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Differential Roles of Glycogen Synthase Kinase-3 Isoforms in the Regulation of Transcriptional Activation

2006; Elsevier BV; Volume: 281; Issue: 41 Linguagem: Inglês

10.1074/jbc.m607468200

1083-351X

Min‐Huei Liang, De‐Maw Chuang,

Kruppel-like factors research

Glycogen synthase kinase-3 (GSK-3) exists as two structurally similar isoforms, α and β, whose activities are negatively regulated by serine phosphorylation but positively controlled by tyrosine phosphorylation. We used GSK-3 isoform-specific small interfering RNAs, dominant negative mutants, and pharmacological inhibitors to search for the differential roles for both GSK-3 isoforms in regulating transcriptional activation in cultured rat cerebral cortical neurons. GSK-3α and GSK-3β were shown to have differentially regulated transactivation such that GSK-3α silencing/inhibition was more robust than GSK-3β silencing/inhibition in causing cAMP-responsive element- and NF-κB-dependent transactivation. Moreover, protein-DNA array studies identified two novel GSK-3-regulated transcription factors, early growth response 1 and Smad3/4, which were oppositely affected by GSK-3α or GSK-3β silencing or inhibition. Taken together, our results underscore critical variations in the function and regulation of GSK-3α and GSK-3β. The development of GSK-3 isoform-specific inhibitors is thus crucial for therapeutic intervention of GSK-3-related neuropathological conditions. Glycogen synthase kinase-3 (GSK-3) exists as two structurally similar isoforms, α and β, whose activities are negatively regulated by serine phosphorylation but positively controlled by tyrosine phosphorylation. We used GSK-3 isoform-specific small interfering RNAs, dominant negative mutants, and pharmacological inhibitors to search for the differential roles for both GSK-3 isoforms in regulating transcriptional activation in cultured rat cerebral cortical neurons. GSK-3α and GSK-3β were shown to have differentially regulated transactivation such that GSK-3α silencing/inhibition was more robust than GSK-3β silencing/inhibition in causing cAMP-responsive element- and NF-κB-dependent transactivation. Moreover, protein-DNA array studies identified two novel GSK-3-regulated transcription factors, early growth response 1 and Smad3/4, which were oppositely affected by GSK-3α or GSK-3β silencing or inhibition. Taken together, our results underscore critical variations in the function and regulation of GSK-3α and GSK-3β. The development of GSK-3 isoform-specific inhibitors is thus crucial for therapeutic intervention of GSK-3-related neuropathological conditions. Gkycogen synthase kinase 3 (GSK-3) 2The abbreviations used are: GSK, glycogen synthase kinase; AP-1, activator protein 1; CRE, cAMP-responsive element; CREB, CRE-binding protein; EGR-1, early growth response-1; GRE, glucocorticoid-responsive element; NF-κB, nuclear factor of κB; siRNA, small interfering RNA; TCF, T-cell factor; DIV, days in vitro. has a central role in regulating cellular function, structure, and survival by coordinated control of phosphorylation of GSK-3 itself and its substrates, the formation of substrate-specific GSK-3 binding/scaffolding protein complexes, and GSK-3 subcellular distribution (reviewed in Refs. 1Bijur G.N. Jope R.S. Neuroreport. 2003; 14: 2415-2419Crossref PubMed Scopus (192) Google Scholar, 2Doble B.W. Woodgett J.R. J. Cell Sci. 2003; 116: 1175-1186Crossref PubMed Scopus (1767) Google Scholar). Among the substrates phosphorylated by GSK-3 are a wide spectrum of transcription factors, including CREB, NF-κB, AP-1, heat shock factor 1, β-catenin, T-cell factor (TCF)/lymphoid enhancer factor, p53, and Bax (reviewed in Ref. 3Jope R.S. Bijur G.N. Mol. Psychiatry. 