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• W2010466342 abstract "Previous studies have shown that small interfering RNA knockdown and pharmacological inhibition of inositol 1,4,5-trisphosphate receptors (IP3Rs) stimulate autophagy. We have investigated autophagy in chicken DT40 cell lines containing targeted deletions of all three IP3R isoforms (triple knock-out (TKO) cells). Using gel shifts of microtubule-associated protein 1 light chain 3 as a marker of autophagy, we find that TKO cells have enhanced basal autophagic flux even under nutrient-replete conditions. Stable DT40 cell lines derived from TKO cells containing the functionally inactive D2550A IP3R mutant did not suppress autophagy in the same manner as wild-type receptors. This suggests that the channel function of the receptor is important in its regulatory role in autophagy. There were no marked differences in the phosphorylation state of AMP-activated protein kinase, Akt, or mammalian target of rapamycin between wild-type and TKO cells. The amount of immunoprecipitated complexes of Bcl-2-Beclin-1 and Beclin-1-Vps34 were also not different between the two cell lines. The major difference noted was a substantially decreased mTORC1 kinase activity in TKO cells based on decreased phosphorylation of S6 kinase and 4E-BP1. The discharge of intracellular stores with thapsigargin stimulated mTORC1 activity (measured as S6 kinase phosphorylation) to a greater extent in wild-type than in TKO cells. We suggest that basal autophagic flux may be negatively regulated by IP3R-dependent Ca2+ signals acting to maintain an elevated mTORC1 activity in wild-type cells and that Ca2+ regulation of this enzyme is defective in TKO cells. The protective effect of a higher autophagic flux in cells lacking IP3Rs may play a role in the delayed apoptotic response observed in these cells. Previous studies have shown that small interfering RNA knockdown and pharmacological inhibition of inositol 1,4,5-trisphosphate receptors (IP3Rs) stimulate autophagy. We have investigated autophagy in chicken DT40 cell lines containing targeted deletions of all three IP3R isoforms (triple knock-out (TKO) cells). Using gel shifts of microtubule-associated protein 1 light chain 3 as a marker of autophagy, we find that TKO cells have enhanced basal autophagic flux even under nutrient-replete conditions. Stable DT40 cell lines derived from TKO cells containing the functionally inactive D2550A IP3R mutant did not suppress autophagy in the same manner as wild-type receptors. This suggests that the channel function of the receptor is important in its regulatory role in autophagy. There were no marked differences in the phosphorylation state of AMP-activated protein kinase, Akt, or mammalian target of rapamycin between wild-type and TKO cells. The amount of immunoprecipitated complexes of Bcl-2-Beclin-1 and Beclin-1-Vps34 were also not different between the two cell lines. The major difference noted was a substantially decreased mTORC1 kinase activity in TKO cells based on decreased phosphorylation of S6 kinase and 4E-BP1. The discharge of intracellular stores with thapsigargin stimulated mTORC1 activity (measured as S6 kinase phosphorylation) to a greater extent in wild-type than in TKO cells. We suggest that basal autophagic flux may be negatively regulated by IP3R-dependent Ca2+ signals acting to maintain an elevated mTORC1 activity in wild-type cells and that Ca2+ regulation of this enzyme is defective in TKO cells. The protective effect of a higher autophagic flux in cells lacking IP3Rs may play a role in the delayed apoptotic response observed in these cells. It is well established that Ca2+ has an important regulatory role in controlling apoptosis (1Orrenius S. Zhivotovsky B. Nicotera P. Nat. Rev. Mol. Cell Biol. 2003; 4: 552-565Crossref PubMed Scopus (2406) Google Scholar, 2Joseph S.K. Hajnóczky G. Apoptosis. 