Bcl-2 regulates proapoptotic calcium signals by interacting with the inositol 1,4,5-trisphophate receptor

Bcl-2 is the founding member of a large family of apoptosis regulating proteins. The antiapoptotic protein Bcl-2 inhibits Ca2+ release from the endoplasmic reticulum (ER). One proposed mechanism involves an interaction of Bcl-2 with the inositol 1, 4, 5-trisphosphate receptor (IP3R) Ca 2+ channel localized with Bcl-2 on the ER. Here we document Bcl-2-IP3R interaction within cells by FRET and identify a Bcl-2 interacting region in the regulatory and coupling domain of the IP3R. A peptide (peptide 2) based on this IP3R sequence displaced Bcl-2 from the IP3R and reversed Bcl-2-mediated inhibition of IP3R channel activity in vitro, IP3-induced ER Ca2+ release in permeabilized cells, and cell permeable IP3 ester-induced Ca 2+ elevation in intact cells. This peptide also reversed Bcl-2's inhibition of T cell receptor-induced Ca2+ elevation and apoptosis. Furthermore, peptide 2 enhances ABT-737-induced cell death in chronic lymphocytic leukemia cells. The interaction of Bcl-2 with IP3R's contributes to the regulation of proapoptotic Ca2+ signals by Bcl-2. We also investigated the region of Bcl-2 responsible for interaction with the IP3R. Based on results of coimmunoprecipitation and GST pull-down experiments the BH4 domain of Bcl-2 is necessary for interaction with the IP3R. A synthetic peptide corresponding to the BH4 domain of Bcl-2 interacts with the same IP3R domain as full length Bcl-2. TAT-BH4, formed by fusing a peptide corresponding to the BH4 domain of Bcl-2 with the protein transduction domain of HIV TAT, enters cells and functions like full length Bcl-2, to inhibit cytoplasmic Ca2+ elevation and apoptosis induced by T cell receptor (TCR) activation. Two experimental findings establish that these actions of TAT-BH4 are mediated through interaction with the IP3R. First, TAT-BH4 inhibits Ca2+ elevation induced by a cell permeant IP3 ester. Second, peptide 2 that blocks Bcl-2-IP3R interaction reverses the inhibitory effect of TAT-BH4 on both Ca2+ elevation and apoptosis following TCR activation. In summary, these findings indicate that the BH4 domain of Bcl-2 is both necessary and sufficient for interaction with the IP3R. The Bcl-2-IP3R interaction is a potential therapeutic target in diseases associated with Bcl-2's inhibition of cell death.