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• W1976378506 abstract "The role of inositol 1,4,5-trisphosphate receptors (IP3R) in caspase-3 activation and cell death was investigated in DT40 chicken B-lymphocytes stably expressing various IP3R constructs. Both full-length type-I IP3R and a truncated construct corresponding to the caspase-3 cleaved “channel-only” fragment were able to support staurosporine (STS)-induced caspase-3 activation and cell death even when the IP3R construct harbored a mutation that inactivates the pore of the Ca2+ channel (D2550A). However, a full-length wild-type IP3R did not promote caspase-3 activation when the 159-amino acid cytosol-exposed C-terminal tail was deleted. STS caused an increase in cytosolic free Ca2+ in DT40 cells expressing wild-type or pore-dead IP3R mutants. However, in the latter case all the Ca2+ increase originated from Ca2+ entry across the plasma membrane. Caspase-3 activation of pore-dead DT40 cells was also more sensitive to extracellular Ca2+ chelation when compared with wild-type cells. STS-mediated release of cytochrome c into the cytosol and mitochondrial membrane potential depolarization could also be observed in DT40 cells lacking IP3Rs or containing the pore-dead mutant. We conclude that nonfunctional IP3Rs can sustain apoptosis in DT40 lymphocytes, because they facilitate Ca2+ entry mechanisms across the plasma membrane. Although the intrinsic ion-channel function of IP3Rs is dispensable for apoptosis induced by STS, the C-terminal tail of IP3Rs appears to be essential, possibly reflecting key protein-protein interactions with this domain. The role of inositol 1,4,5-trisphosphate receptors (IP3R) in caspase-3 activation and cell death was investigated in DT40 chicken B-lymphocytes stably expressing various IP3R constructs. Both full-length type-I IP3R and a truncated construct corresponding to the caspase-3 cleaved “channel-only” fragment were able to support staurosporine (STS)-induced caspase-3 activation and cell death even when the IP3R construct harbored a mutation that inactivates the pore of the Ca2+ channel (D2550A). However, a full-length wild-type IP3R did not promote caspase-3 activation when the 159-amino acid cytosol-exposed C-terminal tail was deleted. STS caused an increase in cytosolic free Ca2+ in DT40 cells expressing wild-type or pore-dead IP3R mutants. However, in the latter case all the Ca2+ increase originated from Ca2+ entry across the plasma membrane. Caspase-3 activation of pore-dead DT40 cells was also more sensitive to extracellular Ca2+ chelation when compared with wild-type cells. STS-mediated release of cytochrome c into the cytosol and mitochondrial membrane potential depolarization could also be observed in DT40 cells lacking IP3Rs or containing the pore-dead mutant. We conclude that nonfunctional IP3Rs can sustain apoptosis in DT40 lymphocytes, because they facilitate Ca2+ entry mechanisms across the plasma membrane. Although the intrinsic ion-channel function of IP3Rs is dispensable for apoptosis induced by STS, the C-terminal tail of IP3Rs appears to be essential, possibly reflecting key protein-protein interactions with this domain. Apoptosis is an essential process required for normal development and tissue homeostasis and can be activated by diverse stimuli, including cytotoxic drugs, DNA damage, irradiation, withdrawal of growth factors, and activation of death receptors by ligands such as tumor necrosis factor or Fas. A requirement for elevated levels of Ca2+ has been implicated in many of these models of apoptosis (1Orrenius S. Zhivotovsky B. Nicotera P. Nat. Rev. Mol. Cell Biol. 2003; 4: 552-565Crossref PubMed Scopus (2377) Google Scholar, 2Rizzuto R. Pinton P. Ferrari D. Chami M. Szabadkai G. Magalhaes P.J. Di Virgilio F. Pozzan T. Oncogene. 