2002; 7: S35-S45Crossref PubMed Google Scholar). Dysregulation of GSK-3-mediated signaling pathways and substrate phosphorylation has been implicated in the pathophysiology of mood disorders (4O'Brien W.T. Harper A.D. Jove F. Woodgett J.R. Maretto S. Piccolo S. Klein P.S. J. Neurosci. 2004; 24: 6791-6798Crossref PubMed Scopus (388) Google Scholar, 5Gould T.D. Einat H. Bhat R. Manji H.K. Int. J. Neuropsychopharmacol. 2004; 7: 387-390Crossref PubMed Scopus (281) Google Scholar), schizophrenia (6Emamian E.S. Hall D. Birnbaum M.J. Karayiorgou M. Gogos J.A. Nat. Genet. 2004; 36: 131-137Crossref PubMed Scopus (791) Google Scholar), Alzheimer disease, type II diabetes, and cancer, among others (2Doble B.W. Woodgett J.R. J. Cell Sci. 2003; 116: 1175-1186Crossref PubMed Scopus (1767) Google Scholar). GSK-3 is an evolutionary conserved, constitutively active, serine/threonine kinase that consists of two distinct isoforms, α and β, in mammals (7Woodgett J.R. EMBO J. 1990; 9: 2431-2438Crossref PubMed Scopus (1154) Google Scholar). Their activities are regulated by serine and tyrosine phosphorylation (8Wang Q.M. Fiol C.J. DePaoli-Roach A.A. Roach P.J. J. Biol. Chem. 1994; 269: 14566-14574Abstract Full Text PDF PubMed Google Scholar, 9Grimes C.A. Jope R.S. Prog. Neurobio. 2001; 65: 391-426Crossref PubMed Scopus (1317) Google Scholar). GSK-3α and GSK-3β are highly homologous within their kinase domains and display similar biochemical and substrate properties (2Doble B.W. Woodgett J.R. J. Cell Sci. 2003; 116: 1175-1186Crossref PubMed Scopus (1767) Google Scholar). However, some structural differences between the two isoforms exist in their N- and C-terminal regions, and a number of reports suggest that the regulation and functions of both GSK-3 isoforms are not always identical. For example, GSK-3α, but not GSK-3β, is regulated by protein kinase C isozymes (10Goode N. Hughes K. Woodgett J.R. Parker P.J. J. Biol. Chem. 1992; 267: 16878-16882Abstract Full Text PDF PubMed Google Scholar), whereas GSK-3β is more potent than GSK-3α in phosphorylating phosphatase inhibitor 2 (11Wang Q.M. Park I.K. Fiol C.J. Roach P.J. DePaoli-Roach A.A. Biochemistry. 1994; 33: 143-147Crossref PubMed Scopus (66) Google Scholar). The disruption of the GSK-3β isoform in mice results in embryonic lethality, and the GSK-3α isoform is unable to compensate for the loss of GSK-3β (12Hoeflich K.P. Luo J. Rubie E.A. Tsao M.S. Jin O. Woodgett J.R. Nature. 2000; 406: 86-90Crossref PubMed Scopus (1223) Google Scholar). Additionally, transfection and small interfering RNA (siRNA) studies suggest that GSK-3α is involved in the processing of β-amyloid precursor protein to form Aβ1–40 and Aβ1–42 peptides, whereas the role of GSK-3β is unclear (13Phiel C.J. Wilson C.A. Lee V.M. Klein P.S. Nature. 2003; 423: 435-439Crossref PubMed Scopus (1091) Google Scholar, 14Su Y. Ryder J. Li B. Wu X. Fox N. Solenberg P. Brune K. Paul S. Zhou Y. Liu F. Ni B. Biochemistry. 2004; 43: 6899-6908Crossref PubMed Scopus (289) Google Scholar). Moreover, differential transcriptional and translational regulation for these two isoforms might be due to variable expression of GSK-3α and GSK-3β in different mammalian tissues (7Woodgett J.R. EMBO J. 1990; 9: 2431-2438Crossref PubMed Scopus (1154) Google Scholar). Transcriptional activation regulates the expression of many downstream genes of signaling pathways directly by GSK-3 through phosphorylation or indirectly as a consequence of GSK-3 activity changes. Although differential regulations of transcription factors by the two isoforms of GSK-3 were proposed (11Wang Q.M. Park I.K. Fiol C.J. Roach P.J. DePaoli-Roach A.A. Biochemistry. 