Phosphorylation Of cyclic-AMP Response Element-binding Protein Research Articles

Regulation of cyclic AMP response-element binding-protein (CREB) by Gq/11-protein-coupled receptors in human SH-SY5Y neuroblastoma cells

Human SH-SY5Y neuroblastoma cells have been used to investigate mechanisms involved in CREB phosphorylation after activation of two endogenously expressed Gq/11-protein-coupled receptors, the M3 muscarinic acetylcholine (mACh) and B2 bradykinin receptors. Stimulation with either methacholine or bradykinin resulted in maximal increases in CREB phosphorylation within 1min, with either a rapid subsequent decrease (bradykinin) to basal levels, or a sustained response (methacholine). Inhibitor studies were performed to assess the involvement of a number of potential kinases in signalling to CREB phosphorylation. Removal of extracellular Ca2+, inhibition of Ca2+/calmodulin-dependent protein kinase II and down-regulation of protein kinase C (PKC) resulted in reduced CREB phosphorylation after both M3 mACh and B2 bradykinin receptor activation. In contrast, inhibition of MEK1/2 by U0126 resulted in significantly reduced CREB phosphorylation levels after B2 bradykinin, but not M3 mACh receptor activation. In addition, we demonstrate that maintained phosphorylation of CREB is necessary for CRE-dependent gene transcription as the M3 mACh, but not the B2 bradykinin receptor activates both a recombinant CRE-dependent reporter gene, and the endogenous c-Fos gene. These data highlight the involvement of multiple, overlapping signalling pathways linking these endogenous Gq/11-coupled metabotropic receptors to CREB and emphasize the importance of the duration of signalling pathway activation in converting a CREB phosphorylation event into a significant change in transcriptional activity.

Differential Expression of Apoptotic Proteins Following Hypoxia-induced CREB Phosphorylation in the Cerebral Cortex of Newborn Piglets

The present study investigates the correlation between the hypoxia-induced phosphorylation of cyclic AMP response element binding protein and the expression of apoptotic proteins (proapoptotic proteins Bax and Bad and antiapoptotic proteins Bcl-2 and Bcl-xl) during hypoxia in the cerebral cortex of newborn piglets. Piglets were divided into normoxic (Nx) and hypoxic (Hx, FiO(2)=0.06 for 1 h) groups. Cerebral tissue hypoxia was documented by ATP and phosphocreatine (PCr) levels. Ser(133) phosphorylation of cyclic AMP response element binding (CREB) protein was determined by Western blot analysis using a specific anti-phosphorylated Ser(133)-CREB protein antibody. The expression of apoptotic proteins was determined by using specific anti-Bax, anti-Bad, anti-Bcl-2 and anti-Bcl-xl antibodies. ATP and PCr values (mumoles/g brain) in Hx were significantly different from Nx (ATP: 4.40 +/- 0.39 in Nx vs. 1.19 +/- 0.44 in Hx, P<0.05 vs. Nx; PCr: 3.60 +/- 0.40 in Nx vs. 0.70 +/- 0.31 in Hx, P<0.05 vs. Nx). Ser(133) phosphorylated CREB protein (OD x mm(2)) was 74.55 +/- 4.75 in Nx and 127.13 +/- 19.36 in Hx (P<0.05 vs. Nx). The expression of proapoptotic proteins Bax and Bad increased and strongly correlated with the increase in CREB protein phosphorylation (correlation coefficient r=0.82 and r=0.85, respectively). The expression of antiapoptotic proteins Bcl-2 and Bcl-xl did not show correlation with CREB protein phosphorylation. We conclude that cerebral hypoxia results in differential regulation of CREB protein-mediated expression of proapoptotic and antiapoptotic proteins in the cerebral cortex of newborn piglets. We propose that the increased expression of proapoptotic vs antiapoptotic genes will lead to an increased potential for apoptotic programmed cell death in the Hx newborn brain.

