title: Evaluation of the BH3-only protein Puma as a direct Bak activator.
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.
authors.affiliation: From the Division of Oncology Research and.
authors.forename: Haiming
authors.initials: H
authors.lastname: Dai
authors.forename: Yuan-Ping
authors.initials: YP
authors.lastname: Pang
authors.forename: Marina
authors.initials: M
authors.lastname: Ramirez-Alvarado
authors.forename: Scott H
authors.initials: SH
authors.lastname: Kaufmann
mesh_terms: D000818:Animals; D051017:Apoptosis Regulatory Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D002470:Cell Survival / Q000502:physiology; D006801:Humans; D008081:Liposomes / Q000737:chemistry; D051379:Mice; D008928:Mitochondria / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary; D011518:Proto-Oncogene Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D025521:Tumor Suppressor Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D050998:bcl-2 Homologous Antagonist-Killer Protein / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D051017:Apoptosis Regulatory Proteins; C494772:BAK1 protein, human; C438378:BBC3 protein, human; C494773:Bak1 protein, mouse; D008081:Liposomes; C488588:PUMA protein, mouse; D011518:Proto-Oncogene Proteins; D025521:Tumor Suppressor Proteins; D050998:bcl-2 Homologous Antagonist-Killer Protein
keywords: Apoptosis; Bak; Bcl-2 Family Proteins; Cell Death; Mitochondrial Apoptosis; Molecular Cell Biology; Oligomerization; Puma