2007; 12: 951-968Crossref PubMed Scopus (135) Google Scholar, 3Pinton P. Giorgi C. Siviero R. Zecchini E. Rizzuto R. Oncogene. 2008; 27: 6407-6418Crossref PubMed Scopus (839) Google Scholar). Inositol 1,4,5-trisphosphate receptors (IP3R) 2The abbreviations used are: IP3Rinositol 1,4,5-trisphosphate receptormTORmammalian target of rapamycinBAPTA-AM1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis(acetoxymethyl ester)ERendoplasmic reticulumAbantibodySTSstaurosporineTKOtriple knock-outWTwild typeAMPKAMP kinase. participate in this pathway at several levels. First, they provide a conduit for the transfer of Ca2+ between the ER and the mitochondria to sensitize the mechanism that facilitates the release of cytochrome c from the mitochondria (4Szalai G. Krishnamurthy R. Hajnóczky G. EMBO J. 1999; 18: 6349-6361Crossref PubMed Scopus (424) Google Scholar). Second, IP3Rs interact with, and are regulated by, several proteins that modify apoptotic pathways, including the anti-apoptotic proteins Bcl-2/Bcl-XL (5Rong Y.P. Aromolaran A.S. Bultynck G. Zhong F. Li X. McColl K. Matsuyama S. Herlitze S. Roderick H.L. Bootman M.D. Mignery G.A. Parys J.B. De Smedt H. Distelhorst C.W. Mol. Cell. 2008; 31: 255-265Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar, 6Li C. Wang X. Vais H. Thompson C.B. Foskett J.K. White C. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 12565-12570Crossref PubMed Scopus (134) Google Scholar), cytochrome c (7Boehning D. Patterson R.L. Sedaghat L. Glebova N.O. Kurosaki T. Snyder S.H. Nat. Cell Biol. 2003; 5: 1051-1061Crossref PubMed Scopus (537) Google Scholar, 8Boehning 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), and Akt kinase (9Khan M.T. Wagner 2nd, L. Yule D.I. Bhanumathy C. Joseph S.K. J. Biol. Chem. 2006; 281: 3731-3737Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 10Szado T. Vanderheyden V. Parys J.B. De Smedt H. Rietdorf K. Kotelevets L. Chastre E. Khan F. Landegren U. Söderberg O. Bootman M.D. Roderick H.L. Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 2427-2432Crossref PubMed Scopus (210) Google Scholar, 11Marchi S. Rimessi A. Giorgi C. Baldini C. Ferroni L. Rizzuto R. Pinton P. Biochem. Biophys. Res. Commun. 2008; 375: 501-505Crossref PubMed Scopus (102) Google Scholar). Finally, with certain apoptotic stimuli (e.g. staurosporine), IP3Rs support apoptosis independently of the channel function of the receptor via a mechanism that may be linked to a direct role of IP3Rs in activating Ca2+ entry mechanisms across the plasma membrane (12Khan M.T. Bhanumathy C.D. Schug Z.T. Joseph S.K. J. Biol. Chem. 2007; 282: 32983-32990Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). inositol 1,4,5-trisphosphate receptor mammalian target of rapamycin 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis(acetoxymethyl ester) endoplasmic reticulum antibody staurosporine triple knock-out wild type AMP kinase. Macroautophagy is a proteolytic process in which cytoplasmic constituents (including organelles) are sequestered within double-membraned vesicles (autophagosomes) that ultimately fuse with lysosomes leading to the degradation of their contents (13Eskelinen E.L. Int. Rev. Cell Mol. Biol. 2008; 266: 207-247Crossref PubMed Scopus (128) Google Scholar). A major physiological regulator of this process is nutrient supply, although the process is also regulated by various hormones and can be dysregulated under pathological conditions (14Mizushima N. Klionsky D.J. Annu. Rev. Nutr. 2007; 27: 19-40Crossref PubMed Scopus (626) Google Scholar). The complicated steps involved in autophagosome formation and lysosome fusion involve multiple proteins and regulation by many different inputs, including the activities of the mTOR pathway and class III phosphatidylinositol 3-kinase. There have been several reports