2003; 22: 8619-8627Crossref PubMed Scopus (388) Google Scholar, 3Hajnoczky G. Davies E. Madesh M. Biochem. Biophys. Res. Commun. 2003; 304: 445-454Crossref PubMed Scopus (391) Google Scholar). Several key enzymes activated during apoptosis such as endonucleases (4Nagata S. Exp. Cell Res. 2000; 256: 12-18Crossref PubMed Scopus (734) Google Scholar), phospholipase-A2 (5Taketo M.M. Sonoshita M. Biochim. Biophys. Acta. 2002; 1585: 72-76Crossref PubMed Scopus (124) Google Scholar), and calpains (6Goll D.E. Thompson V.F. Li H. Wei W. Cong J. Physiol. Rev. 2003; 83: 731-801Crossref PubMed Scopus (2342) Google Scholar) are known to be stimulated by Ca2+. However, the exact steps in apoptotic cascades that are affected by Ca2+ and the mechanisms resulting in the perturbation of Ca2+ homeostasis are poorly understood. Elevations of cytosolic Ca2+ ([Ca2+]i) are achieved in cells by mobilization of intracellular Ca2+ stores and/or enhancement of Ca2+ entry across the plasma membrane. Inositol 1,4,5-trisphosphate receptors (IP3Rs) 2The abbreviations used are: IP3R, inositol 1,4,5-trisphosphate receptor; ER, endoplasmic reticulum; CHAPS, (3-[3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; Cyt c, cytochrome c; aa, amino acid(s); Ab, antibody; mAb, monoclonal antibody; STS, staurosporine; HA, hemagglutinin; TKO, triple knock-out; CO, channel-only; BAPTA-AM, 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis (acetoxymethyl ester). 2The abbreviations used are: IP3R, inositol 1,4,5-trisphosphate receptor; ER, endoplasmic reticulum; CHAPS, (3-[3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; Cyt c, cytochrome c; aa, amino acid(s); Ab, antibody; mAb, monoclonal antibody; STS, staurosporine; HA, hemagglutinin; TKO, triple knock-out; CO, channel-only; BAPTA-AM, 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis (acetoxymethyl ester). are a family of three intracellular Ca2+-release channels, mainly located in the ER membrane, which are primarily responsible for the agonist-mediated release of Ca2+ from intracellular stores. A number of different experimental approaches have implicated IP3Rs as pro-apoptotic regulators. The targeted deletion of all three IP3R isoforms in the chicken DT-40 lymphocyte cell line renders the cells more resistant to apoptotic stimuli induced by anti-IgM or staurosporine (STS) (7Sugawara H. Kurosaki M. Takata M. Kurosaki T. EMBO J. 1997; 16: 3078-3088Crossref PubMed Scopus (375) Google Scholar, 8Assefa Z. Bultynck G. Szlufcik K. Nadif K.N. Vermassen E. Goris J. Missiaen L. Callewaert G. Parys J.B. De Smedt H. J. Biol. Chem. 2004; 279: 43227-43236Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). Decreasing the expression of the type-III (9Khan A.A. Soloski M.J. Sharp A.H. Schilling G. Sabatini D.M. Li S. Ross C.A. Snyder S.H. Science. 1996; 273: 503-506Crossref PubMed Scopus (240) Google Scholar) or the type-I (10Jayaraman T. Marks A.R. Mol. Cell. Biol. 1997; 17: 3005-3012Crossref PubMed Scopus (231) Google Scholar) IP3R isoforms has been shown to confer resistance to various apoptotic stimuli in Jurkat T-cell lines. A selective role for the type-III IP3R in apoptosis was also reported in a study using small interference RNA to suppress expression of individual IP3R isoforms (11Mendes C.C. Gomes D.A. Thompson M. Souto N.C. Goes T.S. Goes A.M. Rodrigues M.A. Gomez M.V. Nathanson M.H. Leite M.F. J. Biol. Chem. 2005; 280: 40892-40900Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar). However, mice deficient in both type-II and III IP3R isoforms do not show defects in developmental apoptosis suggesting the presence of significant redundancy in the requirement for individual IP3R isoforms (12Futatsugi A. Nakamura T. Yamada M.K. Ebisui E. Nakamura K. Uchida K. Kitaguchi T. Takahashi-Iwanaga H. Noda T. Aruga J. Mikoshiba K. Science. 