Central Role of Glycogen Synthase Kinase-3β in Endoplasmic Reticulum Stress-induced Caspase-3 Activation
2002; Elsevier BV; Volume: 277; Issue: 47 Linguagem: Inglês
10.1074/jbc.m206047200
ISSN1083-351X
AutoresLing Song, Patrizia De Sarno, Richard S. Jope,
Tópico(s)Genetics and Neurodevelopmental Disorders
ResumoStress of the endoplasmic reticulum (ER), which is associated with many neurodegenerative conditions, can lead to the elimination of affected cells by apoptosis through only partially understood mechanisms. Thapsigargin, which causes ER stress by inhibiting the ER Ca2+-ATPase, was found to not only activate the apoptosis effector caspase-3 but also to cause a large and prolonged increase in the activity of glycogen synthase kinase-3β (GSK3β). Activation of GSK3β was obligatory for thapsigargin-induced activation of caspase-3, because inhibition of GSK3β by expression of dominant-negative GSK3β or by the GSK3β inhibitor lithium blocked caspase-3 activation. Thapsigargin treatment activated GSK3β by inducing dephosphorylation of phospho-Ser-9 of GSK3β, a phosphorylation that normally maintains GSK3β inactivated. Caspase-3 activation induced by thapsigargin was blocked by increasing the phosphorylation of Ser-9-GSK3β with insulin-like growth factor-1 or with the phosphatase inhibitors okadaic acid and calyculin A, but the calcineurin inhibitors FK506 and cyclosporin A were ineffective. Insulin-like growth factor-1, okadaic acid, calyculin A, and lithium also protected cells from two other inducers of ER stress, tunicamycin and brefeldin A. Thus, ER stress activates GSK3β through dephosphorylation of phospho-Ser-9, a prerequisite for caspase-3 activation, and this process is amenable to pharmacological intervention. Stress of the endoplasmic reticulum (ER), which is associated with many neurodegenerative conditions, can lead to the elimination of affected cells by apoptosis through only partially understood mechanisms. Thapsigargin, which causes ER stress by inhibiting the ER Ca2+-ATPase, was found to not only activate the apoptosis effector caspase-3 but also to cause a large and prolonged increase in the activity of glycogen synthase kinase-3β (GSK3β). Activation of GSK3β was obligatory for thapsigargin-induced activation of caspase-3, because inhibition of GSK3β by expression of dominant-negative GSK3β or by the GSK3β inhibitor lithium blocked caspase-3 activation. Thapsigargin treatment activated GSK3β by inducing dephosphorylation of phospho-Ser-9 of GSK3β, a phosphorylation that normally maintains GSK3β inactivated. Caspase-3 activation induced by thapsigargin was blocked by increasing the phosphorylation of Ser-9-GSK3β with insulin-like growth factor-1 or with the phosphatase inhibitors okadaic acid and calyculin A, but the calcineurin inhibitors FK506 and cyclosporin A were ineffective. Insulin-like growth factor-1, okadaic acid, calyculin A, and lithium also protected cells from two other inducers of ER stress, tunicamycin and brefeldin A. Thus, ER stress activates GSK3β through dephosphorylation of phospho-Ser-9, a prerequisite for caspase-3 activation, and this process is amenable to pharmacological intervention. Impaired function of the endoplasmic reticulum (ER), 1The abbreviations used are: ER, endoplasmic reticulum; AD, Alzheimer's disease; GSK3β, glycogen synthase kinase-3β; IGF-1, insulin-like growth factor-1; PARP, poly(ADP-ribose) polymerase; PP1, protein phosphatase 1; PP2A, protein phosphatase 2A; PP2B, protein phosphatase 2B; PIPES, 1,4-piperazinediethanesulfonic acid.1The abbreviations used are: ER, endoplasmic reticulum; AD, Alzheimer's disease; GSK3β, glycogen synthase kinase-3β; IGF-1, insulin-like growth factor-1; PARP, poly(ADP-ribose) polymerase; PP1, protein phosphatase 1; PP2A, protein phosphatase 2A; PP2B, protein phosphatase 2B; PIPES, 1,4-piperazinediethanesulfonic acid. commonly referred to as ER stress, is an important factor in the neuropathology of a wide variety of neurological disorders (reviewed in Refs. 1Paschen W. Doutheil J. J. Cereb. Blood Flow Metab. 