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• W2035174076 abstract "N-methyl-d-aspartate receptors (NMDARs) are critical for neuronal plasticity and survival, whereas their excessive activation produces excitotoxicity and may accelerate neurodegeneration. Here, we report that stimulation of NMDARs in cultured rat hippocampal or cortical neurons and in the adult mouse brain in vivo disinhibited glycogen synthase kinase 3β (GSK3β) by protein phosphatase 1(PP1)-mediated dephosphorylation of GSK3β at the serine 9 residue. NMDA-triggered GSK3β activation was mediated by NMDAR that contained the NR2B subunit. Interestingly, GSK3β inhibition reduced inhibitory phosphorylation of the PP1 inhibitor 2 (I2) and attenuated serine 9 dephosphorylation by PP1. These data suggest existence of a feedback loop between GSK3β and PP1 that results in amplification of PP1 activation by GSK3β. In addition, GSK3β inhibition decreased PP1-mediated dephosphorylation of the cAMP-response element-binding protein (CREB) at the serine 133 residue in NMDA-stimulated neurons. Conversely, overexpression of GSK3β abolished non-NR2B-mediated activation of CRE-driven transcription. These data suggest that cross-talk between GSK3β and PP1 contributes to NR2B NMDAR-induced inhibition of CREB signaling by non-NR2B NMDAR. The excessive activation of NR2B-PP1-GSK3β-PP1 circuitry may contribute to the deficits of CREB-dependent neuronal plasticity in neurodegenerative diseases. N-methyl-d-aspartate receptors (NMDARs) are critical for neuronal plasticity and survival, whereas their excessive activation produces excitotoxicity and may accelerate neurodegeneration. Here, we report that stimulation of NMDARs in cultured rat hippocampal or cortical neurons and in the adult mouse brain in vivo disinhibited glycogen synthase kinase 3β (GSK3β) by protein phosphatase 1(PP1)-mediated dephosphorylation of GSK3β at the serine 9 residue. NMDA-triggered GSK3β activation was mediated by NMDAR that contained the NR2B subunit. Interestingly, GSK3β inhibition reduced inhibitory phosphorylation of the PP1 inhibitor 2 (I2) and attenuated serine 9 dephosphorylation by PP1. These data suggest existence of a feedback loop between GSK3β and PP1 that results in amplification of PP1 activation by GSK3β. In addition, GSK3β inhibition decreased PP1-mediated dephosphorylation of the cAMP-response element-binding protein (CREB) at the serine 133 residue in NMDA-stimulated neurons. Conversely, overexpression of GSK3β abolished non-NR2B-mediated activation of CRE-driven transcription. These data suggest that cross-talk between GSK3β and PP1 contributes to NR2B NMDAR-induced inhibition of CREB signaling by non-NR2B NMDAR. The excessive activation of NR2B-PP1-GSK3β-PP1 circuitry may contribute to the deficits of CREB-dependent neuronal plasticity in neurodegenerative diseases. Glutamate is a major excitatory neurotransmitter in the central nervous system. In addition to its role in synaptic transmission, glutamate can also induce neuronal plasticity and promote neuronal survival during development. The latter effects are mediated by a subtype of glutamate ionotropic receptors, N-methyl-d-aspartate receptors (NMDARs) 2The abbreviations used are: NMDARN-methyl-d-aspartate receptorGSK3βglycogen synthase kinase-3βPP1protein phosphatase 1NMDAN-methyl-d-aspartateNR2BNMDAR subunit 2BCREBcAMP response element-binding proteinCREcAMP response elementHDHuntington diseasePKAcAMP-dependent protein kinaseI1inhibitor-1I2inhibitor-2ADAlzheimer diseasepSerphosphoserineERKextracellular signal-regulated kinase 1/2RSKribosomal S6 kinaseMK-801dizocilpineAPVdl-amino-5-phosphonovalerateQAquinolinic acidOAokadaic acidANOVAanalysis of variance 2The abbreviations used are: NMDARN-methyl-d-aspartate receptorGSK3βglycogen synthase kinase-3βPP1protein phosphatase 1NMDAN-methyl-d-aspartateNR2BNMDAR subunit 2BCREBcAMP response element-binding proteinCREcAMP response elementHDHuntington diseasePKAcAMP-dependent protein kinaseI1inhibitor-1I2inhibitor-2ADAlzheimer diseasepSerphosphoserineERKextracellular signal-regulated kinase 1/2RSKribosomal S6 kinaseMK-801dizocilpineAPVdl-amino-5-phosphonovalerateQAquinolinic acidOAokadaic acidANOVAanalysis of variance (1Westbrook G.L. Curr. Opin. Neurobiol. 