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. 2001 Sep;78(6):1219-32.
doi: 10.1046/j.1471-4159.2001.00495.x.

CREB DNA binding activity is inhibited by glycogen synthase kinase-3 beta and facilitated by lithium

C A Grimes  1 , R S Jope
Affiliations

CREB DNA binding activity is inhibited by glycogen synthase kinase-3 beta and facilitated by lithium

C A Grimes et al. J Neurochem. 2001 Sep.

Abstract

The regulatory influences of glycogen synthase kinase-3 beta (GSK3 beta) and lithium on the activity of cyclic AMP response element binding protein (CREB) were examined in human neuroblastoma SH-SY5Y cells. Activation of Akt (protein kinase B) with serum-increased phospho-serine-9-GSK3 beta (the inactive form of the enzyme), inhibited GSK3 beta activity, and increased CREB DNA binding activity. Inhibition of GSK3 beta by another paradigm, treatment with the selective inhibitor lithium, also increased CREB DNA binding activity. The inhibitory regulation of CREB DNA binding activity by GSK3 beta also was evident in differentiated SH-SY5Y cells, indicating that this regulatory interaction is maintained in non-proliferating cells. These results demonstrate that inhibition of GSK3 beta by serine-9 phosphorylation or directly by lithium increases CREB activation. Conversely, overexpression of active GSK3 beta to 3.5-fold the normal levels completely blocked increases in CREB DNA binding activity induced by epidermal growth factor, insulin-like growth factor-1, forskolin, and cyclic AMP. The inhibitory effects due to overexpressed GSK3 beta were reversed by treatment with lithium and with another GSK 3beta inhibitor, sodium valproate. Overall, these results demonstrate that GSK3 beta inhibits, and lithium enhances, CREB activation.