2005; 309: 2232-2234Crossref PubMed Scopus (251) Google Scholar). The intrinsic pathway of apoptosis involves the release of cytochrome c (Cyt c) from the mitochondrial intermembrane space and the initiation of a cascade of events that ultimately leads to the activation of caspase-3. Although the exact transport pathway utilized for Cyt c release during apoptosis or necrosis is controversial, it is well established that enhanced accumulation of Ca2+ by the mitochondria sensitizes the Cyt c release pathway to stimulation by apoptotic agents (3Hajnoczky G. Davies E. Madesh M. Biochem. Biophys. Res. Commun. 2003; 304: 445-454Crossref PubMed Scopus (391) Google Scholar, 13Szabadkai G. Rizzuto R. FEBS Lett. 2004; 567: 111-115Crossref PubMed Scopus (123) Google Scholar). The close proximity of ER to mitochondria suggests that the Ca2+ channel function of IP3Rs could be important in modulating the kinetics and sensitivity of apoptotic pathways (3Hajnoczky G. Davies E. Madesh M. Biochem. Biophys. Res. Commun. 2003; 304: 445-454Crossref PubMed Scopus (391) Google Scholar, 13Szabadkai G. Rizzuto R. FEBS Lett. 2004; 567: 111-115Crossref PubMed Scopus (123) Google Scholar, 14Joseph S.K. Hajnoczky G. Apoptosis. 2007; 12: 951-968Crossref PubMed Scopus (135) Google Scholar). Further evidence for the pro-apoptotic role of IP3Rs is indicated by the finding that a number of key components of apoptotic cascades appear to interact with and regulate IP3R function. The anti-apoptotic proteins Bcl-2 and Bcl-xL have been shown to bind IP3Rs, but the studies differ as to whether this results is an inhibition or activation of IP3R channel activity (15Chen R. Valencia I. Zhong F. McColl K.S. Roderick H.L. Bootman M.D. Berridge M.J. Conway S.J. Holmes A.B. Mignery G.A. Velez P. Distelhorst C.W. J. Cell Biol. 2004; 166: 193-203Crossref PubMed Scopus (345) Google Scholar, 16White C. Li C. Yang J. Petrenko N.B. Madesh M. Thompson C.B. Foskett J.K. Nat. Cell Biol. 2005; 7: 1021-1028Crossref PubMed Scopus (354) Google Scholar). Mouse embryonic fibroblast cell lines obtained from Bax/Bak double knock-out mice show an increased phosphorylation of the IP3R and an enhanced Ca2+ leak across the ER membrane (17Oakes S.A. Scorrano L. Opferman J.T. Bassik M.C. Nishino M. Pozzan T. Korsmeyer S.J. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 105-110Crossref PubMed Scopus (385) Google Scholar). This has been attributed to the effect of unrestrained Bcl-2 on IP3Rs (17Oakes S.A. Scorrano L. Opferman J.T. Bassik M.C. Nishino M. Pozzan T. Korsmeyer S.J. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 105-110Crossref PubMed Scopus (385) Google Scholar). The type-I IP3R isoform contains a consensus caspase-3 cleavage site and has been shown to be a substrate for this enzyme in various models of apoptosis (8Assefa Z. Bultynck G. Szlufcik K. Nadif K.N. Vermassen E. Goris J. Missiaen L. Callewaert G. Parys J.B. De Smedt H. J. Biol. Chem. 2004; 279: 43227-43236Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar, 18Hirota J. Furuichi T. Mikoshiba K. J. Biol. Chem. 1999; 274: 34433-34437Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 19Haug L.S. Walaas I. Ostvold A.C. J. Neurochem. 2000; 75: 1852-1861Crossref PubMed Scopus (29) Google Scholar, 20Diaz F. Bourguignon L.Y. Cell Calcium. 