1994; 4: 337-346Crossref PubMed Scopus (78) Google Scholar). On the other hand, excessive activation of NMDARs causes excitotoxic cell death that underlies neuron loss triggered by hypoxia, epilepsy, or neurotrauma (2Olney J.W. Neurobiol. Aging. 1994; 15: 259-260Crossref PubMed Scopus (75) Google Scholar). N-methyl-d-aspartate receptor glycogen synthase kinase-3β protein phosphatase 1 N-methyl-d-aspartate NMDAR subunit 2B cAMP response element-binding protein cAMP response element Huntington disease cAMP-dependent protein kinase inhibitor-1 inhibitor-2 Alzheimer disease phosphoserine extracellular signal-regulated kinase 1/2 ribosomal S6 kinase dizocilpine dl-amino-5-phosphonovalerate quinolinic acid okadaic acid analysis of variance N-methyl-d-aspartate receptor glycogen synthase kinase-3β protein phosphatase 1 N-methyl-d-aspartate NMDAR subunit 2B cAMP response element-binding protein cAMP response element Huntington disease cAMP-dependent protein kinase inhibitor-1 inhibitor-2 Alzheimer disease phosphoserine extracellular signal-regulated kinase 1/2 ribosomal S6 kinase dizocilpine dl-amino-5-phosphonovalerate quinolinic acid okadaic acid analysis of variance It has been recently proposed that NMDARs may produce diverse outcomes depending on their subunit composition. Whereas the NR2A-containing NMDARs stimulate CRE-driven transcription and enhance neuronal survival, the NR2B NMDARs may inhibit CRE-mediated transcription and trigger excitotoxic cell death (3Hardingham G.E. Fukunaga Y. Bading H. Nat. Neurosci. 2002; 5: 405-414Crossref PubMed Scopus (1346) Google Scholar). Decreases in CRE-mediated transcription may play a role in neurodegeneration. For instance, Huntington disease (HD)-associated mutant forms of huntingtin inhibit transcription of CRE-regulated genes, which are required for proper neuronal function and long term neuronal survival (4Nucifora Jr., F.C. Sasaki M. Peters M.F. Huang H. Cooper J.K. Yamada M. Takahashi H. Tsuji S. Troncoso J. Dawson V.L. Dawson T.M. Ross C.A. Science. 2001; 291: 2423-2428Crossref PubMed Scopus (942) Google Scholar, 5Mantamadiotis T. Lemberger T. Bleckmann S.C. Kern H. Kretz O. Martin Villalba A. Tronche F. Kellendonk C. Gau D. Kapfhammer J. Otto C. Schmid W. Schutz G. Nat. Genet. 2002; 31: 47-54Crossref PubMed Scopus (571) Google Scholar, 6Jiang H. Nucifora Jr., F.C. Ross C.A. DeFranco D.B. Hum. Mol. Genet. 2003; 12: 1-12Crossref PubMed Scopus (136) Google Scholar). Activation of CRE-driven transcription by NMDARs depends on phosphorylation of CREB at Ser133 (7Vanhoutte P. Bading H. Curr. Opin. Neurobiol. 2003; 13: 366-371Crossref PubMed Scopus (186) Google Scholar, 8Lonze B.E. Ginty D.D. Neuron. 2002; 35: 605-623Abstract Full Text Full Text PDF PubMed Scopus (1739) Google Scholar). Stimulation of NR2A NMDAR results in increased levels of phospho-Ser133 (pSer133) whereas stimulation of NR2B NMDAR reduces pSer133 (3Hardingham G.E. Fukunaga Y. Bading H. Nat. Neurosci. 2002; 5: 405-414Crossref PubMed Scopus (1346) Google Scholar). The latter effect may be because of activation of protein phosphatase 1 (PP1) that has been shown to target pSer133 in NMDAR-stimulated hippocampal or cerebellar neurons (9Sala C. Rudolph-Correia S. Sheng M. J. Neurosci. 2000; 20: 3529-3536Crossref PubMed Google Scholar, 10Kopnisky K.L. Chalecka-Franaszek E. Gonzalez-Zulueta M. Chuang D.M. Neuroscience. 