Evaluation of the BH3-only protein Puma as a direct Bak activator

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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.

Original languageEnglish (US)
Pages (from-to)89-99
Number of pages11
JournalJournal of Biological Chemistry
Volume289
Issue number1
DOIs
StatePublished - Jan 3 2014

Fingerprint

Puma
Oligomerization
Noxae
Proteins
Membranes
Mitochondria
Surface plasmon resonance
Liposomes
BH3 Interacting Domain Death Agonist Protein
bcl-2 Homologous Antagonist-Killer Protein
Chemical activation
Cells
Surface Plasmon Resonance
Immunoblotting
Cell Survival
Mutation

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology
  • Molecular Biology

Cite this

Evaluation of the BH3-only protein Puma as a direct Bak activator. / Dai, Haiming; Pang, Yuan-Ping; Ramirez-Alvarado, Marina; Kaufmann, Scott H.

In: Journal of Biological Chemistry, Vol. 289, No. 1, 03.01.2014, p. 89-99.

Research output: Contribution to journalArticle

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