2000; 27: 315-328Crossref PubMed Scopus (43) Google Scholar). The C-terminal “channel-only” fragment produced from caspase-3 cleavage has been reported to form a constitutively open channel and to contribute to the Ca2+ leak pathway across ER membranes (8Assefa Z. Bultynck G. Szlufcik K. Nadif K.N. Vermassen E. Goris J. Missiaen L. Callewaert G. Parys J.B. De Smedt H. J. Biol. Chem. 2004; 279: 43227-43236Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar, 21Nakayama T. Hattori M. Uchida K. Nakamura T. Tateishi Y. Bannai H. Iwai M. Michikawa T. Inoue T. Mikoshiba K. Biochem. J. 2004; 377: 299-307Crossref PubMed Scopus (78) Google Scholar). Cyt c released from the mitochondria has been found to bind and activate IP3Rs by decreasing the feedback inhibition of these channels by Ca2+ (22Boehning D. Patterson R.L. Sedaghat L. Glebova N.O. Kurosaki T. Snyder S.H. Nat. Cell Biol. 2003; 5: 1051-1061Crossref PubMed Scopus (534) Google Scholar). Finally, IP3Rs have been found to be substrates for the important anti-apoptotic regulatory protein Akt kinase (23Khan M.T. Wagner L. Yule D.I. Bhanumathy C.D. Joseph S.K. J. Biol. Chem. 2006; 281: 3731-3737Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). The highly conserved phosphorylation site for Akt kinase, as well as the binding sites for Bcl-xL and Cyt c, are all localized to the short, cytosol-exposed C-terminal tail of the receptor (16White C. Li C. Yang J. Petrenko N.B. Madesh M. Thompson C.B. Foskett J.K. Nat. Cell Biol. 2005; 7: 1021-1028Crossref PubMed Scopus (354) Google Scholar, 23Khan M.T. Wagner L. Yule D.I. Bhanumathy C.D. Joseph S.K. J. Biol. Chem. 2006; 281: 3731-3737Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar, 24Boehning D. van Rossum D.B. Patterson R.L. Snyder S.H. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 1466-1471Crossref PubMed Scopus (108) Google Scholar). With the exception of Akt kinase phosphorylation, a common feature of all the aforementioned modes of IP3R regulation during apoptotic signaling is that they are considered to modulate the Ca2+ channel function of the intact or caspase-cleaved IP3R ion channel. To better understand the role of IP3Rs in regulating apoptosis we have examined the effect of introducing an ion channel-defective IP3R mutant into the IP3R DT-40 triple knock-out lymphocytes. Surprisingly, this cell line continued to respond to cytotoxic apoptotic stimuli in a similar manner as wild-type DT-40 cells. Our studies reveal an ion-channel independent role of IP3Rs in apoptosis, which may involve protein-protein interactions, possibly with the C-terminal tail of the receptor. Reagents—Pfu polymerase was from Stratagene (Madison, WI). Protogel-stabilized acrylamide solution was from National Diagnostic (Atlanta, GA). Horseradish peroxidase-conjugated secondary antibodies were purchased from Amersham Biosciences. Enhanced chemiluminescent substrate was obtained from Pierce. Mouse anti-chicken IgM (clone-M4) was from Southern Biotech (Birmingham, AL). RPMI 1640 culture media and G418 sulfate (Geneticin) were from Mediatech (Herndon, VA). Staurosporine, cyclosporine, FK506, and CHAPS were purchased from Sigma. DEVD-7-aminomethyl coumarin was purchased from Alexis (San Diego, CA). For cell viability measurements we used the CellTitre 96 Aqueous One solution cell proliferation assay purchased from Promega (Madison, VI). Antibodies—The Ab against the C terminus of the type-I IP3R (CT-1 Ab) has been described previously (25Joseph S. Samanta S. J. Biol. Chem. 1993; 268: 6477-6486Abstract Full Text PDF PubMed Google Scholar). The antibody was further affinity-purified using the peptide coupled to Ultralink beads as described by the manufacturer (Pierce). Anti-cytochrome c (Cyt c) mAb was from Zymed Labs (San Francisco, CA) and anti-HA mAb was purchased from Roche Applied Science. Anti-tubulin mAb was