2003; 116: 425-435Crossref PubMed Scopus (78) Google Scholar). PP1 activity is regulated by phosphorylation. For instance, PKA-mediated phosphorylation of a PP1 inhibitor 1 (I1) increases its potency to block PP1 (11Cohen P. Annu. Rev. Biochem. 1989; 58: 453-508Crossref PubMed Scopus (2151) Google Scholar, 12Oliver C.J. Shenolikar S. Front. Biosci. 1998; 3: D961-D972Crossref PubMed Google Scholar). Also, overexpression of I1 with a mutation that mimicked phosphorylation by PKA enhanced learning and learninginduced CRE transcription (13Genoux D. Haditsch U. Knobloch M. Michalon A. Storm D. Mansuy I.M. Nature. 2002; 418: 970-975Crossref PubMed Scopus (405) Google Scholar). On the other hand, phosphorylation of PP1 inhibitor 2 (I2) at threonine 72 (Thr72) reduces PP1 inhibition (11Cohen P. Annu. Rev. Biochem. 1989; 58: 453-508Crossref PubMed Scopus (2151) Google Scholar, 12Oliver C.J. Shenolikar S. Front. Biosci. 1998; 3: D961-D972Crossref PubMed Google Scholar). The kinases that phosphorylate Thr72 include GSK3β, ERK1/2, and CDKs (14Hemmings B.A. Resink T.J. Cohen P. FEBS Lett. 1982; 150: 319-324Crossref PubMed Scopus (136) Google Scholar, 15Wang Q.M. Guan K.L. Roach P.J. DePaoli-Roach A.A. J. Biol. Chem. 1995; 270: 18352-18358Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 16Leach C. Shenolikar S. Brautigan D.L. J. Biol. Chem. 2003; 278: 26015-26020Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). Glycogen synthase kinase 3β (GSK3β) is a protein kinase that is upregulated in the brains of Alzheimer disease (AD) patients where it phosphorylates Tau and may contribute to β-amyloid-induced neuronal death (17Takashima A. Noguchi K. Sato K. Hoshino T. Imahori K. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7789-7793Crossref PubMed Scopus (379) Google Scholar, 18Pei J.J. Tanaka T. Tung Y.C. Braak E. Iqbal K. Grundke-Iqbal I. J. Neuropathol. Exp. Neurol. 1997; 56: 70-78Crossref PubMed Scopus (324) Google Scholar, 19Shiurba R.A. Ishiguro K. Takahashi M. Sato K. Spooner E.T. Mercken M. Yoshida R. Wheelock T.R. Yanagawa H. Imahori K. Nixon R.A. Brain Res. 1996; 737: 119-132Crossref PubMed Scopus (65) Google Scholar, 20Bhat R. Xue Y. Berg S. Hellberg S. Ormo M. Nilsson Y. Radesater A.C. Jerning E. Markgren P.O. Borgegard T. Nylof M. Gimenez-Cassina A. Hernandez F. Lucas J.J. Diaz-Nido J. Avila J. J. Biol. Chem. 2003; 278: 45937-45945Abstract Full Text Full Text PDF PubMed Scopus (460) Google Scholar). Inhibition of GSK3β protects against cell death induced by overexpression of mutant huntingtin, suggesting a possible role in HD (21Carmichael J. Sugars K.L. Bao Y.P. Rubinsztein D.C. J. Biol. Chem. 2002; 277: 33791-33798Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar). In healthy neurons, the basal activity of GSK3β is limited by inhibition through phosphorylation at Ser9 (22Grimes C.A. Jope R.S. Prog. Neurobiol. 2001; 65: 391-426Crossref PubMed Scopus (1312) Google Scholar, 23Frame S. Cohen P. Biochem. J. 2001; 359: 1-16Crossref PubMed Scopus (1275) Google Scholar). Ser9 kinases include Akt, ERK1/2/p90RSK, p70S6 kinase, PKA, and protein kinase C (22Grimes C.A. Jope R.S. Prog. Neurobiol. 2001; 65: 391-426Crossref PubMed Scopus (1312) Google Scholar, 23Frame S. Cohen P. Biochem. J. 2001; 359: 1-16Crossref PubMed Scopus (1275) Google Scholar) whereas PP1 and protein phosphatase 2A (PP2A) are implicated in dephosphorylation of Ser9 (24Welsh G.I. Proud C.G. Biochem. J. 1993; 294: 625-629Crossref PubMed Scopus (351) Google Scholar, 25Sutherland C. Leighton I.A. Cohen P. Biochem. J. 1993; 296: 15-19Crossref PubMed Scopus (755) Google Scholar, 26Zhang F. Phiel C.J. Spece L. Gurvich N. Klein P.S. J. Biol. Chem. 2003; 278: 33067-33077Abstract Full Text Full Text PDF PubMed Scopus (383) Google Scholar). Interestingly, Zhang et al. (26Zhang F. Phiel C.J. Spece L. Gurvich N. Klein P.S. J. Biol. Chem. 2003; 278: 33067-33077Abstract Full Text Full Text PDF PubMed Scopus (383) Google Scholar) showed that in several cell lines, GSK3β engaged in a positive feedback loop with PP1 (26Zhang F. Phiel C.J. Spece L. Gurvich N. Klein P.S. J. Biol. Chem. 2003; 278: 33067-33077Abstract Full Text Full Text PDF PubMed Scopus (383) Google Scholar). This occurred through GSK-mediated increase of I2 phosphorylation, PP1 activation, PP1-mediated dephosphorylation of pSer9, and further activation of GSK3β (26Zhang F. Phiel C.J. Spece L. Gurvich N. Klein P.S. J. Biol. Chem. 2003; 278: 33067-33077Abstract Full Text Full Text PDF PubMed Scopus (383) Google Scholar). As the mechanism of NR2B-mediated inhibition of CRE transcription is not fully understood, we investigated a possibility that CREB dephosphorylation following NR2B stimulation is regulated by GSK3β. We report the NR2B NMDAR-triggered activation of GSK3β that was mediated by PP1. We also show that GSK3β further amplified PP1 activity and enhanced CREB dephosphorylation by PP1. Materials—Plasmids containing rat GSK3β wild-type and CRE-Luc (CRE-luciferase reporter) have been previously described (27Dominguez I. Itoh K. Sokol S.Y. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8498-8502Crossref PubMed Scopus (286) Google Scholar, 28Impey S. Obrietan K. Wong S.T. Poser S. Yano S. Wayman G. Deloulme J.C. Chan G. Storm D.R. Neuron. 1998; 21: 869-883Abstract Full Text Full Text PDF PubMed Scopus (771) Google Scholar). The EF1α.LacZ construct was from Invitrogen. The polyclonal anti-GSK3β antibody used for immunoprecipitation was from Santa Cruz Biotechnology. The rabbit antiphospho-Ser9 GSK3β polyclonal antibody was from BIOSOURCE, International (Camarillo, CA); the mouse antiGSK3β/α monoclonal and rabbit anti-CREB monoclonal antibodies were from Upstate Biotechnology, Inc. The anti-Akt, anti-ERK1/2, antiphospho-Ser473 Akt, anti-CREB pSer133 antibodies were from Cell Signaling (Beverly, MA); the antiphospho-ERK1/2 antibody (anti-ACTIVETM MAPK pAb) was purchased from Promega; the antiphosphoSer202 Tau (clone AT8) was from Autogen Bioclear (Mile Elm, UK). The sheep anti-I2 antibody was a gift from Dr. David Brautigan, University of Virginia. The anti-NR2A, -NR2B, and -NR2C antibodies were provided by Dr. Anthone Dunah, Harvard Medical School. Okadaic acid (OA) and tautomycin were purchased from Calbiochem, (San Diego, CA). FK506 (Tacrolimus) was from A. G. Scientific (San Diego, CA). SB216763 was from Tocris Bioscience (Ellisville, MO). All other reagents were from Sigma. Cell Culture and Transfection—Cortical or hippocampal neurons were prepared from newborn Sprague-Dawley rats as described (29Hetman M. Kanning K. Smith-Cavanaugh J.E. Xia Z. J. Biol. Chem. 1999; 274: 22569-22580Abstract Full Text Full Text PDF PubMed Scopus (504) Google Scholar). Culture medium was Basal Medium Eagle (BME) supplemented with 10% heat-inactivated bovine calf serum (Hyclone, Logan, Ut), 35 mm glucose, 1 mm l-glutamine, 100 units/ml of penicillin, 0.1 mg/ml streptomycin. Cell-plating densities were 2 × or 0.8 × 106 per 35-mm plate for cortical or hippocampal neurons, respectively. Cytosine arabinoside (2.5 μm) was added to cultures on the second day after seeding (DIV2) to inhibit the proliferation of non-neuronal cells. Additional glucose (4.5 mm) was added at DIV2 and then at DIV6. Cells were used for experiments at DIV6–8. Transient transfections were performed on DIV4 using the Lipofectamine 2000 reagent (Invitrogen) (30Hetman M. Hsuan S.L. Habas A. Higgins M.J. Xia Z. J. Biol. Chem. 2002; 277: 49577-49584Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). Drug Treatment—Dizocilpine