1994; 33: 143-147Crossref PubMed Scopus (66) Google Scholar, 12Hoeflich K.P. Luo J. Rubie E.A. Tsao M.S. Jin O. Woodgett J.R. Nature. 2000; 406: 86-90Crossref PubMed Scopus (1223) Google Scholar, 13Phiel C.J. Wilson C.A. Lee V.M. Klein P.S. Nature. 2003; 423: 435-439Crossref PubMed Scopus (1091) Google Scholar), the distinct mechanisms and endogenous targets of such regulations remain to be investigated. In the present study, we employed transfection of GSK-3 isoform-specific siRNA, wild-type GSK-3α and -3β, and their dominant negative mutants to address their differential regulation of transactivation in cultured rat cerebral cortical neurons. The efficacies of both GSK-3 isoforms in regulating various transcription factors were compared, and new GSK-3-targeted transcription factors oppositely regulated by these two isoforms were identified. Rat Primary Cerebral Cortical Culture and Cell Culture for Human SH-SY5Y Neuroblastoma Cells—Cerebral cortical neurons prepared from 18-day-old embryonic rats were cultured as described previously (15Hashimoto R. Hough C. Nakazawa T. Yamamoto T. Chuang D.M. J. Neurochem. 2002; 80: 589-597Crossref PubMed Scopus (263) Google Scholar) with modifications. Briefly, dissociated cortical cells were collected by centrifugation at 80 × g and seeded at a density of 1 × 106 cells/cm2 on poly-d-lysine (Sigma)-coated culture plates. Cortical neurons were used for siRNA transfection after 9 days in vitro (DIV) unless otherwise indicated. Human neuroblastoma SH-SY5Y cells were routinely maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and 1% l-glutamine. Cells with a passage number ranging from 102 to 105 were used in the studies. Plasmid DNA—Reporter plasmids pEgr1-Luc and pSmad3/4-Luc were obtained from Panomics (Redwood city, CA). Reporter construct pRL-TK was purchased from Promega (Madison, WI). TCF reporter plasmid and mutant Tcf reporter plasmid (TOPflash and FOPflash, respectively) were purchased from Upstate (Charlottesville, VA). Mercury Pathway Profiling Luciferase System 1 (Clontech, Palo Alto, CA) including reporter constructs pAP-1-Luc, pCRE-Luc, pGRE-Luc, pNF-κB-Luc, and pHSE-Luc was used in conjunction with the silencing of GSK-3 isoform in studies of transcriptional regulation. Wild-type GSK-3α and its dominant negative mutant (hGSK3α/pMT2 and hGSK3α/pMT2-KR, respectively) were obtained from Dr. Peter S. Klein (University of Pennsylvania) with the kind permission of Dr. James R. Woodgett (Ontario Cancer Institute, Toronto, Canada). Wild-type GSK-3β (pAdTrack-CMV-GSK-3β) was supplied by Dr. Ratan V. Bhat (AstraZeneca R&D, Södertälje, Sweden). Two dominant negative mutants (pAdTrack-CMV-GSK-3β-K85R and pAdTrack-CMV-GSK-3β-R96A) for GSK-3β were generated using the QuikChange II site-directed mutagenesis kit (Stratagene, La Jolla, CA). The GSK-3β wild-type and two dominant negative mutants were subsequently subcloned into pEGFP-C1 vector (Clontech) to obtain the three plasmids used in this study, pEGFP-GSK-3β, pEGFP-GSK-3β-K85R, and pEGFP-GSK-3β-R96A. siRNA Preparation and GSK-3 Inhibitors—Several siRNAs targeting either isoform of GSK-3 were designed according to GenBank™ accession numbers (NM_017344 for rat GSK-3α and NM_032080 for rat GSK-3β; NM_019884 for human GSK-3α and NM_002093 for human GSK-3β) and chemically synthesized (Dharmacon, Chicago, IL). The target sequences for the rat-specific GSK-3 siRNAs are as follows: αP747, TCACTTTACCAAGGCCAAG; αP1269, CTTCAGTCCTGGTGAACTT; αP1375, GCTTTAACTGAGACTCAGA; βP555, CCTCTTGCTGGATCCTGAT; βP514, GGTCTACCTTAACCTGGTG; βP1093, GAACTGTCAAGTAACCCAC. The siRNA mixture specific to either GSK-3 isoform consists of the three species of siRNAs described