from Invitrogen-Molecular Probes (Eugene, OR). DNA Constructs—The rat type I IP3R construct containing a Kozac sequence and subcloned into pcDNA 3.1 has been described previously (26Boehning D. Joseph S.K. J. Biol. Chem. 2000; 275: 21492-21499Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). The splice variants used in this study were SI(-), SII(+), and SIII(-). The channel-only (aa 1892–2749) portion of IP3R was generated using PCR. The forward and reverse primers used were 5′-TCGAATTCCCACCATGCGGGATGCCCCATCCCGAAAG-3′ and 5′-GCTTATGGTTTCTAGATTCGCG-3′. These primers encoded EcoR1/XbaI sites (underlined), which were utilized to clone the PCR product into similarly digested pcDNA 3.1 vector. The product was confirmed by automated sequencing (Nucleic acid Facility, Thomas Jefferson University, Philadelphia, PA). The S2681A and S2681E point mutants in full-length IP3R were constructed as described earlier (23Khan M.T. Wagner L. Yule D.I. Bhanumathy C.D. Joseph S.K. J. Biol. Chem. 2006; 281: 3731-3737Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). The S2681A, S2681E, and the D2550A pore mutation in full-length receptors were transferred to the channel-only IP3R construct using BstbI/XbaI restriction sites. Constructs encoding deletions in the C-terminal tail were generated as described by Schug and Joseph (27Schug Z.T. Joseph S.K. J. Biol. Chem. 2006; 281: 24431-24440Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). Cells and Stable Transfection—DT40 cells (wild-type and triple knock-out (TKO) of IP3R isoforms) were a kind Gift of Dr. T. Kurosaki (Kansai Medical University, Moriguchi, Japan). Stable DT40 cells expressing the rat type I IP3R were a gift from Dr. Kevin Foskett (University of Pennsylvania, PA). DT40 cells were grown in RPMI 1640 media supplemented with 10% fetal bovine serum, 1% chicken serum, and 100 units/ml penicillin, 100 μg/ml streptomycin, and maintained at 37 °C in 5% CO2 atmosphere. The stable cell lines expressing various IP3R mutants were prepared by electroporation of 0.5 ml of DT40 TKO cells (106 cells/ml) with 40 μg of XbaI linearized DNA using a Gene pulsar apparatus (Bio-Rad, 340 V and 950 microfarads). The cells were grown in 30 ml of RPMI for 24 h and were then serially diluted in a volume of 1:10, 1:100, and 1:1000 in RPMI containing 1.5 mg/ml G418. The diluted cells were transferred to 96-well plates and incubated for 1–2 weeks in a CO2-incubator at 37 °C. Positive clones were identified by screening for expression of IP3Rs by immunoblotting. Measurement of Caspase Activation in DT40 Cells—For fluorometric assays, DT40 cells were collected and lysed in a buffer containing 20 mm Tris/Hepes, pH 7.4, 0.1% CHAPS, 5 mm EDTA. Lysates containing 10 μg of protein were incubated with 25 μm DEVD-aminomethyl coumarin as substrate for 30 min at 37 °C. Fluorescence of the aminomethyl coumarin product was measured at 380 nm excitation and 460 nm emission wavelength. Flow cytometry detection of caspase-3 was carried out according to manufacturer's instructions using a caspase-3 reagent (FLICA, Invitrogen-Molecular Probes). After apoptosis induction, the DT40 cells were analyzed on a Flow cytometer with 488 nm excitation using 530 nm band pass and 670 nm long pass emission filters. Cell Viability Assays—The assay using CellTitre 96 Aqueous One reagent from Promega was done as described by the manufacturer with some modifications. Briefly, 0.5 × 106 DT40 cells were grown in 1 ml of media (in 24-well plates). The cells were treated with STS for 6 h, 20 μl of CellTitre reagent was added to each well, and the mixture was incubated for another 2 h. For a blank, 20 μl of CellTitre reagent was added to 1 ml of RPMI