maleate (MK-801), ifenprodil, Ro-25-6981, NBQX, CNQX, FK506, tautomycin, SB216763, and okadaic acid were dissolved in dimethyl sulfoxide (Me2SO). The final concentration of Me2SO in the medium was 0.2–0.4%. NMDA and glutamate were dissolved in culture medium. For experiments with tautomycin and SB216763, cells were placed in serum-free culture medium (BME supplemented with 35 mm glucose, 1 mm l-glutamine, 100 units/ml of penicillin, 0.1 mg/ml streptomycin, and 2.5 μm cytosine arabinoside). Tautomycin or SB216763 were added 1 or 2 h before NMDA, respectively. All other drug inhibitors were added 30 min before the stimulation of NMDAR with NMDA or glutamate. Injections of Quinolinic Acid—Adult male FVB mice (6–8 weeks old, weighing 20 g) (n = 12) were deeply anesthetized with freshly prepared 4.0 mg of Avertin (tribromoethanol) (31Papaioannou V.E. Fox J.G. Lab. Anim. Sci. 1993; 43: 189-192PubMed Google Scholar) per 10 g of body weight dissolved in 0.2 ml of 1.25% (v/v) 2-methyl-2-butanol (Sigma-Aldrich) in saline. These mice were stereotactically injected using a 10-μl Hamilton syringe with 1 μl of saline containing 0 or 30 nmol of quinolinic acid (cat. P63204, Sigma-Aldrich) at 4 coordinates (all from Bregma): for striatal injections 0.7 mm rostral, 1.9 mm lateral (left and right), 2.5 mm ventral, and for hippocampal injections 2.0 mm caudal, 1.5 mm lateral (left and right), 1.6 mm ventral. The injection of quinolinic acid was performed over 2 min, and the injection needle was retained in position for 2 min before and after injection. Mice were decapitated 1 h after injection and dissected on ice. The striata, hippocampi, and cerebellum were isolated, frozen on dry ice, and kept at –80 °C for future protein extraction. GSK3β Kinase Assay—GSK3β activity was quantitated using an immune complex kinase assay as described previously (32Hetman M. Cavanaugh J.E. Kimelman D. Xia Z. J. Neurosci. 2000; 20: 2567-2574Crossref PubMed Google Scholar). Western Analysis and Immunostaining—Western blot analysis were performed as described (29Hetman M. Kanning K. Smith-Cavanaugh J.E. Xia Z. J. Biol. Chem. 1999; 274: 22569-22580Abstract Full Text Full Text PDF PubMed Scopus (504) Google Scholar, 33Xia Z. Dudek H. Miranti C.K. Greenberg M.E. J. Neurosci. 1996; 16: 5425-5436Crossref PubMed Google Scholar, 34Xia Z. Dickens M. Raingeaud J. Davis R.J. Greenberg M.E. Science. 1995; 270: 1326-1331Crossref PubMed Scopus (5033) Google Scholar). To detect the phosphorylation shift of I2 by Western blotting, 15% polyacrylamide gels were used. For all other epitopes, proteins were separated on 10% gels. Promoter Assays—Promoter assays were performed as described (35Impey S. Mark M. Villacres E.C. Poser S. Chavkin C. Storm D.R. Neuron. 1996; 16: 973-982Abstract Full Text Full Text PDF PubMed Scopus (507) Google Scholar). Briefly, 0.2 × 106 cells were plated onto each well of a 24-well plate coated with poly-d-lysine. At DIV4-DIV5, neurons were co-transfected with the CRE-Luc (0.8 μg/4 wells) and EF1αLacZ DNA (0.55 μg/4 wells) with or without GSK3β (0.12–0.2 μg/4 wells) expression plasmid. Three days after transfection neurons were treated with NMDA for 20 h. The luciferase activity was determined using a luciferase assay kit (Promega) and normalized to β-galactosidase activity that was assayed using a kit from Promega. Statistical Analysis—Statistical analysis of the data was performed using one-way analysis of variance (ANOVA). NMDAR Stimulation Activates GSK3β—To test the possibility that GSK3β participates in signaling activated by NMDARs, we evaluated the effects of NMDA on GSK3β activity. As others have shown that reduced levels of the inhibitory GSK3β phosphorylation at serine 9 correlate with its activation (22Grimes C.A. Jope R.S. Prog. Neurobiol. 