above and was used in all silencing experiments. An siRNA control (CONTROL non-targeting siRNA 1; Dharmacon) was used as an RNA interference control for all siRNA transfection experiments in both rat neurons and human neuroblastoma cells. Two structurally similar ATP-competitive GSK-3 inhibitors, SB216763 and SB41286, were purchased from Tocris (Ellisville, MO). Transfection Assays and Dual Luciferase Reporter Assay—Rat primary cortical cultures were evenly plated and cultured for 9 DIV before transfection for 48 h with 80 nm siRNA using 8 μl of siPORT Amine transfection reagent (Ambion) in one ml of the transfection mixture. Transfection of luciferase reporter constructs and co-transfections of reporter plasmids with GSK-3 expression vectors (wild-type or dominant negative mutants) into primary cortical neurons were established using an Amaxa Nucleofector (Amaxa, Cologne, Germany) in conjunction with a rat neuron Nucleofector kit (Amaxa) at the time of cell plating. Co-transfections of luciferase reporter plasmids and GSK-3 expression vectors or siRNAs into SH-SY5Y cells were carried out using Lipofectamine 2000 (Invitrogen). Dual luciferase assay was performed using the dual luciferase reporter assay system (Promega). Routinely, dual reporter-transfected cortical neurons were lysed in passive lysis buffer (Promega) and subjected to the detection of firefly luciferase activity and, subsequently, Renilla luciferase activity, which serves as an internal control. Luciferase activities were determined by using a plate-reading luminometer (Packard Bioscience Company, Downers Grove, IL), and the activities of the experimental reporter were normalized to the activities of the background Renilla luciferase. Transcription Factor-DNA Array I Analysis—Cortical neurons at 9 DIV were transfected with siRNA for GSK-3α or GSK-3β for 48 h. Cells were collected and nuclear extracts were prepared using a nuclear extraction kit (Panomics) for analysis with the TranSignal™ protein-DNA array I (Panomics). The procedures were performed according to the manufacturer's protocol with slight modification in the horseradish peroxidase detection process in which DNA Thunder™ Chemiluminescence Reagent Plus (PerkinElmer) was used. Quantitative array results showing more than 2-fold alterations in transcription factor-DNA binding were selected as positive for further confirmation using the reporter activity assay. Immunoblotting Analysis—Cortical neurons were washed twice with 5 ml of phosphate-buffered saline and harvested by scraping into cell lysis buffer (Cell Signaling Technology, Beverly, MA) supplemented with a protease inhibitor mixture (Roche Diagnostics). Protein concentrations were determined by BCA protein assay reagent kit (Pierce). Antibodies against PAI-1, β-actin, β-catenin, and Bcl-2 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Antibody against GSK-3α was obtained from Upstate. Antibodies against GSK-3α, pCREB(Ser133), and CREB were purchased from Cell Signaling Technology (Beverly, MA). ECL Plus Western blotting detection system (Amersham Biosciences) was used to obtain Western blot signals, and autoradiographs were scanned. Intensity of immunoblot signals was analyzed using Scion Image software (Scion Image for Windows, NIH). Statistical Analysis—All results presented are mean ± S.E. from three to four independent experiments, with three or four replicates for each data point. The results were analyzed for statistical significance in GraphPad InStat (GraphPad, San Diego, CA) by Student's t test or one-way analysis of variance. In analysis of variance, a Q-Q plot was adopted for normal distribution tests. We investigated whether the GSK-3 isoforms are