Here we have used FRET measurements to document Bcl-2-IP3R interaction within intact cells and have mapped the site of Bcl-2 interaction on the IP3R, thereby developing an inhibitory peptide used to establish the importance of the Bcl-2-IP3R interaction in regulating Ca2+
signals. This peptide, referred to as peptide 2, corresponds to a 20 amino acid sequence in subdomain 3a1 of the IP3R, a sequence that displays a high degree of homology in all three IP3R isoforms. The GST-IP3R domains used to map the Bcl-2 binding region represent well-folded structural components retaining significant IP3-induced Ca2+
release activity in vitro as long as they remain assembled (Uchida et al., 2003
; Yoshikawa et al., 1999
). Thus, the interaction between Bcl-2 and subdomain 3a1 identified in our experiments likely corresponds to the interaction occurring in the natural situation. That this IP3R subdomain is critical for the interaction of Bcl-2 with IP3Rs is substantiated by the ability of peptide 2 to inhibit this interaction in GST pull-down and coimmunoprecipitation experiments employing both Bcl-2(+) WEHI7.2 cells and endogenous Bcl-2-expressing Jurkat cells. Moreover, this action of peptide 2 in Jurkat cells is consistent with evidence, also presented here, that endogenously expressed Bcl-2 interacts with endogenously expressed IP3Rs. These findings alleviate concern that the Bcl-2-IP3R interaction might be dependent upon overexpression of Bcl-2 at high levels.
Domain 3a1, where Bcl-2 interacts, is located in the regulatory and coupling domain of the IP3R, which transfers the ligand binding signal from the N-terminal IP3 binding domain to the C-terminal channel domain. This domain also functions to keep the inactivated IP3R channel closed (Bezprozvanny, 2005
; Nakayama et al., 2004
; Szlufcik et al., 2006
) and contains many target sites for regulators of IP3R activity (Patterson et al., 2004
; Foskett et al., 2007
). Moreover, recent electron microscopic findings suggested that conformational changes within the IP3R type 1 regulatory and coupling domain switch IP3R states between a “windmill”-like and “square”-like structure, which may represent the “open” and “closed” channel, respectively (Hamada et al., 2003
; Serysheva et al., 2003
; Taylor et al., 2004
). Thus, it is interesting to speculate that the binding of Bcl-2 in this region may regulate this conformational change, thereby regulating IP3R channel opening.
The development of peptide 2 as an inhibitor of Bcl-2-IP3R interaction provided a useful tool with which to investigate the fundamental mechanism by which Bcl-2 inhibits Ca2+ release from the ER, at a molecular level. To this end, we employed peptide 2 in a multiplicity of experimental strategies to remove any doubt that the interaction of Bcl-2 with IP3Rs contributes to Bcl-2’s inhibition of IP3-mediated ER Ca2+ release. First, peptide 2 reversed Bcl-2’s inhibition of IP3R channel opening in vitro. Second, peptide 2, when introduced into cells using Chariot reagent, reversed Bcl-2’s inhibition of TCR-mediated Ca2+ elevation both in Bcl-2(+) WEHI7.2 cells and in Jurkat cells. These findings indicate that Bcl-2 inhibits proapoptotic Ca2+ signals at least in part through interaction with the IP3R. Third, special care was taken to control for the outside possibility that peptide 2 might act in cells at some other level than the IP3R itself (e.g., the TCR signaling pathway upstream of the IP3R). For this purpose, we demonstrated that peptide 2 antagonized both unidirectional 45Ca2+ efflux from the ER in permeabilized cells and IP3 ester-induced Ca2+ elevation in intact cells. Thus, the present findings provide strong evidence that the Bcl-2-IP3R interaction is an important component of the process through which Bcl-2 inhibits IP3-mediated Ca2+ release from the ER.