2001; 65: 391-426Crossref PubMed Scopus (1312) Google Scholar, 23Frame S. Cohen P. Biochem. J. 2001; 359: 1-16Crossref PubMed Scopus (1275) Google Scholar), we measured the levels of phosphoSer9 (pSer9) in NMDA-stimulated neurons. Western blot analysis with an antibody specific for pSer9 revealed decreased levels of this phosphorylation in cultured neurons that were stimulated with NMDA (Fig. 1, A and B). A 60% decrease was evident in both hippocampal and cortical neurons 20 min after addition of 100 μm NMDA (Fig. 1, A and B). The reduced pSer9 levels were observed up to 6 h after initiation of the treatment (Fig. 1, A and B). Dose response experiments showed that the decrease in pSer9 was triggered by NMDA concentrations as low as 10 μm (Fig. 1C). Also, glutamate, a physiological ligand of NMDAR, reduced pSer9 in cortical neurons (Fig. 1D). The glutamate effect was mediated by NMDAR as NMDAR, but not non-NMDA receptor antagonists, prevented the pSer9 decline (Fig. 1D). These data suggest that NMDAR stimulation in cultured cortical or hippocampal neurons reduced the pSer9-dependent inhibition of GSK3β. As it was reported that GSK3β may be activated through the phosphorylation at Tyr216 residue (36Bhat R.V. Shanley J. Correll M.P. Fieles W.E. Keith R.A. Scott C.W. Lee C.M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 11074-11079Crossref PubMed Scopus (354) Google Scholar), we evaluated NMDA effects on pTyr216 levels. Phosphorylation of Tyr216 was not affected by NMDA in cortical or hippocampal neurons (Fig. 1E). This suggests that NMDAR signaling does not involve modulation of pTyr216. To test whether NMDAR stimulation in the adult brain in vivo can also produce a decrease in GSK3β pSer9 levels, we injected a NMDAR agonist, quinolinic acid (QA) into neostriatum and hippocampus of adult mice. Intrastriatal injections of QA produce a pathology with a striking resemblance to HD (37Brouillet E. Conde F. Beal M.F. Hantraye P. Prog. Neurobiol. 1999; 59: 427-468Crossref PubMed Scopus (365) Google Scholar). Intrahippocampal QA injections result in seizures and loss of pyramidal neurons (38Schwarcz R. Brush G.S. Foster A.C. French E.D. Exp. Neurol. 1984; 84: 1-17Crossref PubMed Scopus (140) Google Scholar). As a negative control we used cerebellum that is far from the forebrain injection sites. One hour after QA administration, there was a significant decline of pSer9 levels that reached 39 or 51% of control values in striata or hippocampi, respectively (Fig. 2). The cerebellar pSer9 levels in QA-treated mice were similar to those in control animals (Fig. 2). These data indicate that NMDAR stimulation in the adult mouse brain rapidly activates GSK3β by dephosphorylation of pSer9. To verify that the NMDAR-triggered decrease of pSer9 was accompanied by an elevation of GSK3β activity, we performed a GSK3β immunoprecipitation kinase assay. In cultured hippocampal neurons, the kinase activity of GSK3β increased as early as 5 min after 100 μm NMDA treatment (159% increase, p < 0.01, Fig. 3A). The maximal increase of 172% was seen at 20 min after addition of 100 μm NMDA (p < 0.001, Fig. 3A). The elevated GSK activity was still present at 60 min (128%, p < 0.05, Fig. 3A) and declined to control values 3 h after treatment. The reduction of GSK3β activity was despite the persistent dephosphorylation of pSer9 (Fig. 1A). This discrepancy is likely because of decreased levels of the total GSK3β protein observed at 1 and 3 h after NMDA stimulation (Fig. 1A). The reduction of GSK3β levels may be caused by the degradation of this protein during excitotoxicity, which is induced by 100 μm NMDA. 3A. Habas, E. Szatmari, and M. Hetman, unpublished observation. To determine if stimulation of NMDAR increased GSK3β activity in intact neurons, we evaluated the effects of NMDA on Tau phosphorylation at the Ser202 residue. This phosphorylation has been shown to be carried out by GSK3β (39Cross D.A. Culbert A.A. Chalmers K.A. Facci L. Skaper S.D. Reith A.D. J. Neurochem. 