suggesting that Ca2+ regulates this pathway. Hoyer-Hansen et al. (15Høyer-Hansen M. Bastholm L. Szyniarowski P. Campanella M. Szabadkai G. Farkas T. Bianchi K. Fehrenbacher N. Elling F. Rizzuto R. Mathiasen I.S. Jäättelä M. Mol. Cell. 2007; 25: 193-205Abstract Full Text Full Text PDF PubMed Scopus (893) Google Scholar) showed that agents that elevated Ca2+ in MCF-7 cells increased the formation of autophagosomes and that this was blocked by treatment with the intracellular Ca2+ chelator BAPTA-AM. However, others have reported that blocking Ca2+ elevations (e.g. with L-type Ca2+ channel antagonists) can enhance autophagy suggesting that Ca2+ has an inhibitory effect on autophagy (16Williams A. Sarkar S. Cuddon P. Ttofi E.K. Saiki S. Siddiqi F.H. Jahreiss L. Fleming A. Pask D. Goldsmith P. O'Kane C.J. Floto R.A. Rubinsztein D.C. Nat. Chem. Biol. 2008; 4: 295-305Crossref PubMed Scopus (663) Google Scholar). In addition, the depletion of intracellular stores with thapsigargin has been reported to have both a stimulatory (15Høyer-Hansen M. Bastholm L. Szyniarowski P. Campanella M. Szabadkai G. Farkas T. Bianchi K. Fehrenbacher N. Elling F. Rizzuto R. Mathiasen I.S. Jäättelä M. Mol. Cell. 2007; 25: 193-205Abstract Full Text Full Text PDF PubMed Scopus (893) Google Scholar, 17Criollo A. Maiuri M.C. Tasdemir E. Vitale I. Fiebig A.A. Andrews D. Molgó J. Díaz J. Lavandero S. Harper F. Pierron G. di Stefano D. Rizzuto R. Szabadkai G. Kroemer G. Cell Death Differ. 2007; 14: 1029-1039Crossref PubMed Scopus (268) Google Scholar) and inhibitory (16Williams A. Sarkar S. Cuddon P. Ttofi E.K. Saiki S. Siddiqi F.H. Jahreiss L. Fleming A. Pask D. Goldsmith P. O'Kane C.J. Floto R.A. Rubinsztein D.C. Nat. Chem. Biol. 2008; 4: 295-305Crossref PubMed Scopus (663) Google Scholar, 18Gordon P.B. Holen I. Fosse M. Røtnes J.S. Seglen P.O. J. Biol. Chem. 1993; 268: 26107-26112Abstract Full Text PDF PubMed Google Scholar, 19Brady N.R. Hamacher-Brady A. Yuan H. Gottlieb R.A. FEBS J. 2007; 274: 3184-3197Crossref PubMed Scopus (108) Google Scholar) effect on autophagy. Manipulations designed to change the levels of IP3 in cells (e.g. addition of myo-inositol or Li+) alter the rate of starvation-induced autophagy (17Criollo A. Maiuri M.C. Tasdemir E. Vitale I. Fiebig A.A. Andrews D. Molgó J. Díaz J. Lavandero S. Harper F. Pierron G. di Stefano D. Rizzuto R. Szabadkai G. Kroemer G. Cell Death Differ. 2007; 14: 1029-1039Crossref PubMed Scopus (268) Google Scholar). A specific role for IP3Rs in the autophagic process was suggested by the finding that small interfering RNA knockdown or pharmacological blockade of the IP3R with xestospongin B led to an enhancement of autophagy (17Criollo A. Maiuri M.C. Tasdemir E. Vitale I. Fiebig A.A. Andrews D. Molgó J. Díaz J. Lavandero S. Harper F. Pierron G. di Stefano D. Rizzuto R. Szabadkai G. Kroemer G. Cell Death Differ. 2007; 14: 1029-1039Crossref PubMed Scopus (268) Google Scholar). These data suggest that IP3Rs negatively modulate autophagy. However, it is unclear if this involves the channel function of the IP3R, because some effective agents, such as xestospongin B, had no detectable effects on cytosolic or ER luminal Ca2+(17Criollo A. Maiuri M.C. Tasdemir E. Vitale I. Fiebig A.A. Andrews D. Molgó J. Díaz J. Lavandero S. Harper F. Pierron G. di Stefano D. Rizzuto R. Szabadkai G. Kroemer G. Cell Death Differ. 2007; 14: 1029-1039Crossref PubMed Scopus (268) Google Scholar). Disruption of the single IP3R gene in Dictyostelium discoideum impairs an autophagic death pathway (20Lam D. Kosta A. Luciani M.F. Golstein P. Mol. Biol. Cell. 2008; 19: 691-700Crossref PubMed Scopus (65) Google Scholar). The specific autophagic signaling pathway(s) modulated by IP3Rs remains to be identified. DT40 chicken B-cell lines containing targeted deletion of all three IP3R isoforms (TKO) show a markedly delayed cell death response to various apoptotic stimuli (6Li C. Wang X. Vais H. Thompson C.B. Foskett J.K. White C. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 12565-12570Crossref PubMed Scopus (134) Google Scholar, 12Khan M.T. Bhanumathy C.D. Schug Z.T. Joseph S.K. J. Biol. Chem. 2007; 282: 32983-32990Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar, 21Sugawara H. Kurosaki M. Takata M. Kurosaki T. EMBO J. 1997; 16: 3078-3088Crossref PubMed Scopus (375) Google Scholar). We considered the possibility that adaptive changes in autophagy may have occurred in these cells thereby providing a useful experimental system to investigate the role of IP3Rs in autophagy. In this study, we show that TKO cells have a markedly enhanced rate of autophagy compared with wild-type cells, even under nutrient-replete conditions. The suppression of autophagy required the Ca2+ channel function of the IP3R and was not observed in cell lines transfected with the pore-inactivating D2550A mutant. Several key factors that regulate autophagy were compared in wild-type and TKO cell lines and were not found to be significantly different. These included the activity of AMP and Akt kinase. The differences in basal autophagy could also not be accounted for by altered levels of Beclin-1-Vps34 complexes. Instead, our experiments suggest that altered activity of the mTORC1 complex may be one potential mechanism by which IP3R-mediated Ca2+ fluxes could regulate the autophagic pathway. RPMI 1640 culture media and G418 sulfate (Geneticin) were obtained from Cellgro-Mediatech (Herndon, VA). Staurosporine, rapamycin, bafilomycin, and okadaic acid were purchased from Sigma. Protogel-stabilized acrylamide solution was from National Diagnostics (Atlanta, GA). Nitrocellulose membrane (0.45 μm) was from Bio-Rad. Polyvinylidene difluoride membrane (Immobilon-P, 0.45 μm) was purchased from Millipore (Bedford, MA). The following antibodies were obtained from Cell Signaling (MA): LC3B rabbit polyclonal Ab; phospho-mTOR (Ser-2448) rabbit polyclonal Ab; phospho-mTOR (Ser-2481) rabbit polyclonal Ab; total mTOR rabbit polyclonal Ab; phospho-4E-BP1 (Thr-37/46) (236B4) rabbit monoclonal Ab; phospho-Akt (Ser-473) (587F11) mouse monoclonal Ab; total Akt (5G3) mouse monoclonal Ab; phospho-p70 S6 kinase (Thr-389) (108D2) rabbit monoclonal Ab; total p70 S6 kinase rabbit polyclonal Ab; Beclin-1 rabbit polyclonal Ab; phospho-AMPKα (Thr-172) rabbit polyclonal Ab; and total AMPKα rabbit polyclonal Ab. Mouse anti-Bcl-2 monoclonal Ab was purchased from BD Transduction Laboratories. The rabbit polyclonal calnexin Ab has been described previously (22Joseph S.K. Boehning D. Bokkala S. Watkins R. Widjaja J. Biochem. J. 1999; 342: 153-161Crossref PubMed Scopus (20) Google Scholar). Wild-type (WT) DT40 cells and IP3R triple knock-out (TKO) cells were the kind gifts 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). DT40 cells were grown in RPMI 1640 media supplemented with 10% fetal bovine serum, 1% chicken serum, 100 units/ml penicillin, 100 units/ml streptomycin and maintained at 37 °C in 5% CO2 atmosphere. The stable cell line containing the inactivating D2550A pore mutation was prepared as described previously (12Khan M.T. Bhanumathy C.D. Schug Z.T. Joseph S.K. J. Biol. Chem. 2007; 282: 32983-32990Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). DT40 cells were seeded at a density of 104 cells/ml and were used for experiments after 24 h. There were no significant differences in the growth rate of the individual cell lines (data not shown). The DT40 cells were treated with staurosporine or rapamycin and then centrifuged at 1,000 × g for 10 min. The cells were washed once in ice-cold phosphate-buffered saline and then lysed in a medium containing 1% Triton X-100 and 50 mm Tris/HCl, pH 7.8, 150 mm NaCl, 2 mm sodium orthovanadate, 10 mm sodium pyrophosphate, 20 mm NaF, 5 nm okadaic acid, and a 1× dilution of a complete protease inhibitor mixture (Roche Diagnostics). The lysates were cleared by centrifugation at 10,000 × g. Protein samples (25 μg) were run on 15% polyacrylamide gels and transferred on polyvinylidene difluoride membranes. Immunoblotting was done using the LC3B antibody. Only the lower band of the LC3 doublet (LC3-II) was quantitated because variable immunodetection and efficiency of transfer of the upper band (LC3-I) has been observed (23Mizushima N. Yoshimori T. Autophagy. 