differentially involved in the regulation of transcription factors known to be modulated by GSK-3. Isoform-specific rat GSK-3 siRNAs were designed and synthesized. Western blotting was performed to assess the silencing effects of siRNA for GSK-3α or GSK-3β. Transfection of cortical neurons with different amounts of siRNA cocktails for either GSK-3α or GSK-3β resulted in knockdown of their respective proteins 48 h after transfection, whereas protein levels of the non-targeted GSK-3 isoforms were unaffected (Fig. 1A). Treatment of cortical neurons with 80 nm siRNA cocktails for GSK-3α and GSK-3β caused 76 ± 14% (n = 5) and 81 ± 11% (n = 5) protein depletion of 3α and 3β isoforms, respectively. Thus, the magnitudes of protein reduction by specific siRNA treatments are similar for these two GSK-3 isoforms. As measured by luciferase reporter assay, silencing GSK-3α and GSK-3β with their specific siRNAs was found to cause 7.5- and 3-fold activation of CRE-responsive transcription, respectively (Fig. 1B). NF-κB-responsive transcription was also more robustly activated by GSK-3α silencing than by GSK-3β silencing (6-fold versus 2-fold). Transcription of other responsive cis-element-containing reporter constructs, including TCF, AP-1, GRE, and HSE, was activated to various extents by GSK-3 silencing. Their degrees of activation were similar between the two isoforms using siRNA for either GSK-3α or GSK-3β. By using dominant negative mutants of GSK-3α and GSK-3β, the differential activation of CRE- and NF-κB-dependent transcription by isoform-specific GSK-3 gene silencing was confirmed (Fig. 1C). Thus, a GSK-3α dominant negative mutant produced a greater transcriptional activation of CREB and NF-κB than two GSK-3β dominant negative mutants (GSK-3β K85R and GSK-3β R96A) (Fig. 1C). Conversely, transfection with either wild-type GSK-3α or wild-type GSK-3β resulted in the reduction of both CRE- and NF-κB-dependent transcriptional activities. Transfection of cortical neurons with isoform-specific siRNAs for 48 h caused a modest increase in protein levels of pCREB(Ser133) and Bcl-2, a target gene of CRE-dependent transcription, in a concentration-dependent manner (Fig. 1D). Moreover, the increase of protein levels of both pCREB-(Ser133) and Bcl-2 was slightly greater in cortical neurons transfected with 80 nm GSK-3α siRNA compared with transfection with GSK-3β siRNA. These results are thus compatible with our observations indicating a higher magnitude of CRE-dependent transactivation resulting from GSK-3α gene silencing. On the other hand, a similar magnitude of a dose-dependent increase in the levels of β-catenin, a transcription factor associated with TCF or lymphoid enhancer factor 1 to regulate TCF-dependent transactivation, was observed in cortical neurons transfected with siRNA for either GSK-3 isoform (Fig. 1D). These results further support our observations that showed similar effects of protein depletion of either GSK-3 isoform on TCF-dependent transactivation (Fig. 1B). Inhibition of both GSK-3 isoforms by two ATP-competitive GSK-3 inhibitors, SB216763 and SB415286, at various concentrations was carried out to examine the up-regulating effects of inhibitors on CRE- and NF-κB-dependent transactivation. As shown in Fig. 2A, we observed increased CRE-dependent transcription with a similar magnitude in a concentration-dependent manner. Comparable results were obtained for NF-κB-dependent transactivation (Fig. 2B). Treatment with GSK-3 inhibitors (3 μm SB216763 and 10 μm SB415286) resulted in modest activation of TCF-, AP-1-, GRE-, and HSE-responsive transcription in cortical neurons, supporting a regulatory role for GSK-3 in these four signaling pathways (Fig. 