2001; 77: 94-102Crossref PubMed Scopus (342) Google Scholar). In cortical neurons, phospho-Tau levels increased by 42% at 5 min after treatment with 100 μm NMDA (p < 0.01) suggesting GSK3β activation. At later time points, phospho-Tau levels decreased (data not shown), which is consistent with reports that PP2B or PP1/PP2A dephosphorylate Tau after NMDAR stimulation (40Fleming L.M. Johnson G.V. Biochem. J. 1995; 309: 41-47Crossref PubMed Scopus (86) Google Scholar, 41Adamec E. Mercken M. Beermann M.L. Didier M. Nixon R.A. Brain Res. 1997; 757: 93-101Crossref PubMed Scopus (34) Google Scholar). Therefore, activation of Tau phosphatases may antagonize the effects of GSK3β on Ser202. Together, these data indicate that activation of NMDAR can increase the kinase activity of GSK3β. GSK3β Activation Is a Specific Response to Stimulation of NR2B NMDARs—NMDARs are heterotetramers of two NR1 and two NR2 subunits (1Westbrook G.L. Curr. Opin. Neurobiol. 1994; 4: 337-346Crossref PubMed Scopus (78) Google Scholar, 42Mori H. Mishina M. Neuropharmacology. 1995; 34: 1219-1237Crossref PubMed Scopus (584) Google Scholar). There is one gene for NR1 subunit and four genes for NR2 subunits including NR2A, -B, -C, and -D. We found that NR2A, NR2B, and NR2C were expressed in cultured cortical or hippocampal neurons (Fig. 4A), whereas NR2D expression was undetectable (data not shown). This suggests that cultured neurons possess diverse NMDARs that may trigger distinct responses to NMDA. Therefore, we determined which NMDARs may mediate GSK3β activation by NMDA in neurons. The NMDA-induced decrease in GSK3β pSer9 levels was abolished by an NR2B-selective NMDAR blockers, ifenprodil or Ro-256981 (Fig. 4, B and C). The effect was present in both hippocampal and cortical neurons and was identical to that of APV, which is a non-selective NMDAR antagonist (Fig. 4, B and C). This suggests that pSer9 dephosphorylation in NMDA-treated neurons is mediated by NMDARs that contain the NR2B subunit. NMDARs act as ion channels, which upon activation increase intracellular Ca2+ concentration ([Ca2+]i) (42Mori H. Mishina M. Neuropharmacology. 1995; 34: 1219-1237Crossref PubMed Scopus (584) Google Scholar). Because the rising [Ca2+]i is critical for mobilization of NMDAR-activated signaling pathways (42Mori H. Mishina M. Neuropharmacology. 1995; 34: 1219-1237Crossref PubMed Scopus (584) Google Scholar), we tested whether Ca2+ influx by routes other than NMDAR can reduce pSer9 levels. Treatment with 55 mm KCl in the presence of a NMDAR antagonist, APV (100 μm) increases [Ca2+]i through the voltage-gated calcium channels (VGCC) (43Kudo Y. Ogura A. Br. J. Pharmacol. 1986; 89: 191-198Crossref PubMed Scopus (202) Google Scholar). Indeed, hippocampal neurons treated for 60 min with KCl plus AVP showed activation of ERK1/2 signaling, indicating a rise in [Ca 2+]i (Fig. 4D). However, this treatment did not significantly affect pSer9 levels (Fig. 4D), suggesting that the NMDA-mediated decrease in pSer9 is a specific response to stimulation of NR2B NMDAR and not a general Ca2+-mediated response. PP1 Mediates NMDAR-induced GSK Activation—The NMDA-triggered decrease in pSer9 levels may be caused either by inhibition of Ser9 kinases or by stimulation of Ser9 phosphatases. As a first step to differentiate between these two possibilities, we examined the activity of Akt, the main GSK3β Ser kinase in cultured neurons (30Hetman M. Hsuan S.L. Habas A. Higgins M.J. Xia Z. J. Biol. Chem. 2002; 277: 49577-49584Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). Because in some cell types ERK1/2 