2007; 3: 542-545Crossref PubMed Scopus (1953) Google Scholar). Electron microscopy of DT40 cells was carried out as described by Csordas et al. (24Csordás G. Renken C. Várnai P. Walter L. Weaver D. Buttle K.F. Balla T. Mannella C.A. Hajnóczky G. J. Cell Biol. 2006; 174: 915-921Crossref PubMed Scopus (966) Google Scholar). Autophagy was measured by immunoblotting for the microtubule-associated light chain 3 (LC3) protein. The covalent modification of the cytosolic LC3-I form with phosphatidylethanolamine generates the LC3-II form, which migrates more rapidly on SDS-PAGE (25Kabeya Y. Mizushima N. Yamamoto A. Oshitani-Okamoto S. Ohsumi Y. Yoshimori T. J. Cell Sci. 2004; 117: 2805-2812Crossref PubMed Scopus (1110) Google Scholar). The specific association of LC3-II with autophagosomes has led to this protein being widely used as a marker of autophagy (23Mizushima N. Yoshimori T. Autophagy. 2007; 3: 542-545Crossref PubMed Scopus (1953) Google Scholar). Fig. 1A shows a comparison of LC3-II levels detected by immunoblotting in lysates from wild-type and TKO DT40 cells grown under nutrient-replete conditions. The data show that basal steady-state levels of LC3-II are low in wild-type cells but are ∼4-fold elevated in the TKO cells (Fig. 1B). The induction of apoptosis with staurosporine (STS) over a 6-h period enhanced the levels of LC3-II in wild-type cells and decreased the elevated levels of LC3-II in TKO cells. To determine whether the Ca2+ channel function of IP3Rs was important in mediating its inhibitory effects on autophagy, we utilized a stable DT40 cell line expressing the “pore-dead” D2550A mutant of the rat type I IP3R (26Boehning D. Joseph S.K. J. Biol. Chem. 2000; 275: 21492-21499Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). The expression level of type I IP3R in the mutant has previously been shown to be comparable with wild-type cells (12Khan M.T. Bhanumathy C.D. Schug Z.T. Joseph S.K. J. Biol. Chem. 2007; 282: 32983-32990Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). The pore-dead cell line also showed elevated levels of LC3-II that were decreased by STS treatment as observed with the TKO cells (Fig. 1, A and B). The wild-type DT40 cells used as a control contain all three chicken IP3R isoforms. Levels of LC3-II in pore-dead cells were also elevated when compared with a stable DT40 cell line expressing the wild-type rat type I IP3R (Fig. 1C). We conclude that the channel function of the IP3R is required for suppressing autophagy. It has been pointed out that measurements of the steady-state levels of LC3-II may not reflect autophagic flux because the levels are also determined by the degradation of LC3-II upon fusion of autophagosomes with lysosomes (27Tanida I. Minematsu-Ikeguchi N. Ueno T. Kominami E. Autophagy. 2005; 1: 84-91Crossref PubMed Scopus (936) Google Scholar, 28Klionsky D.J. Abeliovich H. Agostinis P. Agrawal D.K. Aliev G. Askew D.S. 