Abstract
Apoptosis is mediated through the extrinsic or intrinsic pathway. Key regulators of the intrinsic apoptotic pathway are the family of B cell lymphoma 2 (Bcl-2) proteins. The activity of the prototypical Bcl-2 protein is usually considered antiapoptotic. However, under some conditions, Bcl-2 associates with the orphan nuclear hormone receptors Nur77 and Nor-1, converting Bcl-2 into a proapoptotic molecule. Expression of Nur77 and Nor-1 is induced by a variety of signals, including those leading to apoptosis. Translocation of Nur77/Nor-1 to mitochondria results in their association with Bcl-2, exposing the Bcl-2 homology (BH) 3 domain and causing apoptosis. However, the molecular details of this interaction are incompletely understood. Here, through extensive Bcl-2 mutagenesis and functional assays, we identified residues within Bcl-2 that are essential for its interaction with Nur77/Nor-1. Although an initial report has suggested that an unstructured loop region between the Bcl-2 BH4 and BH3 domains is required for Bcl-2's interaction with Nur77/Nor-1, we found that it is dispensable for this interaction. Instead, we found important interacting residues at the BH4 domain and crucial interacting residues between the BH1 and BH2 domains. Bcl-2 alanine mutants at this region could no longer interact with Nur77/Nor-1 and could not initiate Nur77/Bcl-2-mediated cell death. However, they still retained their anti-apoptotic capability in two different death assays. These results establish crucial residues in Bcl-2 required for Nur77/Nor-1-mediated apoptosis and point to potential new strategies for manipulating Bcl-2 function.
Highlights
Apoptosis is mediated through the extrinsic or intrinsic pathway
To identify amino acids in B cell lymphoma 2 (Bcl-2) required for its interaction with Nur77 and Nor-1, we initially focused our attention on the loop between the BH4 and BH3 domains
For Nur77, we used a FLAG-tagged Nur77 lacking a DNA-binding domain (Nur77⌬DBD) as described previously [43,44,45]. This allows us to bypass the requirement to stimulate the cells to initiate Nur77 nucleus-tomitochondrion translocation [53]. These constructs were transfected into HEK293T cells and FLAG co-immunoprecipitation (FLAG-IP) assays were performed to test for the mutant Bcl-2/Nur77⌬DBD interaction
Summary
B cell lymphoma 2 (Bcl-2) residues essential for Bcl-2’s apoptosis-inducing interaction with Nur77/Nor-1 orphan steroid receptors. The anti-apoptotic family members, which include Bcl-2 and Bcl-X, contain all four BH domains and can prevent apoptosis by sequestering and inactivating the BH3-only proteins [19, 27] This function requires intact BH1, BH2, and BH4 domains [28, 29]. Strong T cell receptor signals induce Nur and Nor-1 translocation to the mitochondria, which associate with Bcl-2, an event originally described in several cancer cells upon apoptotic stimuli [39, 44] This association leads to a conformational change that exposes Bcl-2’s BH3 domain. We find that mutating a cluster of residues located in an intervening sequence between the BH1 and BH2 domain can abolish Nur and Nor-1 interaction Mutations at this site do not affect the Bcl-2 normal anti-apoptotic function but can block Nur77-mediated apoptosis. Our study further refines the molecular details of Nur77/Nor-1 and Bcl-2 interaction
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