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Figures

Fig. 1
Fig. 1
Serum decreases GSK3β activity and increases CREB DNA binding activity in SH-SY5Y cells. SH-SY5Y cells were serum-starved for 24 h unless stated otherwise. (a) Phospho-serine-473- Akt and total levels of Akt were measured in lysates from cells incubated without or with serum by immunoblot analysis. Quantitative values are expressed as the percent of phosphorylated Akt levels in serum-starved cells. Mean ± SEM, n = 4. (b) Phosphorylated serine-9-GSK3β levels were measured in lysates from cells incubated without or with serum by immunoprecipitating phosphoserine-9-GSK3β and immunoblotting GSK3β. Quantitative values are expressed as the percent of phosphorylated Serine-9-GSK3β in serum-starved cells. Mean ± SEM, n = 3. (c) GSK3β activity was measured by immunoprecipitating GSK3β from cells incubated without or with serum, incubating the immunoprecipitated GSK3β with recombinant human tau and [32P]ATP, and measuring the phosphorylation of tau, as described in Materials and methods. Quantitative values are expressed as the percent of GSK3β activity in serum-starved cells. Mean ± SEM, n = 4. (d) CREB DNA binding activity was measured in nuclear extracts from cells incubated without or with serum and treated without or with lithium (1 mm; 1 h). Quantitative values are expressed as the percent of CREB DNA binding activity in serum-starved cells. Mean ± SEM, n = 4.
Fig. 2
Fig. 2
Supershift analysis of CREB DNA binding activity in SH-SY5Y cells. CREB DNA binding activity was measured by EMSA in nuclear extracts prepared from serum-starved (24 h) SH-SY5Y cells incubated without (basal) or with lithium (1 mm; 1 h). For supershift (ss) analysis, nuclear extracts were incubated with antibody (0.5 µg) to CREB, Fos, or EGR-1 prior to incubation with radiolabeled oligonucleotide.
Fig. 3
Fig. 3
Lithium stimulates CREB DNA binding activity following B27 supplement-withdrawal in differentiated SH-SY5Y cells. SH-SY5Y cells were differentiated to a neuronal-like phenotype by maintaining cells in Neurobasal medium, supplemented with B27, for 3.5 days. B27 supplement was removed 24 h prior to preparation of nuclear extracts. CREB DNA binding activity was measured by EMSA in nuclear extracts prepared from serum-containing or serum-starved cells treated without or with lithium (1 mm; 1 h).
Fig. 4
Fig. 4
EGF, IGF-1, and forskolin stimulate CREB DNA binding activity in serum-starved SH-SY5Y cells. (a) The association of phospho-serine-133-CREB with CBP was measured by immunoprecipitating CBP from serum-starved (24 h) wild-type SH-SY5Y cells treated without or with lithium (10 mm; 30 min), EGF (50 ng/mL; 30 min), or IGF-1 (50 ng/mL; 30 min) and immunoblotting phospho-serine-133-CREB, as described in Materials and methods. (b) CREB DNA binding activity was measured by EMSA in nuclear extracts prepared from SH-SY5Y cells incubated without serum (24 h) and treated without (basal) or with EGF (50 ng/mL; 1 h), IGF-1 (50 ng/mL; 1 h), or forskolin (Fsk; 10 µm; 1 h) following pre-incubation without or with lithium (Li; 1 mm; 1 h). Quantitative values are expressed as the percent of basal CREB DNA binding activity in serum-starved cells. Mean ± SEM, n = 4–6. (c) Supershift (ss) analysis of CREB DNA binding activity. Nuclear extracts prepared from serum-starved (24 h) cells treated without (basal) or with EGF (50 ng/mL; 1 h), IGF-1 (50 ng/mL; 1 h), or forskolin (10 µm; 1 h) were incubated with antibody to CREB (0.5 µg).
Fig. 5
Fig. 5
Stimulation of CREB DNA binding activity is impaired in SH-SY5Y cells overexpressing GSK3β. (a) Cell lysates from wildtype SH-SY5Y cells (wt), vector-transfected SH-SY5Y cells (Vec), and four cell lines stably overexpressing HA-GSK3β (1, 3, 7, and 9) were immunoblotted for GSK3β. (b) Serum-starved (24 h) wild-type SH-SY5Y cells and GSK3β-overexpressing cells were treated without (basal) or with EGF (50 ng/mL; 1 h) or IGF-1 (50 ng/mL; 1 h) and lysates were immunoblotted for phospho-serine-473-Akt. (c) Serum-starved (24 h) wild-type SH-SY5Y cells and GSK3β-overexpressing cells were treated without (basal) or with EGF (50 ng/mL; 1 h), IGF-1 (50 ng/mL; 1 h), or lithium (1 mm; 1 h) and lysates were immunoblotted for phospho-serine-133-CREB. (d) The association of phospho-serine-133-CREB with CBP was measured by immunoprecipitating CBP from serum-starved (24 h) GSK3β-overexpressing SH-SY5Y cells treated without or with lithium (20 mm; 30 min), EGF (50 ng/mL; 30 min), or IGF-1 (50 ng/mL; 30 min) and immunoblotting phospho-serine-133-CREB, as described in the Materials and methods. (e) A representative CREB EMSA is shown obtained with GSK3β-overexpressing cells and quantitative data is shown of CREB DNA binding activity in nuclear extracts prepared from wild-type and GSK3β-overexpressing cells (cell lines 1, 3, 7, and 9) incubated without serum (24 h) and treated without (basal) or with EGF (50 ng/mL; 1 h), IGF-1 (50 ng/mL; 1 h), or forskolin (Fsk; 10µm; 1 h). Quantitative values are expressed as the percent of basal CREB DNA binding activity in serum-starved cells. Mean ± SEM, n = 4–6. (f) EGR-1 DNA binding activity was measured by EMSA in nuclear extracts prepared from wild-type and GSK3β-overexpressing cells incubated without serum (24 h) and treated without (basal) or with EGF (50 ng/mL; 1 h) or IGF-1 (50 ng/mL; 1 h). ns signifies non-specific binding.