media. The supernatants were transferred in Eppendorf tubes and centrifuged on a microcentrifuge for 2 min. The supernatant was read at 490 nm against the media blank. Measurement of [Ca2+]i in DT40 Cells—Changes in cytosolic [Ca2+] in individual DT40 cells plated on coverslips coated with poly-d-lysine was measured by digital imaging fluorescence microscopy as previously described (28Wagner L.E. Li W.H. Yule D.I. J. Biol. Chem. 2003; 278: 45811-45817Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). The cells were loaded with Fura-2 by incubating with 2 μm Fura-2AM in Hepes buffer, pH 7.4 (10 mm Hepes, 120 mm NaCl, 5 mm KCl, 1 mm CaCl2, 1 mm MgCl2, 6 mm glucose, and 1 mm glutamine) for 20 min at room temperature. The coverslips were washed with the same buffer for 10 min to de-esterify Fura-2 and were transferred to a chamber with 1 ml of HEPES buffer and mounted onto the stage of an inverted microscope thermostatically maintained at 37 °C. Fluorescence images with a 4-s delay were recorded alternately at excitation wavelengths of 340 and 380 nm with an emission wavelength of 460–600 nm using a charge-coupled device imaging system. Changes in cytosolic calcium were measured in response to STS or anti-IgM in the presence or absence of extracellular calcium and are expressed as the ratio of Fura-2 fluorescence at 340 nm and 380 nm (F340/F380). Typically 50–60 cells were monitored per experiment, and traces represent the average of three different experiments. Cyt c Release, Annexin V Assays, and Measurement of Mitochondrial Membrane Potential—Cyt c release into the cytosol was measured in DT40 cells after fractionation using the Apoalert cell fractionation kit (Clontech, Mountain View, CA). Western blotting of lysates was carried out on 15% SDS-PAGE gels with immunoblotting for Cyt c using a monoclonal Ab from Zymed Laboratories Inc. Annexin V fluorescein isothiocyanate conjugate (BioSource, Camarillo, CA) was added together with propidium iodide (5 μg/ml), and staining was analyzed on a flow cytometer. Changes in mitochondrial membrane potential were assessed using the MitoProbe™ DiIC1 (5Taketo M.M. Sonoshita M. Biochim. Biophys. Acta. 2002; 1585: 72-76Crossref PubMed Scopus (124) Google Scholar) assay kit as described by the manufacturer (Invitrogen-Molecular Probes). The stain intensity decreases when reagents disrupt mitochondrial membrane potential. The cells were analyzed on a flow cytometer with 633 nm excitation using emission filters for far red (658 nm). Histograms were analyzed using WinMDI Software. STS-induced Caspase-3 Activation and Cell Death in DT40 Cell Lines Containing Functional and Non-functional IP3Rs—DT40 cells containing a targeted deletion of all 3IP3R isoforms (TKO) were transfected with a number of IP3R mutants and stable cells lines were established. Fig. 1A shows the basic architecture of IP3 receptors with an N-terminal ligand-binding domain, a C-terminal channel domain, and an intervening regulatory domain. Fig. 1B shows a detail of the 159-amino acid C-terminal tail of the receptor that projects into the cytosol together with the binding sites for known proteins that interact with this region. The location of the deletion mutants (channel only, 1TM, tail-less) and point mutants (Asp-2550 and Ser-2618) used in this study are also indicated in the figure. Fig. 2A shows the expression of these mutant constructs detected by immunoblotting with an Ab to the C-terminal 18 amino acids of the type-I IP3R (CT-1) or, in the case of the tail-less mutants, with a C-terminal HA tag Ab. The mutants encoding the caspase-3-cleaved fragment, referred to as “channel-only” (CO) mutants (8Assefa Z. Bultynck G. Szlufcik K. Nadif