ramirez-Alvarado,S. Kaufmann

Published 2013 in Journal of Biological Chemistry

ABSTRACT

Background: How the DNA damage-induced proapoptotic protein Puma triggers apoptosis has been unclear. Results: The Puma BH3 domain not only binds the Bak BH3 binding pocket with nanomolar affinity, but mutations affecting this binding alter Bak oligomerization, membrane permeabilization, and killing. Conclusion: Puma is a direct Bak activator. Significance: These observations help resolve a long-standing debate over the Puma role in apoptosis. 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.

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