Caspase 3 cleavage of the inositol 1,4,5-trisphosphate receptor does not contribute to apoptotic calcium release

Abstract

An important role in the regulation of apoptotic calcium release is played by the ubiquitously expressed family of inositol 1,4,5-trisphosphate receptor (IP3R) channels. One model for IP3R activation during apoptosis is cleavage by the apoptotic protease caspase 3. Here we show that early elevations in cytosolic calcium during apoptosis do not require caspase 3 activity. We detected a robust increase in calcium levels in response to staurosporine treatment in primary human fibroblasts and HeLa cells in the presence of the caspase inhibitor Z-VAD, indicating that calcium release during the initiation of apoptosis occurs independently of caspase 3. Similar results were obtained with MCF-7 cells which lack caspase 3 expression. Stable expression of caspase 3 in MCF-7 cells and TAT-based transduction of the active recombinant caspase 3 directly into living MCF-7 cells had marginal effects on the early events leading to cytosolic calcium elevations and irreversible commitment to apoptotic cell death. Significantly, blocking IP3 binding to the IP3R with an IP3 sponge resulted in suppression of staurosporine-induced calcium release and cell death. Collectively, our results suggest that generation of IP3 is sufficient for the initiation of apoptotic calcium signaling, and caspase 3-mediated truncation of IP3R channel is a consequence, not causative, of apoptotic calcium release.

The Bcl-2 Protein Family Member Bok Binds to the Coupling Domain of Inositol 1,4,5-Trisphosphate Receptors and Protects Them from Proteolytic Cleavage

“…It seems entirely plausible that this could result from steric hindrance by Bok of the ability of chymotrypsin and caspase-3 to access their cleavage sites. This inhibitory effect of Bok may well be biologically relevant, as the C-terminal IP 3 R1 fragment resulting from caspase-3 cleavage has been proposed to form an IP 3 -insensitive, "leaky" channel that contributes toward the apoptotic cascade (40,43), although this notion is controversial (44,50,51). Indeed, a very recent study shows that the IP 3 R1 fragments generated by caspase-3 remain associated and form functional channels fully capable of responding to IP 3 and contributing toward apoptosis (44).…”

Fragmented Inositol 1,4,5-Trisphosphate Receptors Retain Tetrameric Architecture and Form Functional Ca2+ Release Channels

“…Moreover, these latter studies concluded that IP 3 R1 was, if at all, a very late caspase substrate during apoptosis. A recent study has also proposed that caspase activity is not required for the apoptotic Ca 2ϩ increase and that IP 3 -mediated Ca 2ϩ signals, rather than the consequences of IP 3 R1 cleavage, were solely responsible for intracellular Ca 2ϩ elevations associated with staurosporine-induced apoptosis in HeLa cells and human primary fibroblasts (44). An additional scenario was proposed by Khan et al (34) in light of evidence that staurosporine-induced increases in [Ca 2ϩ ] i occurred even in DT40 cells expressing the channel-only domain or fulllength IP 3 R1 with an inactivating channel pore mutation.…”

Simvastatin Potently Induces Calcium-dependent Apoptosis of Human Leiomyoma Cells

“…4C). Many apoptotic stimuli induce calcium release very early in the apoptotic program (minutes to hours) (44), and this is useful to discern whether the calcium elevations are causative or a consequence of the apoptotic program (45). To measure the effects of simvastatin early in the apoptotic program, we measured cytosolic calcium continuously for the first 5 h after simvastatin administration using the genetically encoded calcium indicator GCaMP6s (37).…”