K.N. Vermassen E. Goris J. Missiaen L. Callewaert G. Parys J.B. De Smedt H. J. Biol. Chem. 2004; 279: 43227-43236Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar), were expressed as a doublet of bands. These could correspond to glycosylated and non-glycosylated bands (cf. Refs. 21Nakayama T. Hattori M. Uchida K. Nakamura T. Tateishi Y. Bannai H. Iwai M. Michikawa T. Inoue T. Mikoshiba K. Biochem. J. 2004; 377: 299-307Crossref PubMed Scopus (78) Google Scholar and 29Joseph S.K. Boehning D. Pierson S. Nicchitta C.V. J. Biol. Chem. 1997; 272: 1579-1588Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). In initial experiments we measured the time course of cell death induced by 0.5 μm STS in wild-type and TKO cells and found that the biggest differences were evident at earlier time points (<6 h) (data not shown). The time course of caspase-3 activation over this period is shown in Fig. 2B. In agreement with published data (8Assefa Z. Bultynck G. Szlufcik K. Nadif K.N. Vermassen E. Goris J. Missiaen L. Callewaert G. Parys J.B. De Smedt H. J. Biol. Chem. 2004; 279: 43227-43236Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar), caspase-3 activation was substantially slowed in TKO cells. However, 80% of the caspase-3 activation of wild-type cells could be rescued by the D2550A IP3R pore mutant, which has previously been shown to inactivate the channel (30Boehning D. Mak D.O. Foskett J.K. Joseph S.K. J. Biol. Chem. 2001; 276: 13509-13512Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). The absence of Ca2+ mobilization in response to cross-linking of cell surface IgM receptors with anti-IgM Ab in D2550A cells is shown in Fig. 2B (inset). Several other studies have confirmed that this mutant is functionally inactive (31Rossum D.B. Patterson R.L. Kiselyov K. Boehning D. Barrow R.K. Gill D.L. Snyder S.H. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 2323-2327Crossref PubMed Scopus (59) Google Scholar, 32Alzayady K.J. Wojcikiewicz R.J. Biochem. J. 2005; 392: 601-606Crossref PubMed Scopus (27) Google Scholar, 33Dellis O. Dedos S.G. Tovey S.C. Taufiq U.R. Dubel S.J. Taylor C.W. Science. 2006; 313: 229-233Crossref PubMed Scopus (164) Google Scholar). Data on caspase-3 activation in several additional mutant cell lines is summarized in Fig. 2C and corresponding measurements of cell viability are shown in Fig. 2D. The wild-type DT40 cells contain three chicken IP3R isoforms, whereas the mutant cells contain only one IP3R isoform made in the background of the rat type-I IP3R. For comparison, we have also used a stable DT40 cell line expressing the wild-type rat type-I IP3R as a control (kindly given by Kevin Foskett). These cells showed a somewhat higher caspase-3 activation, but their cell death response was the same as wild-type DT40 cells.FIGURE 2STS-induced changes in caspase-3 activity and cell viability in mutant IP3R DT40 cell lines. A, immunoblots of cell lysates prepared from the indicated DT40 cell lines. The reason why the CO constructs express as a doublet is not known but may be due to differential glycosylation. B, a time course of caspase-3 activation in the indicated DT40 cell lines in response to 0.5 μm STS measured fluorometrically in cell lysates. The inset shows cytosolic free Ca2+ changes measured in WT and D2550A Fura-2-loaded cells in response to 5 μg/ml α-IgM. C, the cumulative data of caspase-3 activation in response to 0.5 μm STS for 6 h measured in the indicated cell lines and expressed relative to the activation in WT cells. Data are mean ± S.E. (n = 3–6). *, p < 0.05 relative to WT cells. In Panel D, conditions were as described in C except that cell viability was measured with a Promega 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay kit.