2C). Treatment with these two GKS-3 inhibitors resulted in an increase in β-catenin protein levels, suggesting that inhibition of GSK-3 kinase activity did occur (Fig. 2D). Transfection of cortical neurons with dominant negative mutants of either GSK-3 isoform also enhanced GRE-responsive transcription (data not shown), confirming the inhibitory effect of GSK-3 in GRE-responsive signaling regulation. Next, in an effort to search for novel targets of GSK-3, cortical neurons were transfected with siRNA specific for GSK-3α or GSK-3β, and the nuclear extracts were analyzed for protein-DNA binding activities. Changes of DNA binding activity with transcription factors EGR-1 (also known as nerve growth factor, NGF-1A) and Smad3/4 were noted (Fig. 3). Binding activity of EGR-1-responsive element with EGR-1 was down-regulated by transfection with siRNA for GSK-3α but up-regulated by siRNA for GSK-3β compared with the non-targeting siRNA control. Luciferase reporter assays confirmed that the transcriptional activity of EGR-1 was decreased by GSK-3α siRNA and its dominant negative mutant but increased by GSK-3β siRNA and its dominant negative mutants (Fig. 4A). In contrast, the DNA binding activity of Smad3/4 was up-regulated by GSK-3α siRNA and appeared to be unaffected by GSK-3β siRNA. Moreover, the activity of Smad3/4-responsive transcription was enhanced by GSK-3α siRNA and its dominant negative mutant but slightly reduced by GSK-3β siRNA and its R96A mutant (Fig. 4B). Of interest, treatment with the GSK-3 inhibitors SB216763 and SB415286 resulted in an increase in Smad3/4 transcriptional activities, similar to the effects of GSK-3α. Differential transcriptional regulation of EGR-1 and Smad3/4 by GSK-3 isoforms was also confirmed using isoform-specific siRNA and dominant negative mutants in human neuroblastoma SH-SY5Y cells (Fig. 4C) and further suggests that the opposite effects of these two isoforms in regulating transcription factors are not unique to cortical neurons.FIGURE 4Confirmation of differential regulation of EGR-1 and Smad3/4 transcriptional activities. A, confirmation of EGR-1 transcriptional activation with luciferase reporter assay. Cortical neurons at the time of plating were co-transfected with either 4 μg of pEGR-1-Luc reporter construct and GSK-3 expression vectors (wild-type or mutant) or with 4 μg of a pEGR-1-Luc reporter construct followed by transfection with 80 nm siRNA mixture for GSK-3α or GSK-3β at 9 DIV. The luciferase activities of cell lysates were determined at 11 DIV. B, confirmation of Smad3/4 transcriptional activation with luciferase reporter assay. Cortical neurons were either co-transfected with 4 μg of pSmad3/4-Luc reporter construct and GSK-3 expression vectors and then treated with GSK-3 inhibitors (3 μm SB216763 or 10 μm SB415286) at 6 DIV for 5 days or transfected with 80 nm siRNA mixture for GSK-3α or GSK-3β at 9 DIV. The luciferase activities of cell lysates were determined at 11 DIV. C, confirmation of EGR-1 and Smad3/4 transcriptional regulation in SH-SY5Y human neuroblastoma cells. Cells were either co-transfected with reporter construct and GSK-3 expression vectors or co-transfected with reporter construct and human siRNA cocktails specific to either GSK-3 isoform at 9 DIV. The target sequences for those human-specific siRNA are: αP468, GTGGCTTACACGGACATCA; αP617, GCTGGACCACTGCAATATT; αP1132, CACCAACCCGGGAACAAAT; βP158, GTCAGCTATACAGACACTA; βP385, TCTGGTGCTGGACTATGTT; βP975, CTGCCCGACTAACACCACT. Luciferase activities of cell lysates were determined 48 h after transfection. Values shown represent means ± S.E. of % of control from three independent experiments. D, representative Western blotting analysis of protein levels of PAI-1 expression 48 h after siRNA transfection in cultured cortical neurons. β-actin was used as a protein-loading control. The immunoblot shown is representative of three experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The protein levels of plasminogen activator inhibitor type-1 (PAI-1), a Smad3/4-dependent TGF-β-regulated gene (16Buisson A. Lesne S. Docagne F. Ali C. Nicole O. MacKenzie E.T. Vivien D. Cell. Mol. Neurobiol. 