signaling may increase GSK3β Ser9 phosphorylation (25Sutherland C. Leighton I.A. Cohen P. Biochem. J. 1993; 296: 15-19Crossref PubMed Scopus (755) Google Scholar, 44Stambolic V. Woodgett J.R. Biochem. J. 1994; 303: 701-704Crossref PubMed Scopus (506) Google Scholar, 45Eldar-Finkelman H. Seger R. Vandenheede J.R. Krebs E.G. J. Biol. Chem. 1995; 270: 987-990Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar, 46Shaw M. Cohen P. FEBS Lett. 1999; 461: 120-124Crossref PubMed Scopus (82) Google Scholar), we also studied the status of ERK1/2 after NMDA treatment. The activities of Akt and ERK1/2 were monitored by Western blotting with antibodies specifically recognizing the phosphorylated, activated forms of these kinases. NMDAR stimulation did not affect Akt whereas it activated ERK1/2 at 5, 20, or 60 min after treatment (Fig. 5A). However, pSer9 levels were significantly reduced at these time points (Fig. 1A). Therefore, inhibition of Ser9 kinases including Akt or ERK1/2-activated p90RSK is not a likely explanation for GSK3β activation by NMDA. NMDARs activate several protein phosphatases including PP1, PP2A, and PP2B (47Mulkey R.M. Endo S. Shenolikar S. Malenka R.C. Nature. 1994; 369: 486-488Crossref PubMed Scopus (904) Google Scholar, 48Thiels E. Norman E.D. Barrionuevo G. Klann E. Neuroscience. 1998; 86: 1023-1029Crossref PubMed Scopus (63) Google Scholar). Therefore, we determined the effects of protein phosphatase inhibitors OA, tautomycin, and FK506 on the NMDA-triggered pSer9 decline. If applied to cultured cells, 1 μm OA inhibits PP2A and PP1, whereas at the lower concentrations it acts selectively on PP2A (49Sheppeck J.E. Gauss C.M. Chamberlin A.R. Bioorg. Med. Chem. 1997; 5: 1739-1750Crossref PubMed Scopus (142) Google Scholar). Indeed, OA at concentrations ≥10 nm significantly increased phosphorylation of a recognized PP2A target, ERK1/2 (Ref. 50Sontag E. Fedorov S. Kamibayashi C. Robbins D. Cobb M. Mumby M. Cell. 1993; 75: 887-897Abstract Full Text PDF PubMed Scopus (461) Google Scholar and Fig. 5B). Similarly, OA applied at the concentrations ≥10 nm increased basal pSer9 levels (Fig. 5, C and D). The increase was concentration-dependent reaching maximal values at 1 μm (10.9- or 5.7-fold of control levels in cortical or hippocampal neurons, respectively; Fig. 5, C and D). These data suggest that under basal conditions, Ser9 dephosphorylation may be carried out by both PP2A and PP1. On the other hand, the NMDA-induced reduction in pSer9 levels was abolished by 1 μm OA but not by lower OA concentrations (Fig. 5, C and D). In addition, a PP1-specific inhibitor, tautomycin (51Favre B. Turowski P. Hemmings B.A. J. Biol. Chem. 1997; 272: 13856-13863Abstract Full Text Full Text PDF PubMed Scopus (280) Google Scholar) did not affect the basal pSer9 or pERK1/2 levels (Fig. 5E). In contrast, tautomycin prevented pSer9 decline in NMDA-treated neurons (Fig. 5E). PP2B inhibition by FK506 did not affect pSer9 levels in basal conditions or after NMDAR activation (Fig. 5F). These data suggest that a PP1-like phosphatase activity mediates the NMDA-induced dephosphorylation of pSer9 whereas a PP2A-like phosphatase dephosphorylates pSer9 under basal conditions. GSK3β Enhances PP1 Activation in NMDAR-stimulated Neurons—GSK3β can stimulate PP1 activity by phosphorylation of I2 (11Cohen P. Annu. Rev. Biochem. 1989; 58: 453-508Crossref PubMed Scopus (2151) Goo" @default.
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• W2035174076 date "2005-11-01" @default.
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• W2035174076 title "A Positive Feedback Loop between Glycogen Synthase Kinase 3β and Protein Phosphatase 1 after Stimulation of NR2B NMDA Receptors in Forebrain Neurons" @default.
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