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A 6-h incubation with 10 nm bafilomycin caused a large enhancement in LC3-II levels such that no differences between WT and TKO cells could be distinguished, although the inhibitory effect of STS on LC3-II levels in the TKO cells was still observed (Fig. 1B). Shorter periods of treatment with bafilomycin, which produced submaximal elevations of LC3-II (e.g. after 1 h), still revealed differences between wild-type and TKO cells (Fig. 1D). The basic findings using LC3 immunoblotting were confirmed by analysis of electron microscope images of DT40 cells that showed an enhanced number of debris-containing vacuoles in TKO cells when compared with either wild-type or TKO cells stably expressing the rat type I IP3R (supplemental Fig. S1). These data in DT40 cells are in agreement with previous findings in other cell types that knockdown or inhibition of IP3Rs enhances autophagic flux (17Criollo A. Maiuri M.C. Tasdemir E. Vitale I. Fiebig A.A. Andrews D. Molgó J. Díaz J. Lavandero S. Harper F. Pierron G. di Stefano D. Rizzuto R. Szabadkai G. Kroemer G. Cell Death Differ. 2007; 14: 1029-1039Crossref PubMed Scopus (268) Google Scholar). Fig. 2 depicts the canonical pathway of autophagy together with known sites that could potentially be regulated by Ca2+ based on information in the literature. Because IP3Rs have an established role in supplying Ca2+ to the mitochondria, which is known to stimulate mitochondrial oxidative metabolism, it is possible that IP3R-deficient DT40 cells might have an increased AMP/ATP ratio. This, in turn, could enhance the activity of AMPK that would stimulate autophagy through the intermediary steps shown in Fig. 2. Calmodulin-dependent kinase kinase-β is an upstream activator of AMPK and has also been proposed to be a site where cytosolic Ca2+ elevations could stimulate autophagy (15Høyer-Hansen M. Bastholm L. Szyniarowski P. Campanella M. Szabadkai G. Farkas T. Bianchi K. Fehrenbacher N. Elling F. Rizzuto R. Mathiasen I.S. Jäättelä M. Mol. Cell. 2007; 25: 193-205Abstract Full Text Full Text PDF PubMed Scopus (893) Google Scholar). We therefore compared the activation state of AMPK in wild-type, TKO, and pore-dead DT40 cell lines by measuring phosphorylation of the protein at Thr-172 using a phospho-specific Ab. This rabbit polyclonal Ab recognized a doublet of bands in the chicken cells (Fig. 3) of which only the lower band was recognized by the pan-α-subunit AMPK Ab at the appropriate molecular mass of ∼63 kDa. This AMPK band was not significantly different between wild-type, TKO, and pore-dead cells. STS did not affect the phosphorylation state of the AMPK band but substantially decreased the phosphorylation of the upper unknown band in all three cell lines.FIGURE 3Autophagy is independent of the phosphorylation state of AMPK, Akt, or mTOR. WT, IP3R TKO, and the D2550A pore-inactive (pore-dead) DT40 cells lines were incubated in nutrient-replete medium with serum in the presence or absence of staurosporine (1 μm) for 6 h. After treatment, the cells were lysed in a buffer containing protein phosphatase inhibitors and processed for immunoblotting with the indicated phospho-specific Abs as described under “Materials and Methods.” The data shown is representative of three experiments. * indicates an unknown band.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Activated Akt kinase inhibits autophagy (Fig. 2) (30Degtyarev M. De Mazière A. Orr C. Lin J. Lee B.B. Tien J.Y. Prior W.W. van Dijk S. Wu H. Gray D.C. Davis D.P. Stern H.M. Murray L.J. Hoeflich K.P. Klumperman J. Friedman L.S. Lin K. J. Cell Biol. 2008; 183: 101-116Crossref PubMed Scopus (352) Google Scholar). Analysis of the phosphorylation state of Akt kinase using phospho-specific Ser-473 Ab indicates a modest increase in activation of Akt in TKO and pore-dead cells. However, these changes are in the opposite direction to account for the stimulated autophagy in these cell lines. STS decreased Ser-437 phosphorylation in TKO and pore-dead cell lines but not in wild-type cells. These changes also do not correlate with the different effects of STS on autophagy in these cell lines. The effects of Akt and AMPK on autophagy are mediated through a pathway that involves TSC and Rheb proteins that regulate the activity of the mTORC1 complex (Fig. 2). High levels of mTOR kinase suppress autophagy by mechanisms that have not been fully characterized but include the phosphorylation of Atg1/Ulk kinases that are involved in the induction of autophagosomes (31Pattingre S. Espert L. Biard-Piechaczyk M. Codogno P. Biochimie. 