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PubMed1996

Targeting of the CREB gene leads to up-regulation of a novel CREB mRNA isoform.

To define the role of cAMP signaling in gene control, we have generated mice with a mutation in the cAMP response element binding protein (CREB) gene. Mice carrying this mutation are viable but show an impairment in memory consolidation. In further analysis of these mice, we have found an up-regulation of a CREB isoform that has not been described previously . The new isoform, termed CREB beta, has nearly the same transactivation potential as the other CREB isoforms and is expressed ubiquitously. The up-regulation appears to be due to an increase in alternative splicing or mRNA stability, but not to an increase in transcriptional rate. Due to the relatively low levels of expression in all tissues, the role of this isoform is likely to be minor in the wild-type mouse. However, its dramatic up-regulation in the mutant mouse, together with the specific deficiencies recently observed in these mice, suggest that it has a very specific role in compensating for CREB alpha and delta in some, but not all, areas where CREB function has been implicated. Together with the up-regulation of the cAMP response element modulator protein (CREM) mRNA and protein levels demonstrated previously in CREB mutant mice, we suggest that the up-regulation of CREB beta may also contribute to compensation within the CREB/ATF family of transcription factors, when CREB delta and CREB alpha are absent.

Alternative Splicing↗
PubMed2000

p53 recruitment of CREB binding protein mediated through phosphorylated CREB: a novel pathway of tumor suppressor regulation.

CREB binding protein (CBP) is a 270-kDa nuclear protein required for activated transcription of a large number of cellular genes. Although CBP was originally discovered through its interaction with phosphorylated CREB (pCREB), it is utilized by a multitude of cellular transcription factors and viral oncoproteins. Both CREB and the tumor suppressor p53 have been shown to directly interact with the KIX domain of CBP. Although coactivator competition is an emerging theme in transcriptional regulation, we have made the fortuitous observation that protein kinase A-phosphorylated CREB strongly enhances p53 association with KIX. Phosphorylated CREB also facilitates interaction of a p53 mutant, defective for KIX binding, indicating that CREB functions in a novel way to bridge p53 and the coactivator. This is accomplished through direct interaction between the bZIP domain of CREB and the amino terminus of p53; a protein-protein interaction that is also detected in vivo. Consistent with our biochemical observations, we show that stimulation of the intracellular cyclic AMP (cAMP) pathway, which leads to CREB phosphorylation, strongly enhances both the transcriptional activation and apoptotic properties of p53. We propose that phosphorylated CREB mediates recruitment of CBP to p53-responsive promoters through direct interaction with p53. These observations provide evidence for a novel pathway that integrates cAMP signaling and p53 transcriptional activity.

CREB-Binding Protein↗
PubMed2001

Enhanced CREB phosphorylation in immature dentate gyrus granule cells precedes neurotrophin expression and indicates a specific role of CREB in granule cell differentiation.

Differentiation and maturation of dentate gyrus granule cells requires coordinated interactions of numerous processes. These must be regulated by protein factors capable of integrating signals mediated through diverse signalling pathways. Such integrators of inter and intracellular physiological stimuli include the cAMP-response element binding protein (CREB), a leucine-zipper class transcription factor that is activated through phosphorylation. Neuronal activity and neurotrophic factors, known to be involved in granule cell differentiation, are major physiologic regulators of CREB function. To examine whether CREB may play a role in governing coordinated gene transcription during granule cell differentiation, we determined the spatial and temporal profiles of phosphorylated (activated) CREB throughout postnatal development in immature rat hippocampus. We demonstrate that CREB activation is confined to discrete, early stages of granule cell differentiation. In addition, CREB phosphorylation occurs prior to expression of the neurotrophins BDNF and NT-3. These data indicate that in a signal transduction cascade connecting CREB and neurotrophins in the process of granule cell maturation, CREB is located upstream of neurotrophins. Importantly, CREB may be a critical component of the machinery regulating the coordinated transcription of genes contributing to the differentiation of granule cells and their integration into the dentate gyrus network.

