Advertisement

Inositol 1,4,5-Trisphosphate Receptor Type 1 Is a Substrate for Caspase-3 and Is Cleaved during Apoptosis in a Caspase-3-dependent Manner*

Open AccessPublished:November 26, 1999DOI:https://doi.org/10.1074/jbc.274.48.34433
      The inositol 1,4,5-trisphosphate (IP3) receptor (IP3R), an IP3-gated Ca2+ channel located on intracellular Ca2+ stores, modulates intracellular Ca2+signaling. During apoptosis of the human T-cell line, Jurkat cells, as induced by staurosporine or Fas ligation, IP3R type 1 (IP3R1) was found to be cleaved. IP3R1 degradation during apoptosis was inhibited by pretreatment of Jurkat cells with the caspase-3 (-like protease) inhibitor, Ac-DEVD-CHO, and the caspases inhibitor, z-VAD-CH2DCB but not by the caspase-1 (-like protease) inhibitor, Ac-YVAD-CHO, suggesting that IP3R1 was cleaved by a caspase-3 (-like) protease. The recombinant caspase-3 cleaved IP3R1 in vitro to produce a fragmentation pattern consistent with that seen in Jurkat cells undergoing apoptosis. N-terminal amino acid sequencing revealed that the major cleavage site is 1888DEVD*1892R (mouse IP3R1), which involves consensus sequence for caspase-3 cleavage (DEVD). To determine whether IP3R1 is cleaved by caspase-3 or is proteolyzed in its absence by other caspases, we examined the cleavage of IP3R1 during apoptosis in the MCF-7 breast carcinoma cell line, which has genetically lost caspase-3. Tumor necrosis factor-α- or staurosporine-induced apoptosis in caspase-3-deficient MCF-7 cells failed to demonstrate cleavage of IP3R1. In contrast, MCF-7/Casp-3 cells stably expressing caspase-3 showed IP3R1 degradation upon apoptotic stimuli. Therefore IP3R1 is a newly identified caspase-3 substrate, and caspase-3 is essential for the cleavage of IP3R1 during apoptosis. This cleavage resulted in a decrease in the channel activity as IP3R1 was digested, indicating that caspase-3 inactivates IP3R1 channel functions.
      IP3
      d-myo-inositol 1,4,5-trisphosphate
      IP3R
      IP3 receptor
      IP3R1
      IP3R type 1
      IP3R2
      IP3R type 2
      IP3R3
      IP3R type 3, mAb, monoclonal antibody
      IICR
      IP3-induced Ca2+ release
      TNF-α
      tumor necrosis factor-α
      Apoptosis is an evolutionary conserved form of cell death by which normal cellular development and homeostasis are maintained. This programmed cell death is regulated by a series of biochemical events, namely activation of a family of cysteine proteases, caspases, which in turn cleave specific intracellular proteins resulting in an irreversible commitment to cell death. Among the caspase family, caspase-3 plays a crucial role in execution of apoptosis. Known substrates for caspase-3 (
      • Rudel T.
      • Bokoch G.M.
      ) involve poly(ADP-ribose) polymerase, p21-activated kinase 2 (
      • Rudel T.
      • Bokoch G.M.
      ,
      • Porter A.G.
      • Ng P.
      • Janicke R.U.
      ), gelsolin (
      • Kothakota S.
      • Azuma T.
      • Reinhard C.
      • Klippel A.
      • Tang J.
      • Chu K.
      • McGarry T.J.
      • Kirschner M.W.
      • Koths K.
      • Kwiatkowski D.J.
      • Williams L.T.
      ), DNA-dependent protein kinase catalytic subunit, DNA fragmentation factor 45 kDa subunit (
      • Liu X.
      • Zou H.
      • Slaughter C.
      • Wang X.
      ), and α-fodrin (
      • Cryns V.L.
      • Bergeron L.
      • Zhu H.
      • Li H.
      • Yuan J.
      ,
      • Martin S.J.
      • O'Brien G.A.
      • Nishioka W.K.
      • McGahon A.J.
      • Mahboubi A.
      • Saido T.C.
      • Green D.R.
      ). By analogy of the cleavage site of these substrates, amino acid sequence of DEXD is considered to be a recognition motif of caspase-3.
