Orphan Nuclear Receptor TR3 (Nur77) Binds and Converts the Phenotype of Bcl-B, an Anti-Apoptotic Bcl-2-Family Protein Predominantly Expressed in Human Plasma Cells.

Abstract

Listen

Translate article

TR3 (Nur77) is an orphan member of the steroid/retinoid family of Nuclear Receptors (NRs) that translocates from nucleus to cytosol, where it binds Bcl-2 and converts its phenotype from anti-apoptotic to pro-apoptotic (Li, et al. Science 289: 1159, 2000; Lin, et al CELL 116: 527, 2004). We surveyed TR3 (Nur77) for interactions with all six human anti-apoptotic Bcl-2-family proteins: Bcl-2, Bcl-XL, Mcl-1, Bcl-W, Bfl-1, and Bcl-B. We observed that Bcl-2, Bfl-1, and Bcl-B interact with TR3 as determined by co-immunoprecipitation assays using lysates from transfected cells and by GST pull down assays using GST-Bcl-2-family fusion proteins. In contrast, Bcl-XL, Bcl-W, and Mcl-1 displayed little or no binding to TR3 (Nur77).Co-localization experiments using fluorescent protein tagging and confocal microscopy corroborated these findings, showing co-localization of a fragment of TR3 (Nur77) that accumulates in cytosol (rather than nucleus) [TR3 lacking the DNA-binding domain [DDBD]) with Bcl-2, Bfl-1, and Bcl-B on mitochondria and other intracellular organelles in intact cells, but not co-localization with Bcl-XL, Mcl-1 or Bcl-W. Co-expression of Bcl-2, Bfl-1, or Bcl-B with TR3DDBD by transfection resulted in robust apoptosis induction, while co-expression of Bcl-XL, Bcl-W, or Mcl-1 with TR3DDBD did not. In contrast to results obtained in TR3DDBD co-expression studies, expressing any of the anti-apoptotic Bcl-2-family proteins (Bcl-2, Bfl-1, Bcl-B, Bcl-XL, Mcl-1, Bcl-W) individually resulted in suppression of apoptosis induced by Staurosporine, illustrating the role of TR3 in converting the phenotypes of Bcl-2, Bfl-1 and Bcl-B from protector to killer.Because binding of TR3 (Nur77) to Bcl-B appeared to be strongest among the Bcl-2-family proteins, we focused on this Bcl-B for additional studies. (Ke, N. et al. J. Biol Chem 276: 12481, 2001. Using monospecific antibodies, the endogenous Bcl-B protein was localized in human tissues, revealing predominant expression in plasma cells. Several myeloma cell lines also expressed Bcl-B protein, as determined by immunoblotting. Stimulating myeloma cell line RPMI8226 with ionomycin and phorbol ester TPA (agents that induce TR3 expression and accumulation in cytosol) resulted in association of endogenous TR3 with endogenous Bcl-B, as determined by co-immunoprecipitation experiments. Reducing endogenous Bcl-B levels using small interfering RNA (siRNA) reduced apoptosis induced by transfected TR3DDBD as well as apoptosis induced by a synthetic peptide that mimics TR3.We conclude that cytosolic TR3 (Nur77) interacts selectively with certain anti-apoptotic members of the Bcl-2-family (Bcl-2, Bfl-1, Bcl-B), converting them from protectors to killers. The ability of TR3 (Nur77) to convert Bcl-B suggests a possible novel strategy for triggering apoptosis of Bcl-B-expressing cells, which may be of utility for eradicating long-lived autoantibody-producing plasma cells or for killing malignant myeloma cells. (Supported by NIH GM60554 and SASS Foundation).