Animals↗
PubMed1994

Simian virus 40 small tumor antigen inhibits dephosphorylation of protein kinase A-phosphorylated CREB and regulates CREB transcriptional stimulation.

We report that the small tumor (small-t) antigen of simian virus 40 (SV40) forms complexes with nuclear protein phosphatase 2A (PP2A) and regulates the phosphorylation and transcriptional transactivation function of the cyclic AMP (cAMP)-regulatory element binding protein (CREB). PP2A coimmunoprecipitated with small t from nuclear extracts from HepG2 cells expressing small t or from rat liver nuclear extracts to which recombinant small t was added. Protein phosphatase 1 was not detected in small-t immunoprecipitates. In HepG2 cells expressing small t, dibutyryl-cAMP (Bt2cAMP) stimulated the phosphorylation of CREB 65-fold, whereas CREB phosphorylation was stimulated only 5- to 8-fold by Bt2cAMP in cells not expressing small t. Small t also inhibited the dephosphorylation of cAMP-dependent protein kinase (PKA)-phosphorylated CREB in rat liver nuclear extracts. In cells expressing small t, Bt2cAMP-stimulated transcription from the phosphoenolpyruvate carboxykinase (PEPCK) gene promoter was enhanced over the level of transcription from the PEPCK promoter in cells not expressing small t. Small t also enhanced Bt2cAMP-stimulated transcription from a Gal4-responsive promoter in cells expressing a chimeric protein containing the Gal4 DNA-binding domain linked to the CREB transactivation domain. However, small t did not stimulate transcription either from a 5' deletion mutant of the PEPCK promoter that is not able to bind CREB or from the Gal4-responsive promoter in the absence of the Gal4-CREB protein. These data suggest that small t enhances Bt2cAMP-stimulated gene transcription by inhibiting the dephosphorylation of PKA-phosphorylated CREB by nuclear PP2A. These findings support previous observations that nuclear PP2A is the primary phosphatase that dephosphorylates PKA-phosphorylated CREB.

Animals↗
PubMed1995

An inactivating point mutation demonstrates that interaction of cAMP response element binding protein (CREB) with the CREB binding protein is not sufficient for transcriptional activation.

The cAMP response element binding protein (CREB) mediates transcriptional activation in response to the cAMP signaling pathway. Several recent studies have suggested that phosphorylation-dependent interaction of CREB with a co-activator designated CREB binding protein (CBP) is a crucial step in mediating transcriptional responses to cAMP. In the present study we have determined that replacement of Ser142 of CREB with Asp greatly decreases the ability of the cAMP-dependent protein kinase to activate CREB. As Ser142 is located within the region of CREB that interacts with CBP, it seemed quite likely that mutations at this site might interfere with binding to CBP. However, both in vitro and in vivo protein-protein interaction assays revealed that replacement of Ser142 with Asp does not interfere with the binding of CREB to CBP. These studies argue strongly that although the binding of CREB to CBP is necessary, it is not sufficient for transcriptional responses to cAMP.

Animals↗
PubMed2006

The hepatitis B virus X protein functionally interacts with CREB-binding protein/p300 in the regulation of CREB-mediated transcription.