1999; 19: 1-18Google Scholar, 2Mattson M.P. LaFerla F.M. Chan S.L. Leissring M.A. Shepel P.N. Geiger J.D. 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Hanley M.R. Dawson A.P. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 2466-2470Google Scholar), which blocks sequestration of calcium by the ER, causing increases in the intracellular concentration of calcium, accumulation of unfolded or misfolded proteins, and activation of caspase-3-mediated apoptosis (16Jiang S. Chow S.C. Nicotera P. Orrenius S. Exp. Cell Res. 1994; 212: 84-92Google Scholar, 17Furuya Y. Lundmo P. Short A.D. Gill D.L. Isaacs J.T. Cancer Res. 1994; 54: 6167-6175Google Scholar, 18Treiman M. Caspersen C. Christensen S.B. Trends Pharmacol. Sci. 1998; 19: 131-135Google Scholar). The mechanisms mediating ER stress-induced activation of the apoptosis program remain incompletely elucidated, although both caspase-7 and caspase-12 have been implicated in addition to the crucial effector caspase-3 (6Nakagawa T. Zhu H. Morishima N. Li E. Xu J. Yankner B.A. Yuan J. Nature. 2000; 403: 98-103Google Scholar, 19Nakagawa T. Yuan J. J. 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Andjelkovich M. Hemmings B.A. Nature. 1995; 378: 785-789Google Scholar), have shown that GSK3β promotes the subsequent apoptotic process (35Crowder R.J. Freeman R.S. J. Biol. Chem. 2000; 275: 34266-34271Google Scholar, 36Hetman M. Cavanaugh J.E. Kimelman D. Xia Z. J. Neurosci. 2000; 20: 2567-2674Google Scholar, 37Li M. Wang X. Meintzer M.K. Laessig T. Birnbaum M.J. Heidenreich K.A. Mol. Cell. Biol. 2000; 20: 9356-9363Google Scholar, 38Cross D.A.E. Culbert A.A. Chalmers K.A. Facci L. Skaper S.D. Reith A.D. J. Neurochem. 2001; 77: 94-102Google Scholar, 39Culbert A.A. Brown M.J. Frame S. Hagen T. Cross D.A.E. Bax B. Reith A.D. FEBS Lett. 2001; 507: 288-294Google Scholar). More that just neuronal apoptosis is promoted by GSK3β activity, because this relationship has been demonstrated in a wide variety of cell types, for example in vascular smooth muscle cells (40Hall J.L. Chatham J.C. Eldar-Finkelman H. Bibbons G.H. Diabetes. 2001; 50: 1171-1179Google Scholar), fibroblasts (41Chen S. Guttridge D.C. You Z. Zhang Z. Fribley A. Mayo M.W. Kitajewski J. Wang C.Y. J. Cell Biol. 2001; 152: 87-96Google Scholar), human erythroid progenitors (42Somervaille T.C.P. Linch D.C. Khwaja A. Blood. 2001; 98: 1374-1381Google Scholar), and cardiac cells (43Tong H. Imahashi K. Steenbergen C. Murphy E. Circ. Res. 2002; 90: 377-379Google Scholar). A number of these studies advantageously used lithium, along with other approaches, to identify the contributory effects of GSK3β to apoptosis. Lithium is useful in this regard, because it is a selective inhibitor of GSK3β (44Klein P.S. Melton D.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8455-8459Google Scholar, 45Stambolic V. Ruel L. Woodgett J.R. Curr. Biol. 1996; 6: 1664-1668Google Scholar), a finding substantiated by an examination of 24 kinases, which showed that GSK3β, and the closely related GSK3α, to be the only kinases substantially inhibited by lithium (46Davies S.P. Reddy H. Caivano M. Cohen P. Biochem. J. 2000; 351: 95-105Google Scholar). Extensive studies have clearly documented that several of lithium's prominent effects, such as inhibition of the phosphorylation of the microtubule-associated protein tau, increased levels of β-catenin, and protection from GSK3β-facilitated apoptosis, are directly dependent on lithium's inhibition of GSK3β (24, 47–49; reviewed in Refs. 21Grimes C.A. Jope R.S. Prog. Neurobiol. 2001; 65: 391-426Google Scholar and 50Phiel C.J. Klein P.S. Annu. Rev. Pharmacol. Toxicol. 2001; 41: 789-813Google Scholar). Based on the extensive evidence that GSK3β promotes apoptosis and that ER stress is involved in a variety of neurodegenerative disorders in which apoptosis may contribute to neuronal loss, we investigated whether there is an association between GSK3β activity and apoptotic signaling induced by ER stress. SH-SY5Y human neuroblastoma cells were grown in RPMI 1640 medium (Cellgro, Herndon, VA) supplemented with 5% fetal clone II (HyClone, Logan, UT), 10% horse serum, 2 mm l-glutamine, 100 units/ml penicillin, and 100 μg/ml streptomycin (Invitrogen, Grand Island, NY). SH-SY5Y cell lines were previously described that stably express dominant-negative GSK3β (51Watcharasit P. Bijur G.N. Zmijewski J.W. Song L. Zmijewska A. Chen X. Johnson G.V.W. Jope R.S. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 7951-7955Google Scholar) or stably overexpress active GSK3β at three to four times the normal level (24Bijur G.N. De Sarno P. Jope R.S. J. Biol. Chem. 2000; 275: 7583-7590Google Scholar). Immortalized hippocampal cells (52Mehler M.F. Rozental R. Dougherty M. Spray D.C. Kessler J.A. Nature. 1993; 362: 62-65Google Scholar) (generously provided by Dr. M. F. Mehler, Albert Einstein College of Medicine) were differentiated by incubation for 6 days at 39 °C in Neurobasal media containing B-27 supplement (53Brewer G.J. Torricelli J.R. Evege E.K. Price P.J. J. Neurosci. Res. 1993; 35: 567-576Google Scholar) prior to experimental manipulations. Cells were washed and preincubated in serum-free or B-27-free media overnight before experimental treatments. Where indicated, cells were treated with LiCl (Sigma), insulin-like growth factor-1 (IGF-1, Intergen, Purchase, NY), cyclosporin A, FK506 (Calbiochem, San Diego, CA), thapsigargin, tunicamycin, brefeldin A, okadaic acid, or calyculin A (Alexis, San Diego, CA). For immunoblotting, cells were washed twice with phosphate-buffered saline and lysed with 100 μl of lysis buffer (20 mm Tris, pH 7.5, 150 mm NaCl, 2 mm EDTA, 2 mm EGTA, 1 mm sodium orthovanadate, 50 mm sodium fluoride, 100 μmphenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, 10 μg/ml aprotinin, 5 μg/ml pepstatin A, 1 nm okadaic acid, and 0.2% Nonidet P-40). The lysates were collected in centrifuge tubes, sonicated, and centrifuged at 16,000 × g for 10 min at 4 °C. Protein concentrations were determined using the BCA method (Pierce). Where indicated, cells were fractionated as described previously (54Bijur G.N. Jope R.S. J. Biol. Chem. 2001; 276: 37436-37442Google Scholar). For subcellular fractionation, lysed cells were collected in microcentrifuge tubes, and centrifuged at 2,700 ×g for 10 min at 4 °C. The supernatant containing the cytosol was further centrifuged at 20,800 × g for 15 min at 4 °C to obtain the cytosolic fraction. The nuclei in the pellet were washed three times by gently resuspending the nuclei in 200 μl of wash buffer (10 mm PIPES, pH 6.8, 300 mm sucrose, 3 mm MgCl2, 1 mm EGTA, 25 mm NaCl, 1 mm sodium orthovanadate, 50 mm sodium fluoride, 100 μmphenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, 10 μg/ml aprotinin, and 5 μg/ml pepstatin A) and centrifuging at 2,700 ×g for 5 min at 4 °C. For a final wash, the nuclei were resuspended in 100 μl of wash buffer, layered over a cushion of 1 ml of sucrose buffer (1 m sucrose, 1 mm sodium orthovanadate, 50 mm sodium fluoride, 100 μmphenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, 10 μg/ml aprotinin, and 5 μg/ml pepstatin A), and centrifuged at 2,700 ×g for 10 min. The sucrose buffer containing non-sedimented cellular debris was discarded, and the pellet containing nuclei was washed in 100 μl of lysis buffer and centrifuged at 2,700 ×g for 5 min at 4 °C to remove residual sucrose buffer. Extracts were mixed with Laemmli sample buffer (2% SDS) and placed in a boiling water bath for 5 min. Proteins were resolved in SDS-polyacrylamide gels, transferred to nitrocellulose, and incubated with anti-GSK3β, anti-poly(ADP-ribose) polymerase (PARP), anti-proteolyzed PARP 85-kDa fragment (Pharmingen/Transduction Laboratories, San Diego, CA), anti-phospho-Ser-9-GSK3β, anti-phospho-Ser-473-Akt, anti-total Akt, anti-β-catenin, and anti-active casapse-3 (Cell Signaling, Beverly, MA) antibodies. Immunoblots were developed using horseradish peroxidase-conjugated goat anti-mouse or goat anti-rabbit IgG, followed by detection with enhanced chemiluminescence, and the protein bands were quantitated with a densitometer. Fluorometric assays of caspase-3 activity using the substrate Ac-DEVD-AMC (Alexis) were carried out as described previously (24Bijur G.N. De Sarno P. Jope