Fig. 6
Fig. 6
Growth factor stimulation of CREB DNA binding activity is blocked at multiple time points in SH-SY5Y cells overexpressing GSK3β. (a) CREB DNA binding activity was measured in nuclear extracts prepared from wild-type, vector-transfected, and HAGSK3β-overexpressing SH-SY5Y cells incubated without serum (24 h) and treated without or with EGF (50 ng/mL) or IGF-1 (50 ng/ mL). Quantitative values are expressed as the percent of basal CREB DNA binding activity in serum-starved cells. Mean ± SEM, n = 3–4. (b) GSK3β activity was measured by immunoprecipitating GSK3β, incubating the immunoprecipitated GSK3β with recombinant human tau and [32P]ATP, and measuring the phosphorylation of tau, as described in Materials and methods, in wild-type (wt) cells, or in HA-GSK3β-overexpressing cells (HA-GSK3β) incubated without serum (24 h) and treated without (basal) or with EGF (50 ng/mL; 1 h) or IGF-1 (50 ng/mL; 1 h).
Fig. 7
Fig. 7
Lithium concentration-dependently inhibits GSK3β activity. GSK3β activity was measured by immunoprecipitating GSK3β from serum-starved (24 h) wild-type (wt) and GSK3β-overexpressing SH-SY5Y cells, incubating the immunoprecipitated GSK3β with recombinant human tau, [32P]ATP, and lithium, and measuring the phosphorylation of tau, as described in Materials and methods. Shown is a representative autoradiograph from an experiment with GSK3β-overexpressing cells, and quantitative values from both cell types expressed as the percent of GSK3β activity in the respective celltype in the absence of lithium, and values from GSK3β-overexpressing cells also are shown expressed as the percent of GSK3β activity in wild-type cells. Mean ± SEM, n = 3 experiments.
Fig. 8
Fig. 8
Lithium restores growth factor stimulation of CREB DNA binding activity in SH-SY5Y cells overexpressing GSK3β. (a) CREB DNA binding activity was measured by EMSA in nuclear extracts prepared from GSK3β-overexpressing cells incubated without serum (24 h) and treated without or with EGF (50 ng/mL; 1 h), or IGF-1 (50 ng/mL; 1 h) alone or following pre-incubation with lithium (Li; 20 mm; 1 h). (b) Quantitative values are expressed as the percent of basal CREB DNA binding activity in serum-starved GSK3β-overexpressing cells. Mean ± SEM, n = 4–6. (c) Nuclear extracts prepared from GSK3β-overexpressing cells incubated without serum (24 h) and treated without or with EGF (50 ng/mL; 1 h), or IGF-1 (50 ng/mL; 1 h) alone or following pre-incubation with lithium (Li; 20 mm; 1 h) were immunoblotted for total levels of CREB.
Fig. 9
Fig. 9
Lithium restores cyclic AMP stimulation of CREB DNA binding activity in SH-SY5Y cells overexpressing GSK3β. CREB DNA binding activity was measured by EMSA in nuclear extracts prepared from wild-type (wt) and GSK3β-overexpressing cells incubated without serum (24 h) and treated without (basal) or with diBcAMP (100 µm; 1 h) alone or following pre-incubation with lithium (20 mm; 1 h). Quantitative values are expressed as the percent of basal CREB DNA binding activity in serum-starved cells. Mean ± SEM, n = 2–3.
Fig. 10
Fig. 10
Valproate concentration-dependently inhibits GSK3β activity and restores growth factor and cyclic AMP stimulation of CREB DNA binding activity in SH-SY5Y cells overexpressing GSK3β. (A) GSK3β activity was measured by immunoprecipitating GSK3β from serum-starved (24 h) GSK3β-overexpressing SH-SY5Y cells, incubating the immunoprecipitated GSK3β with recombinant human tau, [32P]ATP, and valproate, and measuring the phosphorylation of tau, as described in Materials and methods. Quantitative values are expressed as the percent of GSK3β activity in serum-starved wildtype SH-SY5Y cells. Mean ± SEM, n = 3 experiments. (b) CREB DNA binding activity was measured by EMSA in nuclear extracts prepared from GSK3β-overexpressing cells incubated without serum (24 h) and treated without or with EGF (50 ng/mL; 1 h), IGF-1 (50 ng/mL; 1 h), or diBcAMP (100 βM; 1 h) alone or following preincubation with sodium valproate (10 mm; 1 h).
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