Protein O-N-Acetylglucosaminylation Modulates Promoter Activities of Cyclic AMP Response Element and Activator Protein 1 and Enhances E-Selectin Expression on HuH-7 Human Hepatoma Cells

High glucose accelerates O-N-acetylglucosaminylation (O-GlcNAcylation) of proteins and causes diabetic complications. In the present study, we found that treatment of HuH-7 human hepatoma cells with high glucose or the protein O-N-acetylglucosaminidase (O-GlcNAcase) inhibitor O-(2-acetoamide-2-deoxy-D-glucopyranosylidene)amino-N-phenylcarbamate (PUGNAc) increased the cell surface expression of E-selectin. A dual luciferase reporter assay indicated that high glucose and PUGNAc suppressed promoter activities of the cyclic AMP response element (CRE) and enhanced those of activator protein 1 (AP-1). Enhanced CRE promoter activities in HuH-7 cells treated with dibutyryl cAMP or co-transfected with a protein kinase A expression vector pFC-PKA that enhances the phosphorylation of CRE binding protein (CREB) were suppressed by PUGNAc. In contrast, PUGNAc further increased the enhanced AP-1 promoter activity in cells transfected with a mitogen-activated protein kinase kinase kinase expression vector pFC-MEKK that enhances c-Jun phosphorylation. Immuno-blotting using an anti-O-GlcNAc antibody revealed that high glucose and PUGNAc accelerated protein O-GlcNAcylation and that there were substantial differences in the O-GlcNAcylated proteins in the cytoplasmic and nuclear fractions. In addition, PUGNAc increased the nuclear import of O-GlcNAcylated CREB. These results suggest that protein O-GlcNAcylation modulates the promoter activities of E-selectin gene, suppression of CRE and enhancement of AP-1, and enhances E-selectin protein expression on hepatocytes.

Nuclear Ca++-influx, Ca++/Calmodulin-Dependent Protein Kinase IV Activity and CREB Protein Phosphorylation during Post-Hypoxic Reoxygenation in Neuronal Nuclei of Newborn Piglets: The Role of Nitric Oxide

The present study tests the hypothesis that post-hypoxic reoxygenation results in an nitric oxide (NO)-mediated increase in nuclear Ca(++)-influx, increased calmodulin kinase (CaM kinase) IV activity, and increased Ser(133) phosphorylation of cyclic AMP response element binding (CREB) protein in neuronal nuclei of the cerebral cortex of newborn piglets. Piglets were divided into normoxic (Nx), hypoxic (Hx, FiO(2) = 0.07 for 1 h), hypoxic with 6 h reoxygenation (Hx + reox), and Hx + reox injected with 7-nitroindazole sodium salt (7-NINA), a nNOS inhibitor, immediately after hypoxia (Hx + 7-NINA). Cerebral tissue hypoxia was documented by ATP and phosphocreatine (PCr) levels. Nuclear Ca(++)-influx was determined using (45)Ca(++) and CaM kinase IV activity determined by (33)P-incorporation into syntide-2. Ser(133) phosphorylation of CREB protein was determined by Western blot analysis using a specific anti-phosphorylated Ser(133)-CREB protein antibody. ATP and PCr values in Hx, Hx + reox, and Hx + 7-NINA were significantly different from Nx (P < 0.05 versus Nx). Ca(++)-influx (pmoles/mg protein/min) was 3.79 +/- 0.91 in Nx; 11.81 +/- 2.54 in Hx (P < 0.05 versus Nx), 16.55 +/- 3.55 in Hx + reox (P < 0.05 versus Nx), and 12.40 +/- 2.93 in Hx + 7-NINA (P = NS versus Hx). CaM kinase IV activity (pmoles/mg protein/min) was 1,220 +/- 76 in Nx, 2,403 +/- 254 in Hx (P < 0.05 versus Nx), 1,971 +/- 147 in Hx + reox (P < 0.05 versus Hx), and 1,939 +/- 125 Hx + 7-NINA (P < 0.05 versus Hx). Ser(133) phosphorylated CREB protein expression (OD x mm(2)) was 87 +/- 2 in Nx, 203 +/- 24 in Hx (P < 0.05 versus Nx), 186 +/- 23 in Hx + reox (P < 0.05 Nx, P = NS versus Hx), and 128 +/- 10 in Hx + 7-NINA (P < 0.05 versus Hx and Hx + reox). The results show that post-Hx administration of 7-NINA prevents the increased nuclear Ca(++)-influx and CREB protein phosphorylation at Ser(133) during reox. We conclude that post-Hx increase in nuclear Ca(++)-influx leading to increased phosphorylation of CREB protein is mediated by NO derived from nNOS. However, hypoxia-induced increase in CaM Kinase IV activity decreased during the post-Hx reox. We propose that hypoxia-induced increase in CaM Kinase IV activity leads to increased phosphorylation of CREB protein and transcription of proapoptotic genes during post-Hx reox resulting in Hx neuronal death.