The hepatitis B virus infects more than 350 million people worldwide and is a leading cause of liver cancer. The virus encodes a multifunctional regulator, the hepatitis B virus X protein (HBx), that is essential for virus replication. HBx is involved in modulating signal transduction pathways and transcription mediated by various factors, notably CREB that requires the recruitment of the co-activators CREB-binding protein (CBP)/p300. Here we investigated the role of HBx and its potential interaction with CBP/p300 in regulating CREB transcriptional activity. We show that HBx and CBP/p300 synergistically enhanced CREB activity and that CREB phosphorylation by protein kinase A was a prerequisite for the cooperative action of HBx and CBP/p300. We further show that HBx interacted directly with CBP/p300 in vitro and in vivo. Using chromatin immunoprecipitation, we provide evidence that HBx physically occupied the CREB-binding domain of CREB-responsive promoters of endogenous cellular genes such as interleukin 8 and proliferating cell nuclear antigen. Moreover expression of HBx increased the recruitment of p300 to the interleukin 8 and proliferating cell nuclear antigen promoters in cells, and this is associated with increased gene expression. As recruitment of CBP/p300 is known to represent the limiting event for activating CREB target genes, HBx may disrupt this cellular regulation, thus predisposing cells to transformation.

CREB-Binding Protein↗
PubMed1996

CREB-1 and CREB-2 immunoreactivity in the rat brain.

This study is focused to learn about the cellular localization of transcription factors binding to the cAMP response element-CREBs-in the brain of normal rats and in animals subjected to excitotoxic cell damage. For this purpose, CREB-1 and CREB-2 immunoreactivity is examined in the developing and adult rat brain under physiological conditions, and following systemic kainic acid (KA) injection at convulsant doses in the adult, as a validated experimental model of excitotoxic injury. CREB-1 immunoreactivity is constitutively expressed in periventricular glia and Bergmann glia, and appears in reactive astrocytes following KA-induced excitotoxic cell damage. In contrast, CREB-2 is constitutively expressed in all neurons of the cerebrum, cerebellum and brain stem in the developing and adult brain. CREB-2 immunoreactivity is not increased following KA excitotoxic cell damage. These results demonstrate that CREB-1 and CREB-2 in the brain of the rat are localized in separate cellular compartments and that their expression is differentially regulated in pathologic states.

Animals↗
PubMed1996

Complex formation between CREB and Tax enhances the binding affinity of CREB for the human T-cell leukemia virus type 1 21-base-pair repeats.

The regulation of human T-cell leukemia virus type 1 (HTLV-1) gene expression is dependent on three cis-acting elements, known as the 21-bp repeats, in the long terminal repeat. Each of the 21-bp repeats contains a nonpalindromic cyclic AMP response element (CRE) sequence which is capable of binding members of the ATF/CREB family of transcription factors. The HTLV-1 transactivator protein Tax is able to markedly stimulate the in vitro binding of CREB to the CRE sites present in each of the 21-bp repeats but not to CRE sites present in cellular promoters. The ability to Tax to stimulate CREB binding to different CRE sites correlates with the ability of Tax to activate gene expression from these sites. We wished to determine how sequence differences between the somatostatin CRE and the 21-bp repeat were involved in this different response to Tax. Scatchard analysis indicated that CREB bound to the somatostatin CRE with a single class of high-affinity binding while CREB bound to the 21-bp repeats with a biphasic binding pattern, indicating the presence of both low- and high-affinity binding. Tax increased the affinity of CREB binding but not that of another ATF/CREB protein, CREB2, to the 21-bp repeat. However, Tax did not increase affinity of binding of CREB to the somatostatin CRE. To determine the mechanism by which Tax increased dCREB binding affinity, immobilized oligonucleotides corresponding to either the 21-bp repeat or the somatostatin CRE were used to demonstrate that Tax formed a highly specific complex with CREB on the 21-bp repeat but not on the somatostatin CRE. These results indicate that formation of a complex between Tax and CREB results in specific high-affinity binding of this ternary complex to the HTLV-1 21 bp repeats.

Base Sequence↗
PubMed1996

An alternatively spliced polycistronic mRNA encoding cyclic adenosine 3',5'-monophosphate (cAMP)-responsive transcription factor CREB (cAMP response element-binding protein) in human testis extinguishes expression of an internally translated inhibitor CREB isoform.