2003; 23: 539-550Crossref PubMed Scopus (83) Google Scholar), was also differentially affected in cultured cortical neurons by GSK-3 isoform-specific siRNA gene silencing, similar to the effects on Smad3/4 signaling (Fig. 4D). Thus, PAI-1 protein levels were up-regulated by GSK-3α siRNA but down-regulated by GSK-3β siRNA (Fig. 4D), even though the effect of transactivation by GSK-3β protein depletion was small in cortical cells (Fig. 4B). In addition, the transcription factor serum-responsive element was detected to be differentially up-regulated by GSK-3α siRNA and GSK-3β siRNA (Fig. 3). Experiments are in progress to verify this effect by reporter analysis. The present study showed that inhibition of GSK-3α was more effective than that of GSK-3β in stimulating CREB- and NF-κB-dependent transcriptional activation. Moreover, we demonstrated differential changes in the transcriptional activation of two transcription factors, EGR-1 and Smad3/4, in response to either GSK-3α or GSK-3β silencing using simultaneous multiple transcription factor/DNA array technology. This is, to our knowledge, the first evidence demonstrating the transcriptional modulation of EGR-1 and Smad3/4 by GSK-3. CREB has an important neurophysiological role, as it regulates the expression of proteins critically involved in synaptic plasticity, neuronal development, and cell fate (reviewed in Refs. 17Silva A.J. Kogan J.H. Frankland P.W. Kida S. Annu. Rev. Neurosci. 1998; 21: 127-148Crossref PubMed Scopus (1209) Google Scholar, 18Shaywitz A.J. Greenberg M.E. Annu. Rev. Biochem. 1999; 568: 821-861Crossref Scopus (1787) Google Scholar). CREB is transcriptionally activated by phosphorylation at Ser-133, a process catalyzed by a number of protein kinases and necessary for GSK-3β-mediated phosphorylation of CREB at Ser-129 (19Fiol C.J. Williams J.S. Chou C.H. Wang Q.M. Roach P.J. Andrisani O.M. J. Biol. Chem. 1994; 26: 32187-32193Abstract Full Text PDF Google Scholar). Several studies have demonstrated that GSK-3β negatively regulates CREB activity (20Bullock B.P. Habener J.F. Biochemistry. 1998; 37: 3795-3809Crossref PubMed Scopus (136) Google Scholar, 21Fang X. Yu S.X. Lu Y. Bast Jr., R.C. Woodgett J.R. Mills G.B. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 11960-11965Crossref PubMed Scopus (642) Google Scholar, 22Grimes C.A. Jope R.S. J. Neurochem. 2001; 78: 1219-1232Crossref PubMed Scopus (340) Google Scholar). Our study not only supports this notion but also suggests that GSK-3α is more robust than GSK-3β in inhibiting CRE-responsive transcriptional regulation. There are conflicting results regarding the functional effects of GSK-3 on the phosphorylation of NF-κB, with some showing activation (12Hoeflich K.P. Luo J. Rubie E.A. Tsao M.S. Jin O. Woodgett J.R. Nature. 2000; 406: 86-90Crossref PubMed Scopus (1223) Google Scholar, 23Deng J. Xia W. Miller S.A. Wen Y. Wang H.Y. Hung M.C. Mol. Carcinog. 