γ-Hydroxybutyrate induces cyclic AMP-responsive element-binding protein phosphorylation in mouse hippocampus: An involvement of GABAB receptors and cAMP-dependent protein kinase activation

γ-Hydroxybutyrate is a widely used recreational drug. Its abuse has been associated with cognitive impairments and development of tolerance and dependence. However, the neural mechanisms underlying these effects remain unclear. In the present study we investigated the possible cellular signaling mechanisms that might mediate γ-hydroxybutyrate’s action. Acute administration of γ-hydroxybutyrate (500 mg/kg, i.p.) was found to cause a rapid and long-lasting increase in the phosphorylation level of the cAMP-responsive element-binding protein in mouse (C57/BL6) hippocampus. Pretreatment with the specific GABAB receptor antagonist [3-[1-(R)-[(3-cyclohexylmethyl)hydroxyphosphinyl]-2-(S)-hydroxy-propyl]amino]ethyl]-benzoic acid (20 mg/kg, i.p.) prevented the action of γ-hydroxybutyrate, confirming a GABAB receptor-mediated mechanism. In addition, acute γ-hydroxybutyrate administration induced a significant increase in cytosolic cAMP-dependent protein kinase activity in the hippocampus, and pretreatment with the cAMP-dependent protein kinase inhibitor H-89 could prevent the effect of γ-hydroxybutyrate on cAMP-responsive element-binding protein phosphorylation, indicating a direct involvement of cAMP-dependent protein kinase in γ-hydroxybutyrate-induced cAMP-responsive element-binding protein phosphorylation. On the other hand, the increased expression of phosphorylated cAMP-responsive element-binding protein was not observed in the hippocampus of mice subjected to repeated γ-hydroxybutyrate exposure, suggesting the development of a γ-hydroxybutyrate-induced desensitization of the signaling pathway leading to cAMP-responsive element-binding protein activation. Since cAMP-responsive element-binding protein activation has been implicated in a variety of neural plasticities, our findings may have revealed a new mechanism underlying γ-hydroxybutyrate-induced neuroadaptations.

Single cell analysis of activity-dependent cyclic AMP-responsive element-binding protein phosphorylation during long-lasting long-term potentiation in area CA1 of mature rat hippocampal-organotypic cultures

Phosphorylation of the transcription factor cyclic AMP (cAMP)-response element-binding protein (CREB) has been implicated in long-term synaptic plasticity and memory, and its activation has been proposed to be required for the maintenance of long-term potentiation (LTP). The previously described temporal dynamics of CREB phosphorylation during the maintenance of LTP showed differences between experimental models. In the present study the level of CREB phosphorylation was evaluated in organotypic hippocampal slices from young adult rats (P25-30) after long-lasting LTP was induced. Immunohistochemistry and confocal imaging were used to determine the ratio between non-phosphorylated and phosphorylated CREB at a single cell resolution, revealing not only the temporal dynamics but also the extent of CREB phosphorylation. The activation of CREB after LTP-induction was compared with cAMP-activation after bath application of forskolin. An increase in cAMP by forskolin resulted in a persistent, uniform increase of the phosphorylated CREB (pCREB/CREB immunofluorescence ratio) in all hippocampal principal neurons. In contrast, the induction of long-lasting LTP in CA1 was accompanied by a local increase in the pCREB/CREB ratio. Both CREB activation and LTP induction in mature cultured slices required N-methyl- d-aspartate (NMDA) receptor activation. CREB phosphorylation continued to increase for 4 h during LTP maintenance. This sustained activation is in contrast to previous observations in acutely prepared slices and supports the hypothesis that CREB plays an important role during the late phases of LTP.

Identification of calcium-dependent and -independent signaling pathways involved in polychlorinated biphenyl-induced cyclic AMP-responsive element-binding protein phosphorylation in developing cortical neurons