Cyclic AMP response element-binding protein (CREB) regulates the expression of cAMP-responsive genes. In the rat testis, several isoforms of CREB arise from alternative exon splicing that occurs cyclically during the 12-day cell association cycles of spermatogenesis. Insertion of alternatively spliced exon W into CREB mRNA during spermatogenesis results in a polycistronic RNA that encodes two novel internally translated CREB repressor isoforms called I-CREBs, consisting of the carboxy-terminal DNA-binding domain devoid of the transactivation domains. Here we report the alternative splicing of an additional novel exon Z in CREB mRNA expressed in human but not in mouse or rat testis. Insertion of exon Z abolishes the synthesis of one of the two inhibitor CREBs due to the introduction of an inframe stop codon within exon Z. We show that exon Z is not spliced into mRNAs in mouse and rat testes due to the evolution of mutations in the splice signals flanking exon Z. These findings suggest that the splicing in of exon Z may be part of a human-specific mechanism to regulate cAMP-dependent regulatory pathways in spermatogenesis by extinguishing the expression of a CREB repressor.

Alternative Splicing↗
PubMed2001

Hexosamine-induced fibronectin protein synthesis in mesangial cells is associated with increases in cAMP responsive element binding (CREB) phosphorylation and nuclear CREB: the involvement of protein kinases A and C.

Hyperglycemia-induced alterations in mesangial (MES) cell function and extracellular matrix protein accumulation are seen in diabetic glomerulopathy. Recent studies have demonstrated that some of the effects of high glucose (HG) on cellular metabolism are mediated by the hexosamine biosynthesis pathway (HBP), in which fructose-6-phosphate is converted to glucosamine 6-phosphate by the rate-liming enzyme glutamine:fructose-6-phosphate amidotransferase (GFA). In this study, we investigated the role of HBP on HG-stimulated fibronectin protein synthesis, a matrix component, in SV-40-transformed rat kidney MES cells. Treatment of MES cells with 25 mmol/l glucose (HG) for 48 h increases cellular fibronectin levels by two- to threefold on Western blots when compared with low glucose (5 mmol/l). Glucosamine (GlcN; 1.5 mmol/l), which enters the hexosamine pathway distal to GFA action, also increases fibronectin synthesis. Azaserine (AZA; 0.5 micromol/l), an inhibitor of GFA, blocks the HG- but not the GlcN-induced fibronectin synthesis. Fibronectin contains cAMP responsive element (CRE) consensus sequences in its promoter and the phosphorylation of CRE-binding protein (CREB) may regulate its expression. On Western blots, HG and GlcN stimulate two- to threefold the phosphorylation of CREB at Ser 133, whereas CREB protein content was unaltered by either HG or GlcN. In addition, nuclear CREB activity was increased by HG and GlcN on gel-shift assays using (32)P-CRE oligonucleotides. AZA impeded the HG-enhanced CREB phosphorylation and CRE binding but had no effect on GlcN-mediated CREB phosphorylation and CRE binding. Pharmacologic inhibition of protein kinase C (PKC) and protein kinase A (PKA), which are involved in hexosamine-mediated matrix production, blocked the CREB phosphorylation and fibronectin synthesis seen in HG and GlcN conditions. We conclude that the effects of HG on fibronectin synthesis in the mesangium are mediated by the HBP possibly via hexosamine regulation of CREB and PKC/PKA signaling pathways. These results support the hypothesis that the HBP is a sensor and regulator of the actions of glucose in the kidney.

Animals↗
PubMed1999

Transcriptional regulation of the transforming growth factor-beta2 promoter by cAMP-responsive element-binding protein (CREB) and activating transcription factor-1 (ATF-1) is modulated by protein kinases and the coactivators p300 and CREB-binding protein.