R.S. J. Biol. Chem. 2000; 275: 7583-7590Google Scholar). For this, fluorometric assays were conducted in 96-well clear bottom plates, and all measurements were carried out in triplicate wells. To each well 200 μl of assay buffer (20 mm HEPES, pH 7.5, 10% glycerol, 2 mm dithiothreitol) was added. Peptide substrates for caspase-3 (Ac-DEVD-AMC) (Alexis Biochemicals, San Diego, CA) were added to each well to a final concentration of 25 ng/μl. When the caspase-3 inhibitor (Ac-DEVD-CHO) was used, it was added at a concentration of 2.5 ng/μl immediately before addition of the caspase-3 substrate. Cell lysates (20 μg of protein) were added to start the reaction. Background fluorescence was measured in wells containing assay buffer, substrate, and lysis buffer without the cell lysate. Assay plates were incubated at 37 °C for 1 h for measurement of caspase-3, and fluorescence was measured on a fluorescence plate reader (Bio-Tek, Winooski, VT) set at 360-nm excitation and 460-nm emission. Caspase activity was calculated as [(mean AMC fluorescence from triplicate wells) − (background fluorescence)]/μg of protein. The activity of GSK3β was measured as described previously (54Bijur G.N. Jope R.S. J. Biol. Chem. 2001; 276: 37436-37442Google Scholar). For this, to immunoprecipitate GSK3β, 100 μg of protein was incubated with 0.75 μg of monoclonal GSK3β antibody overnight at 4 °C with gentle agitation. Extracts were incubated with 30 μl of protein G-Sepharose for 1 h at 4 °C. The immobilized immune complexes were washed twice with immunoprecipitation lysis buffer and twice with kinase buffer (20 mm Tris, pH 7.5, 5 mmMgCl2, and 1 mm dithiothreitol). Kinase activity was measured by mixing immunoprecipitated GSK3β with 25 μl of kinase buffer containing 20 mm Tris, pH 7.5, 5 mm MgCl2, 1 mm dithiothreitol, 250 μm ATP, 1.4 μCi of [γ-32P]ATP (AmershamBiosciences, Arlington Heights, IL), and 0.1 μg/μl recombinant tau protein (Panvera, Madison, WI). The GSK3β inhibitor lithium (20 mm (44Klein P.S. Melton D.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8455-8459Google Scholar)) was added in vitro to confirm that phosphorylation was mediated by GSK3β. The samples were incubated at 30 °C for 15 min, and 25 μl of Laemmli sample buffer (2% SDS) was added to each sample to stop the reaction. Samples were placed in a boiling water bath for 5 min, and proteins were separated in 7.5% SDS-polyacrylamide gels. The gels were vacuum-dried, exposed to a phosphoscreen overnight, and quantitated using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA). The efficiency of GSK3β immunoprecipitation was determined by immunoblotting for GSK3β. Characteristics associated with apoptosis were assessed in human neuroblastoma SH-SY5Y cells after the induction of ER stress with thapsigargin. These parameters included measurements of the activity of the effector caspase, caspase-3, proteolysis of PARP (a classical substrate cleaved by caspase-3), immunoblot detection of the cleavage of procaspase-3 to active caspase-3 fragments, and changes in morphology. Caspase-3 activity, measured by fluorogenic substrate cleavage (24Bijur G.N. De Sarno P. Jope R.S. J. Biol. Chem. 2000; 275: 7583-7590Google Scholar), increased between 2 and 4 h after treatment with 2 μm thapsigargin (Fig.1 A). Similar time courses after thapsigargin treatment were observed in Western blot analyses of the proteolysis of PARP from an intact 116-kDa protein to a stable 85-kDa breakdown product, and the production of 17- and 19-kDa activated caspase-3 (Fig. 1 A). Examination of cells treated with thapsigargin and stained with Hoechst 33342 (24Bijur G.N. De Sarno P. Jope R.S. J. Biol. Chem. 2000; 275: 7583-7590Google Scholar) revealed the characteristic morphology associated with apoptosis, including nuclear condensation and cell shrinkage (data not shown). These results confirm previous reports that thapsigargin causes SH-SY5Y cells to undergo caspase-3-mediated apoptosis (55Nath R. Raser K.J. Hajimohammadreza I. Wang K.K. Biochem. Mol. Biol. Int. 1997; 43: 197-205Google Scholar, 56McGinnis K.M. Whitton M.M. Gnegy M.E. Wang K.K. J. Biol. Chem. 1998; 273: 19993-20000Google Scholar, 57Mukerjee N. McGinnis K.M. Park Y.H. Gnegy M.E. Wang K.K. Arch. Biochem. Biophys. 