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Two non-functional IP3R mutants have previously been shown to support STS and anti-IgM induced apoptosis in DT40 cells (8Assefa Z. Bultynck G. Szlufcik K. Nadif K.N. Vermassen E. Goris J. Missiaen L. Callewaert G. Parys J.B. De Smedt H. J. Biol. Chem. 2004; 279: 43227-43236Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). One of these mutants was deleted in the N-terminal 225 amino acids corresponding to the suppressor domain (Fig. 1A). The other was the CO mutant encoding the C-terminal fragment formed from the receptor after caspase-3 cleavage. The results were interpreted as indicating that the caspase-3-cleaved fragment from both inactive mutants could still induce aCa2+ leak from the ER that was necessary for sustaining apoptosis (8Assefa Z. Bultynck G. Szlufcik K. Nadif K.N. Vermassen E. Goris J. Missiaen L. Callewaert G. Parys J.B. De Smedt H. J. Biol. Chem. 2004; 279: 43227-43236Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). In agreement with this previous study, the cells expressing the CO mutant were sensitive to STS-induced cell death (Fig. 2, C and D). However, the same result was obtained with the CO mutant encoding the pore inactivating D2550A mutant. It is possible that the CO mutant when functioning in an ER Ca2+ leak pathway has completely different pore architecture than a full-length wild-type receptor. Nevertheless, the data also raise the possibility that full-length receptors (or their caspase-3-cleaved fragments) do not contribute directly to ER Ca2+ leak pathways and that they have some other role in regulating apoptosis. The 1TM construct (amino acids 2253–2749) encodes the segment of the receptor that contains the C-terminal channel domain. This construct was also able to support STS-induced caspase-3 activation and cell death (Fig. 2, C and D) indicating that the large segment of the receptor from the caspase-3 cleavage site at amino acids 1892–2253 is not required for apoptosis. However, deletion of 141 or 159 amino acids of the C-terminal cytosolic tail from full-length receptors was sufficient to suppress caspase-3 activation and cell death to the levels observed in TKO cells (Fig. 2, C and D). In independent experiments we have established that the TL-5 and TL-6 mutants are functionally inactive as IP3-gated channels (27Schug Z.T. Joseph S.K. J. Biol. Chem. 2006; 281: 24431-24440Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). Because channel-inactive mutants (e.g. D2550A or CO) can still support cell death, this implies that the C-terminal tail has a unique function in regulating apoptosis induced by STS. Similar findings were made when using anti-IgM as an inducer of apoptosis, notably, caspase-3 activation was supported by the D2550A pore-inactive full-length IP3R or the D2550A CO mutant but not the IP3R lacking the C-terminal tail (supplemental Fig. S1). We have previously shown that IP3Rs are phosphorylated by Akt kinase in the C-terminal tail at serine 2618 without altering IP3-dependent channel activity (23Khan M.T. Wagner L. Yule D.I. Bhanumathy C.D. Joseph S.K. J. Biol. Chem. 2006; 281: 3731-3737Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). The functional role of this phosphorylation is unknown. However, we noted that the ability of IP3Rs to support STS-induced caspase-3 activation was enhanced when the Akt phosphorylation site was inactivated by mutation to alanine (S2618A) as compared with a phosphomimic mutant (S2618E). It was suggested that perhaps important functional responses to Akt kinase phosphorylation could" @default.
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