2008; 90: 313-323Crossref PubMed Scopus (436) Google Scholar). We examined the following two phosphorylation sites on mTOR: Ser-2448 (a site for phosphorylation by several kinases, including Akt (32Sekuliæ A. Hudson C.C. Homme J.L. Yin P. Otterness D.M. Karnitz L.M. Abraham R.T. Cancer Res. 2000; 60: 3504-3513PubMed Google Scholar)) and Ser-2481 (an autophosphorylation site (33Peterson R.T. Beal P.A. Comb M.J. Schreiber S.L. J. Biol. Chem. 2000; 275: 7416-7423Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar)). The basal phosphorylation state of neither site was different in the three different DT40 cell lines (Fig. 3). However, mutation of serines 2448 or 2481 does not alter mTOR kinase activity (32Sekuliæ A. Hudson C.C. Homme J.L. Yin P. Otterness D.M. Karnitz L.M. Abraham R.T. Cancer Res. 2000; 60: 3504-3513PubMed Google Scholar), which is conventionally measured by monitoring the phosphorylation of its substrates. One such substrate is p70 S6 kinase, which is specifically phosphorylated by mTOR on Thr-389 (34Kim D.H. Sarbassov D.D. Ali S.M. King J.E. Latek R.R. Erdjument-Bromage H. Tempst P. Sabatini D.M. Cell. 2002; 110: 163-175Abstract Full Text Full Text PDF PubMed Scopus (2354) Google Scholar). Fig. 4, A and B, shows that there are substantially lower levels of phosphorylated S6 kinase in the TKO and pore-dead cells. We confirmed this finding by examining another mTOR substrate 4E-BP1 using an Ab directed at Ser-65 (35Gingras A.C. Gygi S.P. Raught B. Polakiewicz R.D. Abraham R.T. Hoekstra M.F. Aebersold R. Sonenberg N. Genes Dev. 1999; 13: 1422-1437Crossref PubMed Scopus (1004) Google Scholar). As with S6 kinase, a lower phosphorylation of 4E-BP1 was observed in TKO and pore-dead cells (Fig. 4A). The phosphorylation state of S6 kinase could be restored by stable transfection of the rat type I IP3R into TKO cells and remained elevated in a DT40 cell line containing only the endogenous chicken type I isoform (DKO-1) (Fig. 4C). We suggest that a lower mTOR kinase activity in the IP3R-deficient cell lines could account for their higher basal autophagy. We examined the effects of rapamycin, an inhibitor of mTOR (36Corradetti M.N. Guan K.L. Oncogene. 2006; 25: 6347-6360Crossref PubMed Scopus (328) Google Scholar), on the levels of LC3-II in wild-type and TKO cells (Fig. 5). In both cell lines, 100 nm rapamycin completely inhibited mTOR activity within 2 h as judged by inhibition of phosphorylation of Thr-389 of S6 kinase (Fig. 5A). Rapamycin produced a smaller and slower decrease in Ser-2448 phosphorylation of mTOR itself, consistent with this site being a relatively insensitive indicator of mTOR kinase activity. Rapamycin elevated the levels of LC3-II in wild-type cells with a peak increase at 4 h, which averaged almost 80% of the elevated levels observed in untreated TKO cells (Fig. 5B). Rapamycin addition to TKO cells caused only a small further increase in LC3-II levels. The results with rapamycin are consistent with the hypothesis that differences in the activity of the mTOR kinase pathway are an important determinant in setting the different levels of basal autopha" @default.
• W2010466342 created "2016-06-24" @default.
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• W2010466342 date "2010-05-01" @default.
• W2010466342 modified "2023-10-11" @default.
• W2010466342 title "Role of Inositol Trisphosphate Receptors in Autophagy in DT40 Cells" @default.
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