2000; 379: 337-343Google Scholar). To test if GSK3β is involved in the apoptotic response to ER stress, the activity of GSK3β was assessed in SH-SY5Y cells after thapsigargin treatment. As described previously (54Bijur G.N. Jope R.S. J. Biol. Chem. 2001; 276: 37436-37442Google Scholar), GSK3β activity was measured by immunoprecipitating GSK3β, measuring its catalysis of the phosphorylation of recombinant tau protein, a well-characterized substrate of GSK3β (reviewed in Ref. 58Johnson G.V.W. Hartigan J.A. Alzheimer's Dis. Rev. 1998; 3: 125-141Google Scholar), and confirming that phosphorylation was mediated by immunoprecipitated GSK3β by inclusion of the GSK3β inhibitor lithium (20 mm (44Klein P.S. Melton D.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8455-8459Google Scholar)) in the kinase assay. These measurements revealed that there was a large and prolonged increase in GSK3β activity after thapsigargin treatment (Fig.1 B). GSK3β activity increased within 2 h after exposure of cells to 2 μm thapsigargin and was 277 ± 27% (n = 3) of control levels after 4 h of thapsigargin treatment (Fig. 1 B). In situactivation of GSK3β by thapsigargin treatment was further confirmed by measuring the level of β-catenin. Phosphorylation of cytosolic β-catenin by GSK3β promotes its degradation, whereas inhibition of GSK3β allows the stabilization, accumulation, and nuclear translocation of β-catenin (59Ferkey D.M. Kimelman D. Dev. Biol. 2000; 225: 471-479Google Scholar). An additional pool of β-catenin is sequestered at the plasma membrane, and its stability is unaffected by GSK3β. Treatment of SH-SY5Y cells with thapsigargin caused depletion of cytosolic β-catenin (Fig. 1 C), and a modest reduction of nuclear β-catenin, whereas membrane-bound β-catenin was unaltered. Treatment with lithium to inhibit GSK3β attenuated thapsigargin-induced depletions of cytosolic and nuclear β-catenin. These results are indicative of GSK3β activation by thapsigargin and inhibition by lithium. Thus, thapsigargin treatment caused the activation of GSK3β, a previously unknown response to ER stress, concomitantly with the initiation of apoptotic signaling, which raised the possibility that GSK3β may be involved in the signaling pathway linking ER stress to caspase-3 activation. Considering recent findings that GSK3β can promote apoptosis (reviewed in Ref. 21Grimes C.A. Jope R.S. Prog. Neurobiol. 2001; 65: 391-426Google Scholar), the thapsigargin-induced activation of GSK3β raised the question of whether this is an essential component of the apoptosis signaling cascade that is induced by ER stress leading to activation of caspase-3. To test this, the effects of thapsigargin on caspase-3 activation were examined under conditions where the activity of GSK3β was modified. To test if increased GSK3β activity is stimulatory, thapsigargin-induced PARP proteolysis was compared in control SH-SY5Y cells, vector-transfected cells, and two different stable lines of SH-SY5Y cells that overexpress active GSK3β 3- to 4-fold above the endogenous level of GSK3β, which have been described previously (24Bijur G.N. De Sarno P. Jope R.S. J. Biol. Chem. 2000; 275: 7583-7590Google Scholar). Thapsigargin-induced PARP proteolysis was similar in wild-type and vector-transfected SH-SY5Y cells but was much greater in cells overexpressing GSK3β (Fig.2 A), indicating that increased active GSK3β promotes thapsigargin-induced caspase activation. In opposition to overexpression of GSK3β, thapsigargin-induced PARP proteolysis was examined in SH-SY5Y cells stably expressing a dominant-negative mutant of GSK3β (60Yost C. Torres M. Miller J.R. Huang E. Kimelman D. Moon R.T. Genes Dev. 1996; 10: 1443-1454Google Scholar). Although incubation with 2 μm thapsigargin caused a time-dependent increase in PARP proteolysis in control cells, there was little PARP proteolysis in cells expressing dominant-negative GSK3β (Fig.2 B). To test further if GSK3β is a necessary intermediate in thapsigargin-induced caspase-3 activation, cells were pretreated with lithium, a selective inhibitor of GSK3β (44Klein P.S. Melton D.