Transcription of the transforming growth factor-beta2 (TGF-beta2) gene is dependent on a cAMP-response element/activating transcription factor (CRE/ATF) site that is bound by CREB and ATF-1 as well as an E-box motif that is bound by upstream stimulatory factors 1 and 2 (USF1 and USF2). To identify additional factors involved in the expression of the TGF-beta2 gene, we employed F9 embryonal carcinoma (EC) cells, which express TGF-beta2 only after the cells differentiate. We show that overexpression of the transcription factors, CREB, ATF-1, USF1, and USF2 dramatically increases TGF-beta2 promoter activity in F9-differentiated cells. We further show that the coactivators p300 and CBP up-regulate the TGF-beta2 promoter when CREB and ATF-1 are expressed in conjunction with protein kinases that phosphorylate CREB on serine 133 and ATF-1 on serine 63. Importantly, we identify the presence of serine 133-phosphorylated CREB in the nucleus of F9-differentiated cells but not in the nucleus of F9 EC cells. This phosphorylated form is present in whole cell extracts of both the parental and differentiated cells, suggesting that nuclear accumulation of serine 133-phosphorylated CREB is regulated during differentiation of F9 EC cells and is likely to play an important role in the activation of the TGF-beta2 gene.

Activating Transcription Factor 1↗
PubMed1996

CREB and CREB-binding proteins play an important role in the IE2 86-kilodalton protein-mediated transactivation of the human cytomegalovirus 2.2-kilobase RNA promoter.

The human cytomegalovirus (HCMV) immediate-early region 2 86-kDa protein (IE2 86) is the major transactivator of the promoter for the 2.2-kb class of early RNAs (open reading frame UL 112-113). Previously, we reported that a DNA segment on this promoter between nucleotides (nt) -113 and -59 was critical for activation by IE2 86 in vivo and could be bound by IE2 86 in vitro (R. Schwartz, M. H. Sommer, A. Scully, and D. H. Spector, J. Virol. 68:5613-5622, 1994). With a set of site-specific mutations within nt -84 to -61, we have localized the essential cis-acting sequences to nt -72 to -61, which contain an ATF/CREB-binding site. The IE2 86-binding site between nt -113 and -85 is not essential for activation of the promoter by IE2 86 in transient-expression assays, but its presence can enhance the level of activation mediated through the sequences located between nt -84 and -59. Electrophoretic mobility shift assays with a segment containing nt -84 to -59 and nuclear extracts from human cells permissive for the HCMV infection revealed a complex band pattern. However, by supershift analysis with specific antibodies, we were able to identify CREB as the major ATF/CREB family member in the protein-DNA complexes. Further evidence that CREB is a target for IE2 86-mediated induction, is provided by the finding that IE2 86 activates the somatostatin promoter to high levels. Although the binding of IE2 86 to nonphosphorylated full-length CREB or deltaCREB is minimal, IE2 86 does form complexes with p300 and the CREB-binding protein (CBP), which in turn bind to CREB and can serve as adaptor proteins for CREB function. In addition, the in vivo functional relevance of the interaction between IE2 86 and CBP is indicated by the ability of IE2 86 to enhance transcriptional activation mediated by a GAL4-CBP fusion protein brought to a promoter by GAL4-binding sites.

Activating Transcription Factor 1↗
PubMed1996

Phosphorylation of CREB at Ser-133 induces complex formation with CREB-binding protein via a direct mechanism.

We have characterized a phosphoserine binding domain in the coactivator CREB-binding protein (CBP) which interacts with the protein kinase A-phosphorylated, and hence activated, form of the cyclic AMP-responsive factor CREB. The CREB binding domain, referred to as KIX, is alpha helical and binds to an unstructured kinase-inducible domain in CREB following phosphorylation of CREB at Ser-133. Phospho-Ser-133 forms direct contacts with residues in KIX, and these contacts are further stabilized by hydrophobic residues in the kinase-inducible domain which flank phospho-Ser-133. Like the src homology 2 (SH2) domains which bind phosphotyrosine-containing peptides, phosphoserine 133 appears to coordinate with a single arginine residue (Arg-600) in KIX which is conserved in the CBP-related protein P300. Since mutagenesis of Arg-600 to Gln severely reduces CREB-CBP complex formation, our results demonstrate that, as in the case of tyrosine kinase pathways, signal transduction through serine/threonine kinase pathways may also require protein interaction motifs which are capable of recognizing phosphorylated amino acids.

