Apoptosis Protection by Mcl-1 and Bcl-2 Modulation of Inositol 1,4,5-Trisphosphate Receptor-dependent Ca2+ Signaling

Emily F Eckenrode,Jun Yang,Gopal V Velmurugan,J Kevin Foskett,Carl White

doi:10.1074/jbc.m109.096040

Abstract

Members of the Bcl-2 protein family play a central role in the regulation of apoptosis. An interaction between anti-apoptotic Bcl-x(L) and the endoplasmic reticulum (ER)-localized inositol trisphosphate receptor Ca(2+) release channel (InsP(3)R) enables Bcl-x(L) to be fully efficacious as an anti-apoptotic mediator (White, C., Li, C., Yang, J., Petrenko, N. B., Madesh, M., Thompson, C. B., and Foskett, J. K. (2005) Nat. Cell Biol. 7, 1021-1028). Physiologically, Bcl-x(L) binds to the InsP(3)R to enhance its gating and Ca(2+) signaling. Here we have discovered that structurally related proteins Bcl-2 and Mcl-1 function similarly. Bcl-2, Mcl-1 and Bcl-x(L) bind with comparable affinity to the carboxyl termini of all three mammalian InsP(3)R isoforms with important functional consequences. Stable expression of Bcl-2 or Mcl-1 lowered ER Ca(2+) content and enhanced the rate of InsP(3)-mediated Ca(2+) release in response to submaximal InsP(3) stimulation in permeabilized wild-type DT40 cells but not in cells lacking InsP(3)R. In addition, expression of either Bcl-2 or Mcl-1 enhanced spontaneous InsP(3)R-dependent Ca(2+) oscillations and spiking in intact cells in the absence of agonist stimulation. Bcl-2- and Mcl-1-mediated protection from apoptosis induced by staurosporine or etoposide was enhanced in cells expressing InsP(3)R, demonstrating that their interactions with InsP(3)R enable Bcl-2 and Mcl-1 to be fully efficacious anti-apoptotic mediators. Our data suggest a molecular mechanism that is shared by several anti-apoptotic Bcl-2 proteins that provides apoptosis resistance by direct interactions at the ER with the InsP(3)R that impinges on cellular Ca(2+) homeostasis.

Highlights

In addition to mitochondria, both pro-and anti-apoptotic Bcl-2 proteins localize to the endoplasmic reticulum (ER),2 where they regulate Ca2ϩ fluxes across the ER membrane [5, 6]
Bcl-xL, Bcl-2, and Mcl-1 All Bind with Similar Affinities to the Carboxyl Terminus of All Three InsP3R Isoforms—It was previously demonstrated that Bcl-xL binds to all three mammalian InsP3R isoforms in a region localized to the carboxyl terminus extending from the luminal loop between transmembrane helices 5 and 6 to the carboxyl terminus (TM6ϩC) [8]
The quantities of beads were titrated, and the amounts of GST-InsP3R fragments used for pull-down experiments, as well as the concentrations of Flagtagged Bcl-2 family member proteins expressed in COS-7 cell lysates, were adjusted to equivalent levels

Summary

Introduction

Both pro-and anti-apoptotic Bcl-2 proteins localize to the endoplasmic reticulum (ER),2 where they regulate Ca2ϩ fluxes across the ER membrane [5, 6]. Steady-state [Ca2ϩ]ER was normalized in Bcl-2- and Mcl-1-expressing cells when ER Ca2ϩ uptake was activated in the presence of the InsP3R inhibitor heparin (Fig. 2B). Bcl-2 and Mcl-1 Enhance the Probability of InsP3-dependent [Ca2ϩ]i Oscillations/Spiking—Single cell imaging of Fura-2loaded DT40-WT cells was performed to determine the effects of the interactions of Mcl-1 and Bcl-2 with the InsP3R on cellular Ca2ϩ signaling.

Results

Conclusion