Type 1 inositol‐1,4,5‐trisphosphate receptor is a late substrate of caspases during apoptosis

Abstract

Apoptosis is characterized by the proteolytic cleavage of hundreds of proteins. One of them, the type 1 inositol-1,4,5-trisphosphate receptor (IP(3) R-1), a multimeric receptor located on the endoplasmic reticulum (ER) membrane that is critical to calcium homeostasis, was reported to be cleaved during staurosporine (STS) induced-apoptosis in Jurkat cells. Because the reported cleavage site separates the IP(3) binding site from the channel moiety, its cleavage would shut down a critical signaling pathway that is common to several cellular processes. Here we show that IP(3) R-1 is not cleaved in 293 cells treated with STS, TNFα, Trail, or ultra-violet (UV) irradiation. Further, it is not cleaved in Hela or Jurkat cells induced to undergo apoptosis with Trail, TNFα, or UV. In accordance with previous reports, we demonstrate that it is cleaved in a Jurkat cell line treated with STS. However its cleavage occurs only after poly(ADP-ribose) polymerase (PARP), which cleavage is a hallmark of apoptosis, and p23, a poor caspase-7 substrate, are completely cleaved, suggesting that IP(3) R-1 is a relatively late substrate of caspases. Nevertheless, the receptor is fully accessible to proteolysis in cellulo by ectopically overexpressed caspase-7 or by the tobacco etch virus (TEV) protease. Finally, using recombinant caspase-3 and microsomal fractions enriched in IP(3) R-1, we show that the receptor is a poor caspase-3 substrate. Consequently, we conclude that IP(3) R-1 is not a key death substrate.

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

“…Furthermore, production of the 95 kDa C-terminal fragment was not evident in these experiments (open arrowhead, Fig. 2D), which is consistent with a recently published report [26]. The slow kinetics of IP 3 R-1 degradation in vitro and in intact cells would make it unlikely that caspase 3-mediated truncation of IP 3 R-1 contributes to the rapid mobilization of intracellular calcium seen with staurosporine treatment [14].…”

“…Our results complement a study which was published while this paper was under review demonstrating that the IP 3 R-1 in not cleaved using several apoptotic paradigms, and that it is a poor substrate for caspase 3 in vitro [26]. We confirm and extend these findings in this study to show that caspase 3 does not contribute to apoptotic calcium release.…”

“…Furthermore, caspase 3 activation and cleavage of intracellular substrates is a relatively late event in apoptosis, and we have shown that caspase activities cannot explain the initial changes in intracellular calcium in response to apoptotic stimuli such as staurosporine and Fas ligand [14, 15]. Another group has recently confirmed that cleavage of IP 3 R-1 by caspase occurs late in apoptosis and, therefore, it is not a key event in apoptotic signaling [16]. …”

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

“…For example, IP 3 R1 was shown to undergo caspase 3-dependent cleavage in various cell types, and this cleavage led to the formation of a C-terminal constitutively leaky channel termed a "channel-only domain" that mediates Ca 2ϩ release essential for complete execution of the apoptotic program (22,24,27). In contrast, it has been reported that IP 3 R1 is not cleaved in HEK293, HeLa, and Jurkat cells undergoing apoptosis in response to exposure to staurosporine, TNF␣, Trail, or UV irradiation (43). Moreover, these latter studies concluded that IP 3 R1 was, if at all, a very late caspase substrate during apoptosis.…”

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).…”