2004; 39: 139-146Crossref PubMed Scopus (86) Google Scholar) and others reporting inhibition (24Bournat J.C. Brown A.M. Soler A.P. J. Neurosci. Res. 2000; 61: 21-32Crossref PubMed Scopus (95) Google Scholar). It also should be noted that NF-κB activation is not always associated with survival proteins. A number of studies have demonstrated that NF-κB activation is linked to neuronal death induced by insults such as quinolinic acid excitotoxicity in the rat striatum (25Qin J.Z. Chaturvedi V. Denning M.F. Choubey D. Diaz M.O. Nickoloff B.J. J. Biol. Chem. 1999; 274: 37957-37964Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar) or cerebral ischemia in rodents (26Schneider A. Martin-Villalba A. Weih F. Vogel J. Wirth T. Schwaninger M. Nat. Med. 1999; 5: 554-559Crossref PubMed Scopus (42) Google Scholar). In addition, it has been reported that NF-κB is the only target known to be differentially regulated by two GSK-3 isoforms in a transgenic mouse model (12Hoeflich K.P. Luo J. Rubie E.A. Tsao M.S. Jin O. Woodgett J.R. Nature. 2000; 406: 86-90Crossref PubMed Scopus (1223) Google Scholar). The diversity of the cellular functions of NF-κB and the complexity of the regulation of NF-κB transcriptional activity by GSK-3 underscore the need for further studies to clarify the roles for GSK-3 isoforms in NF-κB regulation. Our results suggest transcription factors Smad3 and EGR-1 as prospective substrates for GSK-3. Detection of potential phosphorylation sites by GSK-3 on EGR-1 and Smad3 using NetPhosK 1.0 server (27Blom N. Sicheritz-Ponten T. Gupta R. Gammeltoft S. Brunak S. Proteomics. 2004; 4: 1633-1649Crossref PubMed Scopus (1501) Google Scholar) further suggests possible direct regulation of these two transcription factors by GSK-3 (data not shown). Nine potential GSK-3β consensus phosphorylation sites of EGR-1 were found to reside on two main domains that are highly rich in serine/threonine residues. These sites are conserved among human, rat, mouse, and frog and constitute substrate recognition sites for GSK-3β. With Smad3, we found one tyrosine and three serine phosphorylation sites conserved among human, rat, mouse, frog, and fruit fly. Whether these two transcription factors are direct GSK-3 substrates and how they are regulated differentially by GSK-3 are currently being explored in our laboratory. EGR-1, a zinc finger transcription factor, has been implicated in diverse biological functions, including cell growth, differentiation (28Harada T. Morooka T. Ogawa S. Nishida E. Nat. Cell Biol. 2001; 3: 453-459Crossref PubMed Scopus (281) Google Scholar, 29Lee J.H. Kim K.T. J. Neurochem. 2004; 91: 634-647Crossref PubMed Scopus (71) Google Scholar), and apoptosis (30Liu C. Rangnekar V.M. Adamson E. Mercola D. Cancer Gene Ther. 1998; 5: 3-28PubMed Google Scholar, 31Levkovitz Y. Baraban J.M. J. Neurosci. 2001; 21: 5893-5901Crossref PubMed Google Scholar, 32Pignatelli M. Luna-Medina R. Perez-Rendon A. Santos A. Perez-Castillo A. Biochem. J. 2003; 373: 739-746Crossref PubMed Scopus (59) Google Scholar). In cerebellar granule cells, the EGR-1 family of transcription factors plays a critical role in apoptosis by facilitating c-Jun activation (31Levkovitz Y. Baraban J.M. J. Neurosci. 2001; 21: 5893-5901Crossref PubMed Google Scholar). EGR-1 was also reported to promote p73-mediated apoptosis in N2A cells (32Pignatelli M. Luna-Medina R. Perez-Rendon A. Santos A. Perez-Castillo A. Biochem. 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Given that aberrant GSK-3 activity has been implicated in the pathophysiology of neurological and neuropsychiatric disorders, the development of isoform-specific inhibition or gene silencing seems to be essential for therapeutic intervention and a better understanding of the pathogenesis of these categories of diseases. We thank Connie Chen for technical assistance and Dr. Michael Rowe and Peter Leeds of the Section for critical reading of the manuscript. We also thank the National Institutes of Health Fellows Editorial Board for superb editorial assistance.