Cyclic AMP (cAMP)-responsive element-binding protein (CREB) is a transcription factor important in developing nervous system cells and is activated by a variety of signaling molecules. Aroclor 1254 (A1254), a polychlorinated biphenyl mixture, perturbs Ca 2+ homeostasis and increases CREB phosphorylation in rat neonatal cortical cell cultures in a time- and concentration-dependent manner. The present experiments determined that the cell type responding to A1254 with Ca 2+ increases and phosphorylated CREB (phospho-CREB) was predominantly of neuronal morphology and microtubule-associated protein (MAP2)-positive phenotype. Similarly, glutamate (100 μM) increased phospho-CREB immunoreactivity selectively in MAP2-immunopositive cells. Using Western blotting and immunocytochemical techniques, we identified key signal transduction pathways operative in phosphorylating CREB in cortical cell cultures and examined their participation in 3 ppm A1254-induced CREB activation. Cortical cultures treated with glutamate, forskolin or the phorbol ester phorbol 12-myristate 13-acetate exhibited robust increases in phospho-CREB. Tetrodotoxin (1 μM) completely inhibited CREB phosphorylation by A1254, suggesting that synaptic activity is involved in A1254-induced CREB activation. Buffering [Ca 2+] i with bis(2-aminophenoxy)ethane- N, N, N′, N′-tetraacetic acid tetrakis(acetoxymethyl) ester in the absence of extracellular Ca 2+ partially inhibited A1254-induced CREB phosphorylation. Inhibition of mitogen-activated protein kinase (10 μM U0126) or protein kinase C (PKC; bisindoylmaleimide, 5 μM) activation did not inhibit A1254-induced CREB phosphorylation. By contrast, inhibition of protein kinase A (PKA) with 100 μM PKA inhibitor peptide, PKI, blocked A1254-induced CREB phosphorylation. Thus, we examined whether A1254 activates PKA by increasing cAMP; 10 μM forskolin, but not A1254, elevated intracellular cAMP levels. These results indicate that in neocortical cells in culture, CREB phosphorylation occurs via Ca 2+-, PKA-, and PKC-dependent pathways. Furthermore, A1254-induced CREB phosphorylation occurs predominantly in neurons, is dependent on synaptic activity and mediated by Ca 2+- and PKA-dependent pathways.

Convergence of Multiple Protein Kinase Pathways of Angiotensin II Type 1 Receptor on cAMP Response Element-Binding Protein (CREB) in Rat Vascular Smooth Muscle Cells

P163 We previously reported a critical role of cyclic AMP response element (CRE) of interleukin-6 gene promoter in the induction of interleukin-6 gene expression by angiotensin II (AngII). We examined signaling pathways that are responsible for AngII-induced phosphorylation of CRE binding protein (CREB) at serine 133 that is a critical marker for the activation in rat vascular smooth muscle cells (VSMC). We performed Western blot analysis using an antibody against the phosphorylated form of CREB. AngII time-dependently induced phosphorylation of CREB with a peak at 5 minutes. The phosphorylation was dose-dependent. The AngII-induced phosphorylation of CREB was blocked by CV11974, an AngII type I receptor (AT1-R) antagonist but not by PD123319, an antagonist of Ang II type 2 receptor, suggesting that AT1-R mediates the phosphorylation of CREB. Inhibition of extracellular signal-regulated protein kinase (ERK) by PD98059 or inhibition of p38 mitogen activated protein kinase (MAPK) by SB203580 partially inhibited AngII-induced CREB phosphorylation. A protein kinase A (PKA) inhibitor, H89, also partially suppressed AngII-induced CREB phosphorylation. However, inhibition of calmodulin dependent kinase II or phosphatydilinositol 3 kinase did not affect Ang II-induced CREB phosphorylation. Transcriptional activation as measured by a CRE/luciferase reporter was increased by 1.7-fold by AngII stimulation. Infection of adenovirus vector expressing dominant negative form of CREB, which the serine residue at 133 is replaced with alanine to VSMC partially suppressed Ang II-induced thymidine incorporation. These findings suggest that AngII activates multiple protein kinase pathways involving two MAPK pathways and PKA, all of which contribute to the activation of CREB. And CREB-dependent gene transcription plays an important role for Ang II-induced mitogenesis. Activation of CREB-dependent gene transcription may play important roles in cardiovascular remodeling process.

Convergence of Multiple Protein Kinase Pathways of Angiotensin II Type 1 Receptor on cAMP Response Element-Binding Protein (CREB) in Rat Vascular Smooth Muscle Cells

P163 We previously reported a critical role of cyclic AMP response element (CRE) of interleukin-6 gene promoter in the induction of interleukin-6 gene expression by angiotensin II (AngII). We examined signaling pathways that are responsible for AngII-induced phosphorylation of CRE binding protein (CREB) at serine 133 that is a critical marker for the activation in rat vascular smooth muscle cells (VSMC). We performed Western blot analysis using an antibody against the phosphorylated form of CREB. AngII time-dependently induced phosphorylation of CREB with a peak at 5 minutes. The phosphorylation was dose-dependent. The AngII-induced phosphorylation of CREB was blocked by CV11974, an AngII type I receptor (AT1-R) antagonist but not by PD123319, an antagonist of Ang II type 2 receptor, suggesting that AT1-R mediates the phosphorylation of CREB. Inhibition of extracellular signal-regulated protein kinase (ERK) by PD98059 or inhibition of p38 mitogen activated protein kinase (MAPK) by SB203580 partially inhibited AngII-induced CREB phosphorylation. A protein kinase A (PKA) inhibitor, H89, also partially suppressed AngII-induced CREB phosphorylation. However, inhibition of calmodulin dependent kinase II or phosphatydilinositol 3 kinase did not affect Ang II-induced CREB phosphorylation. Transcriptional activation as measured by a CRE/luciferase reporter was increased by 1.7-fold by AngII stimulation. Infection of adenovirus vector expressing dominant negative form of CREB, which the serine residue at 133 is replaced with alanine to VSMC partially suppressed Ang II-induced thymidine incorporation. These findings suggest that AngII activates multiple protein kinase pathways involving two MAPK pathways and PKA, all of which contribute to the activation of CREB. And CREB-dependent gene transcription plays an important role for Ang II-induced mitogenesis. Activation of CREB-dependent gene transcription may play important roles in cardiovascular remodeling process.