3T3 Cells↗
PubMed2001

MLL and CREB bind cooperatively to the nuclear coactivator CREB-binding protein.

A fragment of the mixed-lineage leukemia (MLL) gene (Mll, HRX, ALL-1) was identified in a yeast genetic screen designed to isolate proteins that interact with the CREB-CREB-binding protein (CBP) complex. When tested for binding to CREB or CBP individually, this MLL fragment interacted directly with CBP, but not with CREB. In vitro binding experiments refined the minimal region of interaction to amino acids 2829 to 2883 of MLL, a potent transcriptional activation domain, and amino acids 581 to 687 of CBP (the CREB-binding or KIX domain). The transactivation activity of MLL was dependent on CBP, as either adenovirus E1A expression, which inhibits CBP activity, or alteration of MLL residues important for CBP interaction proved effective at inhibiting MLL-mediated transactivation. Single amino acid substitutions within the MLL activation domain revealed that five hydrophobic residues, potentially forming a hydrophobic face of an amphipathic helix, were critical for the interaction of MLL with CBP. Using purified components, we found that the MLL activation domain facilitated the binding of CBP to phosphorylated CREB. In contrast with paradigms in which factors compete for limiting quantities of CBP, these results reveal that two distinct transcription factor activation domains can cooperatively target the same motif on CBP.

Binding Sites↗
PubMed2000

Calmodulin kinase II attenuation of gene transcription by preventing cAMP response element-binding protein (CREB) dimerization and binding of the CREB-binding protein.

Calmodulin Kinase II (CamKII) inhibits the transcription of many CRE-dependent genes, but the mechanism of dominant transcriptional inhibition is unknown. Here we show that phosphorylation of serine 142 in CREB by CamKII leads to dissociation of the CREB dimer without impeding DNA binding capacity. CamKII-modified CREB binds to DNA efficiently as a monomer; however, monomeric CREB is unable to recruit the CREB-binding protein (CBP) even when phosphorylated at serine 133. Thus, CamKII confers a dominant inhibitory effect on transcription by preventing dimerization of CREB, and this mechanism may account for the attenuation of gene expression.

Amino Acid Sequence↗
PubMed1993

Transcription of the rat glucagon gene by the cyclic AMP response element-binding protein CREB is modulated by adjacent CREB-associated proteins.

The cyclic AMP (cAMP) response element (CRE) of the rat glucagon gene (Glu-CRE, 5'-TGACGTCA-3') mediates transcriptional responses to 8-bromo-cAMP and protein kinase A (PKA) in a glucagon-producing hamster islet cell line (InR1G9). By several different DNA-protein binding assays, we show that the transcription factor CREB binds to the CRE octamer and that additional nuclear proteins bind to sequences adjacent to the CRE. Mutation of the Glu-CRE octamer attenuates both the binding of CREB and cAMP-dependent PKA-stimulated transcriptional activity in transient transfection experiments but does not affect the binding of adjacent CREB-associated proteins. Progressive deletions and clustered point mutations of the sequences flanking the Glu-CRE identify sequences (5'-TCATT-3') located both 5' and 3' to the core CRE octamer that bind several proteins. Two proteins with molecular masses of 80 and 100 kDa bind to each of the 5' and 3' TCATT sites. Formation of additional protein-DNA complexes containing 45- and 20-kDa proteins depends upon the integrity of both TCATT sequences. Deletion or point mutation of the TCATT motif located on the 3' side of the CRE octamer results in enhanced transcriptional responses to PKA, suggesting that the CREB-associated proteins decrease the ability of CREB to mediate PKA-stimulated transcription. Results from these studies demonstrate that nucleotides flanking the core CRE octamer can influence the activity of the CRE by serving as binding sites for proteins that modulate the function of CREB and suggest a mechanism to explain why some consensus palindromic CREs are less responsive to cAMP stimulation than others.