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8455-8459Google Scholar, 45Stambolic V. Ruel L. Woodgett J.R. Curr. Biol. 1996; 6: 1664-1668Google Scholar, 46Davies S.P. Reddy H. Caivano M. Cohen P. Biochem. J. 2000; 351: 95-105Google Scholar). Thapsigargin-induced PARP proteolysis was concentration-dependently inhibited in cells pretreated with 1–20 mm lithium (Fig. 2 C). Pretreatment with 20 mm lithium, which we have shown inhibits GSK3βin vitro by ∼80% (49Grimes C.A. Jope R.S. J. Neurochem. 2001; 78: 1219-1232Google Scholar), reduced thapsigargin-induced PARP proteolysis by 60–80%, indicating a close correspondence between inhibition of GSK3β activity and of caspase-3 activation. Furthermore, examination of the lithium concentration-dependent attenuation of thapsigargin-induced PARP proteolysis revealed less protection in cells overexpressing GSK3β than in wild-type or vector-transfected SH-SY5Y cells due to the greater activity of GSK3β (Fig. 2 C), substantiating the conclusion that lithium's protective action is due to inhibition of GSK3β. Taken together, these results indicate that GSK3β is a necessary and regulatory component of thapsigargin-induced signaling leading to activation of caspase-3. Although GSK3β is a constitutively active enzyme, the activity of GSK3β is modulated by phosphorylation, with phosphorylation of Ser-9 decreasing activity and phosphorylation of Tyr-216 increasing activity (reviewed in Ref. 21Grimes C.A. Jope R.S. Prog. Neurobiol. 2001; 65: 391-426Google Scholar). To examine if either of these post-translational modifications of GSK3β was altered by thapsigargin treatment to account for the thapsigargin-induced activation of GSK3β, the phosphorylation state of GSK3β in SH-SY5Y cells was examined by immunoblot analyses. These measurements revealed a time-dependent decrease in phospho-Ser-9-GSK3β immunoreactivity after thapsigargin treatment (Fig.3 A), whereas phospho-Tyr-216-GSK3β immunoreactivity was unaltered (data not shown). Hence, thapsigargin treatment activated GSK3β by reducing the inhibitory Ser-9 phosphorylation of GSK3β. Because Akt (also known as protein kinase B) is a primary kinase responsible for phosphorylating Ser-9 of GSK3β (34Cross D.A. Alessi D.R. Cohen P. Andjelkovich M. Hemmings B.A. Nature. 1995; 378: 785-789Google Scholar), we tested if thapsigargin affected the activation-associated phosphorylation of Ser-473 of Akt. These measurements demonstrated that treatment with thapsigargin greatly decreased phospho-Ser-473-Akt immunoreactivity (Fig. 3 B). Thus, thapsigargin treatment reduced both the inhibitory phosphorylation of Ser-9 on GSK3β and the activating phosphorylation of Ser-473 on Akt, leading to increased GSK3β activity. To further examine the relationship between the activities of Akt, GSK3β, and caspase-3, we tested if receptor-mediated activation of Akt affected the changes in phosphorylation of Akt and GSK3β and caspase-3 activity induced by thapsigargin in SH-SY5Y cells. Administration of insulin-like growth factor-1 (IGF-1), a growth factor, which activates receptors endogenously expressed in SH-SY5Y cells known to activate Akt (61Kurihara S. Hakuno F. Takahashi S. Endocr. J. 2000; 47: 739-751Google Scholar), counteracted the inhibitory effect of thapsigargin on Akt, causing an increase in phospho-Ser-473-Akt (Fig.3 C), blocked the thapsigargin-induced dephosphorylation of phospho-Ser-9-GSK3β, and in the same samples there was virtually complete elimination of thapsigargin-induced PARP proteolysis (Fig.3 C). Thus, countering thapsigargin-induced inactivation of Akt and activation of GSK3β blocked signaling to caspase-3. Considering previous reports that the calcium-activated protein phosphatase 2B (calcineurin; PP2B) can cause apoptosis (62Asai A. Qiu J.-h. Narita Y. Chi S. Saito N. Shinoura N. Hamada H. Kuchino Y. Kirino T. J. Biol. Chem. 1999; 274: 34450-34458Google Scholar) and is activa
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