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.
Background/Aims: The inositol 1,4,5-trisphosphate receptor (IP3R), a ligand-gated Ca2+ channel, plays an important role in the control of intracellular Ca2+. Three isoforms of IP3R have been identified and most cell types express different proportions of these isoforms. The purpose of this study was to investigate how IP3R signalling is involved in the activation of the Ca2+-sensitive transcription factors NFAT and CREB. Methods: Each IP3R isoform expressed in HEK 293A cells was knocked down using selective siRNA. Free intracellular Ca2+ was monitored spectrofluometrically. NFAT and CREB activities were measured with luciferase reporter constructs. Results: IP3R-2-knocked down HEK 293A cells showed a deficient CCh-induced Ca2+ response that could be rescued by co-stimulation with VIP, a cAMP increasing agonist. NFAT transcriptional activity, but not CREB transcriptional activity, was significantly reduced in IP3R-2-knocked down HEK 293A cells. Overexpression of IP3R-1 could fully compensate for IP3R-2 knock down to mobilize Ca2+ and to activate NFAT. Conclusion: Our results show that the knock down of IP3R-2 significantly reduced the intracellular Ca2+ response of HEK 293 cells. This reduced Ca2+ response did not affect the activation of CREB but significantly decreased the activation of NFAT, suggesting that the Ca2+ signals required for the activation of NFAT are stronger than those required for the activation of CREB.
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