Differential signaling of glial cell line-derived neurotrophic factor and brain-derived neurotrophic factor in cultured ventral mesencephalic neurons

In the ventral mesencephalon, two neurotrophic factors, brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor, have been shown previously to have similar effects on the survival of dopaminergic neurons. Here, we compared the signaling mechanisms for brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor, focusing on the mitogen-associated protein kinase and the transcription factor cyclic-AMP responsive element-binding protein. Double-staining experiments indicated that many neurons co-expressed the receptors for glial cell line-derived neurotrophic factor and brain-derived neurotrophic factor, c-RET and TrkB, suggesting that they are responsive to both brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor. Although both brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor induced a rapid phosphorylation of mitogen-associated protein kinase and cyclic-AMP responsive element-binding protein, there were significant differences in the kinetics and pharmacology of the phosphorylation. The phosphorylation of mitogen-associated protein kinase by glial cell line-derived neurotrophic factor was transient; within 2 h, the level of mitogen-associated protein kinase phosphorylation returned to baseline. In contrast, the effect of brain-derived neurotrophic factor was long lasting; the mitogen-associated protein kinase remained phosphorylated for up to 4 h after brain-derived neurotrophic factor treatment. PD098059, a specific inhibitor for mitogen-associated protein kinase kinase, completely blocked the glial cell line-derived neurotrophic factor signaling through mitogen-associated protein kinase, but had no effect on brain-derived neurotrophic factor-induced mitogen-associated protein kinase phosphorylation. Both brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor induced the phosphorylation of cyclic-AMP responsive element-binding protein in the nuclei of ventral mesencephalon neurons. However, PD098059 blocked the cyclic-AMP responsive element-binding protein phosphorylation induced by glial cell line-derived neurotrophic factor, but not that by brain-derived neurotrophic factor. These results indicate that, although both brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor act on ventral mesencephalon neurons, the two factors have different signaling mechanisms, which may mediate their distinctive biological functions.

Effects of aging on light-induced phase-shifting of circadian behavioral rhythms, fos expression and CREB phosphorylation in the hamster suprachiasmatic nucleus.

Aging is associated with a variety of alterations in circadian rhythms, including changes in the response to environmental stimuli. The underlying causes for these age-related changes in the circadian system remain unknown. Recent studies have demonstrated that light induces the expression of Fos and phosphorylation of the cyclic-AMP response element-binding protein in the rodent suprachiasmatic nuclei, the location of a master circadian pacemaker in mammals, suggesting that these transcription factors may mediate the effects of light on the circadian clock. The purpose of this study was to determine the effects of aging upon light-induced phase-shifting of circadian locomotor activity rhythms, Fos protein expression and cyclic-AMP response element-binding protein phosphorylation in the suprachiasmatic nuclei. Young (three to four months) and old (18-22 months) male golden hamsters free-running in constant darkness were exposed to 5-min monochromatic light pulses of different irradiance levels, at circadian time 19, after which either steady-state phase shifts of locomotor activity rhythms were measured, or else immunocytochemistry for Fos or for phospho-cyclic-AMP response element-binding protein was performed. Old hamsters were approximately 20 times less sensitive to the phase-shifting effects of light on the activity rhythm, and the photic irradiance threshold for Fos-like immunoreactivity induction in the suprachiasmatic nuclei was elevated when compared to young animals. Aging was also associated with a deficit in cyclic-AMP response element-binding protein phosphorylation by light. These data indicate that there are dramatic changes in light-activated molecular responses in the suprachiasmatic nuclei of old hamsters, and suggest that these molecular changes may underlie age-related changes in the effects of light on the circadian clock system.