      Inositol 1,4,5-trisphosphate (IP3)1 receptor (IP3R), an IP3-gated Ca2+ channel located on intracellular Ca2+ stores, plays a crucial role in a variety of cell functions, including fertilization, cell proliferation, metabolism, secretion, contraction of smooth muscle, and neural signals (
      • Berridge M.J.
      ,
      • Mikoshiba K.
      ). Molecular cloning studies revealed that there are three types of IP3R: IP3R type 1 (IP3R1), IP3R type 2 (IP3R2), and IP3R type 3 (IP3R3) (
      • Furuichi T.
      • Yoshikawa S.
      • Miyawaki A.
      • Wada K.
      • Maeda N.
      • Mikoshiba K.
      ,
      • Sudhof T.C.
      • Newton C.L.
      • Archer III, B.T.
      • Ushkaryov Y.A.
      • Mignery G.A.
      ,
      • Blondel O.
      • Takeda J.
      • Janssen H.
      • Seino S.
      • Bell G.I.
      ,
      • Yamamoto-Hino M.
      • Sugiyama T.
      • Hikichi K.
      • Mattei M.G.
      • Hasegawa K.
      • Sekine S.
      • Sakurada K.
      • Miyawaki A.
      • Furuichi T.
      • Hasegawa M.
      • Mikoshiba K.
      ). The involvement of IP3Rs during apoptosis has been proposed (
      • Khan A.A.
      • Soloski M.J.
      • Sharp A.H.
      • Schilling G.
      • Sabatini D.M.
      • Li S.H.
      • Ross C.A.
      • Snyder S.H.
      ,
      • Jayaraman T.
      • Marks A.R.
      ,
      • Sugawara H.
      • Kurosaki M.
      • Takata M.
      • Kurosaki T.
      ). Khanet al. (
      • Khan A.A.
      • Soloski M.J.
      • Sharp A.H.
      • Schilling G.
      • Sabatini D.M.
      • Li S.H.
      • Ross C.A.
      • Snyder S.H.
      ) reported that mRNA and protein of IP3R3 increase in B and T lymphocytes in response to anti-IgM antibodies and dexamethasone, respectively. Reduction of IP3R3 expression by antisense construct of IP3R3 cDNA blocked the dexamethasone-induced apoptosis. Jayaraman and Marks (
      • Jayaraman T.
      • Marks A.R.
      ) reported that a stable transformant of the human T-cell line, Jurkat, expressing an antisense cDNA construct of IP3R1 is resistant to apoptotic stimuli, including Fas, dexamethasone, and γ-irradiation, despite the finding that T-cells in IP3R1-deficient mice normally develop and respond to proliferative and death signals (
      • Hirota J.
      • Baba M.
      • Matsumoto M.
      • Furuichi T.
      • Takatsu K.
      • Mikoshiba K.
      ). Sugawara et al. (
      • Sugawara H.
      • Kurosaki M.
      • Takata M.
      • Kurosaki T.
      ) reported that IP3/Ca2+ signaling is involved in B-cell antigen receptor-induced apoptosis in a chick B-cell line, DT40 cells. In their experiments, IP3R-deficient cells showed a reduction in apoptosis in which the degree of resistance depend on the number of IP3Rs depleted, i.e. triple IP3R-deficient cells were more resistant than single IP3R-deficient cells.
      Although it has been demonstrated that IP3Rs are involved in the process of apoptosis, less attention has been directed to the relationship between IP3Rs and caspases. Among the IP3R family, IP3R1 is the most widely expressed in tissues and is recognized as an ubiquitous type of IP3R. In the primary amino acid sequence of IP3R1, there is the DEVD consensus sequence for caspase-3 cleavage at 1889–1892 amino acids of mouse IP3R1, which is conserved among species (1888DEVD rat IP3R1 and 1835DEVD human IP3R1). In the present studies, we asked whether IP3R1 could serve as a substrate of caspase-3 during apoptosis, and we obtained evidence that IP3R1 is a newly identified substrate for caspase-3. Using caspase-3-deficient cells, MCF-7 (
      • Janicke R.U.
      • Sprengart M.L.
      • Wati M.R.
      • Porter A.G.
      ), we found that caspase-3 is essential for the cleavage of IP3R1. In addition, effects of the cleavage by caspase-3 on the IP3R1 channel function were also given attention.

