The trans-membrane domain of Bcl-2α, but not its hydrophobic cleft, is a critical determinant for efficient IP 3 receptor inhibition

// Hristina Ivanova 1 , Abigael Ritaine 2 , Larry Wagner 3 , Tomas Luyten 1 , George Shapovalov 2 , Kirsten Welkenhuyzen 1 , Bruno Seitaj 1 , Giovanni Monaco 1 , Humbert De Smedt 1 , Natalia Prevarskaya 2 , David I. Yule 3 , Jan B. Parys 1 , Geert Bultynck 1 1 Katholieke Universiteit Leuven, Laboratory of Molecular and Cellular Signaling, Department of Cellular and Molecular Medicine and Leuven Cancer Institute (LKI), BE-3000 Leuven, Belgium 2 Inserm U-1003, Equipe Labellisee par la Ligue Nationale Contre le Cancer et LABEX (Laboratoire d'excellence), Universite Lille1, 59655 Villeneuve d'Ascq, France 3 University of Rochester Medical Center School of Medicine and Dentistry, Rochester, NY 14642, USA Correspondence to: Geert Bultynck, email: geert.bultynck@kuleuven.be Keywords: calcium signaling, anti-apoptotic Bcl-2, trans-membrane domain, hydrophobic cleft, IP 3 receptor Received: March 03, 2016      Accepted: July 18, 2016      Published: August 2, 2016 ABSTRACT The anti-apoptotic Bcl-2 protein is emerging as an efficient inhibitor of IP 3 R 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 IP 3 Rs 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 IP 3 R-mediated Ca 2+ release and staurosporine-induced apoptosis. Strikingly, the trans-membrane domain was sufficient for IP 3 R 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 IP 3 R inhibition by (i) positioning the BH4 domain in the close proximity of its binding site on IP 3 R, thus facilitating their interaction; (ii) inhibiting IP 3 R-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 IP 3 R suppression, our findings indicate that ABT-199 does not interfere with IP 3 R regulation by Bcl-2 and its mechanism of action as a cell-death therapeutic in cancer cells likely does not involve Ca 2+ signaling.