Abstract
Bcl-2 is the best characterized member of a large family of proteins that regulate apoptosis. Although it is established that Bcl-2 localized at the mitochondria functions as an anti-apoptotic protein, the function of Bcl-2 at the nucleus remains unclear. Recently we showed that nuclear compartment-associated Bcl-2 inhibits transcription factor activation. Based on this observation, we hypothesized that presence of Bcl-2 at the nucleus may induce rather than protect cells from apoptosis. Here we investigated the putative apoptotic role of nuclear compartment-associated Bcl-2. Additionally, we examined the role of the Bcl-2 BH4 domain in mediating binding to FKBP38, the Bcl-2 mitochondrial chaperone. Our results demonstrate a novel, pro-apoptotic function for nuclear Bcl-2 and identify the Bcl-2 BH4 domain as a key regulator in mediating Bcl-2/FKBP38 binding. These results indicate that Bcl-2 has a dual role as both a protector and a killer and that the ability to switch roles depends on Bcl-2 subcellular localization.
Highlights
The ability of Bcl-2 to protect cells from apoptosis is critically influenced by protein-protein interactions, post-translational
Bcl-2 localizes to multiple organelles, its anti-apoptotic function has predominantly been investigated at the mitochondria, a site in which Bcl-2 is actively transported to via the mitochondrial chaperone protein FKBP38 [14], an atypical member of the FK506-binding immunophilin protein family [15]
Analyses performed on three independent experiments revealed that all cell lines assayed underwent a significant degree of apoptosis upon transient transfection of Bcl-2/YFP or Bcl-2⌬BH4/YFP as compared with cells transfected with the YFP control vector
Summary
Cell culture and general reagents were obtained from Invitrogen, Fisher, and Sigma-Aldrich unless specified otherwise. PMKitNeo (vector) and pMKitNeoBcl-2␣ were kind gifts of Dr S. Mito-Bcl-2 and Mito-Bcl-2-GFP were both kind gifts of Dr C. W. Distelhorst (Case Western Reserve University, Cleveland, OH). Bcl2⌬BH4 was generated by introducing a start codon at amino acid position 25 of parental pMKitNeo-Bcl-2␣ flanked by a XhoI site. The obtained fragment was digested with XhoI/ EcoRI and re-cloned into pMKit-Neo. pEYFP-N1 (YFP) vector was purchased from Clontech (Mountain View, CA). PBcl-2/ YFP was generated by PCR amplification of amino acids 1– 661 of parental pMKitNeo-Bcl-2␣, substituting the stop codon of Bcl-2 with an alanine flanked by an XhoI site. The obtained fragment was digested with XhoI/EcoRI and re-cloned into pEYFP-N1 in-frame with its EYFP coding region
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