One proposed component of this process upon which there is not complete agreement is the role of Bcl-2 in regulating ER Ca2+
concentration, a topic discussed in recent reviews (Distelhorst and Shore, 2004
; Giacomello et al., 2007
; Pinton and Rizzuto, 2006
; Rong and Distelhorst, 2008
). In our earlier work (Chen et al., 2004
) and in the present study, as well as findings in other laboratories (see Introduction), a Bcl-2-imposed decrease in ER Ca2+
concentration was not detected. Nevertheless, the previously reported detection of a Bcl-2-imposed decrease in Ca2+
concentration by others and our present findings regarding the interaction of Bcl-2 with the IP3R are not necessarily mutually exclusive. In fact, recent reports propose a role for Bcl-2-IP3R interaction in regulating ER Ca2+
concentration (Oakes et al., 2005
) and indicate that the ability of Bcl-2 to decrease ER Ca2+
concentration is dependent upon both Bcl-2 phosphorylation state (Bassik et al., 2004
) and which of the three IP3R isoforms is expressed (Li et al., 2007
). Overall, the major focus of the current work is not on reexamining potential effects of Bcl-2 on ER Ca2+
concentration, but on the Bcl-2-IP3R and designing a peptide inhibitor that reverses Bcl-2’s inhibitory effect on calcium release and apoptosis. These findings stand whether or not Bcl-2 decreases ER Ca2+
That peptide 2, when introduced into intact cells, reverses Bcl-2’s inhibition of TCR- or cell-permeable IP3 ester-induced Ca2+
elevation suggests that the Bcl-2-IP3R interaction previously detected by coimmunoprecipitation in cell extracts indeed occurs within intact cells. This conclusion is further substantiated by FRET measurements. Bcl-2 is known to localize to both the ER and mitochondria, but IP3Rs are mainly localized to the ER, and Bcl-2’s inhibitory effect on IP3-mediated Ca2+
elevation is observed in cells where Bcl-2 is selectively targeted to the ER (Chen et al., 2004
). The FRET measurements reported here document an interaction of Bcl-2 with IP3Rs on the ER, although FRET techniques are challenging and have recognized pitfalls and limitations (Piston and Kremers, 2007
). Therefore, in this work we employed two different, independent methods of FRET measurement, acceptor photobleaching in fixed cells and direct FRET measurements in living cells, as complementary methods to determine if Bcl-2 interacts with IP3R in cells. Also, because of the known limitations of both of these methods, a stringent set of controls was included. The most straightforward method to detect FRET is to photobleach the acceptor and to monitor the change in donor emission. For this method the actual FRET signal and the FRET efficiency could be overestimated depending on conditions (e.g., fixation) used (Rizzo et al., 2006
). Thus, the quantification of FRET efficiencies in acceptor photobleaching experiments at present is valid only for establishing the presence or absence of FRET (Karpova et al., 2003
). A number of negative controls reduced the possibility of false-positive FRET. Although live cell FRET by ratiometric imaging is the simplest FRET method, potential artifacts due to the spectral bleed-through have to be considered. The corrective ratiometric method employed in our measurements of live cell FRET has been established to subtract the crosstalk between the fluorescence proteins (see appendix of Erickson et al., 2001
). Though actual FRET values depend on the exact amount of acceptor proteins interacting with donor proteins, it is not necessary for the donor and acceptor concentrations to be equal in both of the methods employed here (Erickson et al., 2001
; Piston and Kremers, 2007
). Nevertheless, according to the experimental conditions, the CFP-Bcl-2 and YFP-IP3R concentrations do not vary dramatically. Our data indicate that donor and acceptor pairs interact in sufficiently close proximity and orientation to allow FRET. With these precautions in mind, both FRET techniques provided positive evidence that Bcl-2 does indeed interact with IP3Rs within cells.
Overall, the findings presented here indicate that Bcl-2 operates on two fronts, blocking BH3-only protein activity and blocking proapoptotic Ca2+ signals. Recently developed Bcl-2 antagonists target the Bcl-2-BH3-only protein interaction, whereas peptide 2 interferes selectively with the Bcl-2-IP3R interaction. Perhaps in the future small-molecule inhibitors of the latter interaction could be developed that mimic the activity of peptide 2 and complement the therapeutic activity of small compounds targeting the interaction of Bcl-2 with BH3-only proteins.