Selective regulation of IP3-receptor-mediated Ca2+ signaling and apoptosis by the BH4 domain of Bcl-2 versus Bcl-Xl

Abstract

Antiapoptotic B-cell lymphoma 2 (Bcl-2) targets the inositol 1,4,5-trisphosphate receptor (IP3R) via its BH4 domain, thereby suppressing IP3R Ca2+-flux properties and protecting against Ca2+-dependent apoptosis. Here, we directly compared IP3R inhibition by BH4-Bcl-2 and BH4-Bcl-Xl. In contrast to BH4-Bcl-2, BH4-Bcl-Xl neither bound the modulatory domain of IP3R nor inhibited IP3-induced Ca2+ release (IICR) in permeabilized and intact cells. We identified a critical residue in BH4-Bcl-2 (Lys17) not conserved in BH4-Bcl-Xl (Asp11). Changing Lys17 into Asp in BH4-Bcl-2 completely abolished its IP3R-binding and -inhibitory properties, whereas changing Asp11 into Lys in BH4-Bcl-Xl induced IP3R binding and inhibition. This difference in IP3R regulation between BH4-Bcl-2 and BH4-Bcl-Xl controls their antiapoptotic action. Although both BH4-Bcl-2 and BH4-Bcl-Xl had antiapoptotic activity, BH4-Bcl-2 was more potent than BH4-Bcl-Xl. The effect of BH4-Bcl-2, but not of BH4-Bcl-Xl, depended on its binding to IP3Rs. In agreement with the IP3R-binding properties, the antiapoptotic activity of BH4-Bcl-2 and BH4-Bcl-Xl was modulated by the Lys/Asp substitutions. Changing Lys17 into Asp in full-length Bcl-2 significantly decreased its binding to the IP3R, its ability to inhibit IICR and its protection against apoptotic stimuli. A single amino-acid difference between BH4-Bcl-2 and BH4-Bcl-Xl therefore underlies differential regulation of IP3Rs and Ca2+-driven apoptosis by these functional domains. Mutating this residue affects the function of Bcl-2 in Ca2+ signaling and apoptosis.

Feedback regulation mediated by Bcl-2 and DARPP-32 regulates inositol 1,4,5-trisphosphate receptor phosphorylation and promotes cell survival

“…We posit that Bcl-2-overexpressing cancer cells may exploit this mechanism to inhibit apoptosis. In WEHI7.2 cells, Bcl-2 overexpression inhibits anti-CD3-induced Ca 2+ elevation, as previously reported (6,7,18,19) (Fig. 1 B and C).…”

“…The Bcl-2 homology domain 4 (BH4) domain of Bcl-2 mediates its interaction with InsP 3 Rs (6,7). This domain also binds the Ca 2+ /calmodulin-activated serine/threonine (Thr) protein phosphatase calcineurin (CaN) (8).…”

“…A 20-amino acid peptide (InsP 3 Rderived peptide, or IDP) corresponding to the Bcl-2 binding site on the InsP 3 R inhibits Bcl-2-InsP 3 R interaction, thus eliminating Bcl-2's control over InsP 3 R-mediated Ca 2+ elevation (6,7). This peptide induces marked Ca 2+ elevation and Ca 2+ -mediated apoptosis in primary chronic lymphocytic leukemia (CLL) cells and in B-cell lymphoma lines, indicating that Bcl-2-InsP 3 R interaction contributes to the apoptosis-resistance characteristic of these lymphoid malignancies (13,14).…”

Biphasic regulation of InsP 3 receptor gating by dual Ca 2+ release channel BH3-like domains mediates Bcl-x L control of cell viability

“…These results demonstrate that channel-gating inhibition by high [Bcl-x L ] requires binding to both the H1 BH3-like domain and a region (3a1) localized in the channel-coupling domain. The BH4 domain of Bcl-2 has been implicated in its binding to the InsP 3 R (35,36). It is possible that the BH4 domain of Bcl-x L also interacts with this channel region, because a synthetic Bcl-x L BH4 peptide reduced high [Bcl-x L ]-mediated channel inhibition (Fig.…”

“…Whereas this interaction also inhibits Ca 2+ release (26), Bok interacts with the channel 500 residues C-terminal to the Bcl-2 binding sequence via its BH4 domain but does not affect Ca 2+ release (29). Conversely, the Bcl-x L BH4 domain may lack this interaction (36). Inhibition of the Bcl-2 BH4 domain interaction with the channel enhanced InsP 3 R-mediated Ca 2+ signals and apoptosis sensitivity in white blood cells (18,35,37).…”