      Acknowledgments

      We thank Dr. M. Miura for the human recombinant CPP32/caspase-3 and for the FLAG-tagged caspase-3 expression construct, pM136, and M. Ohara for helpful comments and language assistance.

      REFERENCES

        • Rudel T.
        • Bokoch G.M.
        Science. 1997; 276: 1571-1574
        • Porter A.G.
        • Ng P.
        • Janicke R.U.
        Bioessays. 1997; 19: 501-507
        • Kothakota S.
        • Azuma T.
        • Reinhard C.
        • Klippel A.
        • Tang J.
        • Chu K.
        • McGarry T.J.
        • Kirschner M.W.
        • Koths K.
        • Kwiatkowski D.J.
        • Williams L.T.
        Science. 1997; 278: 294-298
        • Liu X.
        • Zou H.
        • Slaughter C.
        • Wang X.
        Cell. 1997; 89: 175-184
        • Cryns V.L.
        • Bergeron L.
        • Zhu H.
        • Li H.
        • Yuan J.
        J. Biol. Chem. 1996; 271: 31277-31282
        • Martin S.J.
        • O'Brien G.A.
        • Nishioka W.K.
        • McGahon A.J.
        • Mahboubi A.
        • Saido T.C.
        • Green D.R.
        J. Biol. Chem. 1995; 270: 6425-6428
        • Berridge M.J.
        Nature. 1993; 361: 315-325
        • Mikoshiba K.
        Curr. Opin. Neurobiol. 1997; 7: 339-345
        • Furuichi T.
        • Yoshikawa S.
        • Miyawaki A.
        • Wada K.
        • Maeda N.
        • Mikoshiba K.
        Nature. 1989; 342: 32-38
        • Sudhof T.C.
        • Newton C.L.
        • Archer III, B.T.
        • Ushkaryov Y.A.
        • Mignery G.A.
        EMBO J. 1991; 10: 3199-3206
        • Blondel O.
        • Takeda J.
        • Janssen H.
        • Seino S.
        • Bell G.I.
        J. Biol. Chem. 1993; 268: 11356-11363
        • Yamamoto-Hino M.
        • Sugiyama T.
        • Hikichi K.
        • Mattei M.G.
        • Hasegawa K.
        • Sekine S.
        • Sakurada K.
        • Miyawaki A.
        • Furuichi T.
        • Hasegawa M.
        • Mikoshiba K.
        Receptors Channels. 1994; 2: 9-22
        • Khan A.A.
        • Soloski M.J.
        • Sharp A.H.
        • Schilling G.
        • Sabatini D.M.
        • Li S.H.
        • Ross C.A.
        • Snyder S.H.
        Science. 1996; 273: 503-507
        • Jayaraman T.
        • Marks A.R.
        Mol. Cell. Biol. 1997; 17: 3005-3012
        • Sugawara H.
        • Kurosaki M.
        • Takata M.
        • Kurosaki T.
        EMBO J. 1997; 16: 3078-3088
        • Hirota J.
        • Baba M.
        • Matsumoto M.
        • Furuichi T.
        • Takatsu K.
        • Mikoshiba K.
        Biochem. J. 1998; 333: 615-619
        • Janicke R.U.
        • Sprengart M.L.
        • Wati M.R.
        • Porter A.G.
        J. Biol. Chem. 1998; 273: 9357-9360
        • Sugiyama T.
        • Furuya A.
        • Monkawa T.
        • Yamamoto-Hino M.
        • Satoh S.
        • Ohmori K.
        • Miyawaki A.
        • Hanai N.
        • Mikoshiba K.
        • Hasegawa M.
        FEBS Lett. 1994; 354: 149-154
        • Sugiyama T.
        • Yamamoto-Hino M.
        • Miyawaki A.
        • Furuichi T.
        • Mikoshiba K.
        • Hasegawa M.
        FEBS Lett. 1994; 349: 191-196
        • Yoshikawa F.
        • Iwasaki H.
        • Michikawa T.
        • Furuichi T.
        • Mikoshiba K.
        J. Biol. Chem. 1999; 274: 316-327
        • Nakade S.
        • Rhee S.K.
        • Hamanaka H.
        • Mikoshiba K.
        J. Biol. Chem. 1994; 269: 6735-6742
        • Nakade S.
        • Maeda N.
        • Mikoshiba K.
        Biochem. J. 1991; 277: 125-131
        • Grynkiewicz G.
        • Poenie M.
        • Tsien R.Y.
        J. Biol. Chem. 1985; 260: 3440-3450
        • Wojcikiewicz R.J.H.
        • Oberdorf J.A.
        J. Biol. Chem. 1996; 271: 16652-16655
        • Janicke R.U.
        • Ng P.
        • Sprengart M.L.
        • Porter A.G.
        J. Biol. Chem. 1998; 273: 15540-15545
        • Sakahira H.
        • Enari M.
        • Nagata S.
        Nature. 1998; 391: 96-99
        • Tang D.
        • Kidd V.J.
        J. Biol. Chem. 1998; 273: 28549-28552
        • Hirota J.
        • Michikawa T.
        • Miyawaki A.
        • Furuichi T.
        • Okura I.
        • Mikoshiba K.
        J. Biol. Chem. 1995; 270: 19046-19051