The trans-membrane domain of Bcl-2α, but not its hydrophobic cleft, is a critical determinant for efficient IP3 receptor inhibition

Abstract

The anti-apoptotic Bcl-2 protein is emerging as an efficient inhibitor of IP3R function, contributing to its oncogenic properties. Yet, the underlying molecular mechanisms remain not fully understood. Using mutations or pharmacological inhibition to antagonize Bcl-2's hydrophobic cleft, we excluded this functional domain as responsible for Bcl-2-mediated IP3Rs inhibition. In contrast, the deletion of the C-terminus, containing the trans-membrane domain, which is only present in Bcl-2α, but not in Bcl-2β, led to impaired inhibition of IP3R-mediated Ca2+ release and staurosporine-induced apoptosis. Strikingly, the trans-membrane domain was sufficient for IP3R binding and inhibition. We therefore propose a novel model, in which the Bcl-2's C-terminus serves as a functional anchor, which beyond mere ER-membrane targeting, underlies efficient IP3R inhibition by (i) positioning the BH4 domain in the close proximity of its binding site on IP3R, thus facilitating their interaction; (ii) inhibiting IP3R-channel openings through a direct interaction with the C-terminal region of the channel downstream of the channel-pore. Finally, since the hydrophobic cleft of Bcl-2 was not involved in IP3R suppression, our findings indicate that ABT-199 does not interfere with IP3R regulation by Bcl-2 and its mechanism of action as a cell-death therapeutic in cancer cells likely does not involve Ca2+ signaling.

The selective Bcl-2 inhibitor venetoclax, a BH3 mimetic, does not dysregulate intracellular Ca 2+ signaling

“…Moreover, addition of venetoclax did not alleviate the inhibition of IP 3 R-mediated Ca 2+ flux by Bcl-2 ( fig. 3B), which is consistent with our previous data obtained in COS cells transiently overexpressing Bcl-2 [30].…”

“…BH4 domain and its C-terminal transmembrane domain [28][29][30] HEK293 cells were grown in DMEM containing 4500 mg/L glucose and 50 μg/mL gentamicin. These cells were cultured at 37°C and 5% CO 2 .…”

Constitutive IP3 signaling underlies the sensitivity of B-cell cancers to the Bcl-2/IP3 receptor disruptor BIRD-2

“…Bcl-2 impacts IP 3 Rs by binding with its N-terminal BH4 domain to the central, modulatory domain of the channel [14][15][16]. Furthermore, Bcl-2′s C-terminal transmembrane domain enables efficient IP 3 R inhibition within cells [17]. A cell-permeable peptide tool named Bcl-2/IP 3 R Disruptor-2 (BIRD-2) was developed, capable of stripping Bcl-2 from IP 3 Rs [18].…”

Targeting Bcl-2-IP3 receptor interaction to treat cancer: A novel approach inspired by nearly a century treating cancer with adrenal corticosteroid hormones

“…Nevertheless, the relatively low affinity of inhibition by the BH4 domain (measured in vitro IC50=30μM) (118, Monaco, 2012 #5128) does not appear to explain the potent inhibitory effect of Bcl-2 full-length protein under physiological conditions. Using genetic and pharmacological approaches, Ivanova et al (22) implicate the C-terminal IP 3 R1 domain in Bcl-2 binding and cell death regulation. Furthermore, they demonstrated a direct interaction between a peptide corresponding to the transmembrane domain of Bcl-2 (TMD-Bcl-2) and the purified C-terminal fragment of IP 3 R1.…”

“…Exciting new therapeutic approaches have dramatically changed the therapeutic approach to CLL by targeting B-cell receptor signaling pathways, for example Bruton tyrosine kinase which is targeted by ibrutinib (16, 17) and the anti-apoptotic Bcl-2 protein which is targeted at one of its mechanisms of action by ABT-199/Venetoclax (18, 11). The left half of Figure 1 summarizes recent advances in targeting the interaction of Bcl-2 with IP 3 Rs, using peptide inhibitors or small molecules to induce IP 3 R-mediated Ca 2+ -elevation and cell death as a novel therapeutic approach (19, 20, 21, 22). …”