Animals↗
PubMed2003

Central administration of neuropeptide Y reduces alpha-melanocyte-stimulating hormone-induced cyclic adenosine 5'-monophosphate response element binding protein (CREB) phosphorylation in pro-thyrotropin-releasing hormone neurons and increases CREB phosphorylation in corticotropin-releasing hormone neurons in the hypothalamic paraventricular nucleus.

Neuropeptide Y (NPY) has a potent inhibitory effect on TRH gene expression in the paraventricular nucleus (PVN) and contributes to the fall in circulating thyroid hormone levels during fasting mediated by a reduction in serum leptin levels. Because alpha-MSH activates the TRH gene by increasing the phosphorylation of CREB in the nucleus of these neurons, we raised the possibility that at least one of the mechanisms by which NPY reduces TRH mRNA in hypophysiotropic neurons is by antagonizing the ability of alpha-MSH to phosphorylate CREB. As NPY increases CRH mRNA in the hypothalamus, we further determined whether intracerebroventricular (i.c.v.) administration of NPY regulates the phosphorylation of CREB in hypophysiotropic CRH neurons. NPY [10 micro g in artificial CSF (aCSF)] was administered into the lateral ventricle i.c.v. 30 min before the i.c.v. administration of aCSF or alpha-MSH (10 micro g in aCSF), the latter in a dose previously demonstrated to increase proTRH mRNA and phosphorylate CREB in TRH neurons. By double-labeling immunocytochemistry, only few TRH neurons in the PVN contained phosphoCREB (PCREB) in animals treated only with aCSF (4 +/- 0.2%) or with NPY followed by aCSF (9.7 +/- 2.5), whereas alpha-MSH-infused animals dramatically increased the percentage of TRH neurons containing PCREB (75.3 +/- 6.9%). Pretreatment with NPY before alpha-MSH infusion, however, significantly reduced the percentage of TRH neurons containing PCREB (40.8 +/- 3.5%) compared with alpha-MSH infused animals (P = 0.01). Only 12.2 +/- 0.9% of CRH neurons of the medial parvocellular neurons contained PCREB nuclei in vehicle-treated animals, whereas 30 min following NPY infusion, the number of CRH neurons containing PCREB increased dramatically to 88 +/- 2.9%. Whereas alpha-MSH infusion increased the percentage of CRH neurons that contained PCREB to 56 +/- 2.2% compared with control, animals pretreated with NPY further increased the number of CRH neurons colocalizing with PCREB to 87 +/- 2.5%. These data demonstrate a functional interaction between NPY and alpha-MSH in the regulation of proTRH neurons in the PVN, suggesting that NPY can antagonize alpha-MSH induced activation of the TRH gene by interfering with melanocortin signaling at the postreceptor level, preventing the phosphorylation of CREB. In contrast, NPY infusion increases the phosphorylation of CREB in CRH neurons, indicating that NPY has independent effects on discrete populations of neurons in the PVN, presumably mediated through different signaling mechanisms.

Animals↗
PubMed1995

A novel cyclic AMP response element-binding protein-1 (CREB-1) splice product may down-regulate CREB-1 activity.

In this report we identify novel spliced forms of cyclic AMP (cAMP) response element-binding protein-1 (CREB-1) mRNA. These forms contained an additional 17 nucleotide insert, which we refer to as the beta exon, located between exons 4 and 7 of the delta, and 5 and 7 of the alpha forms of CREB-1 transcript (nomenclature of Ruppert et al. 1992; EMBO Journal 11, 1503-1512). The inclusion of the beta exon led to the generation of mRNAs in which the frame of CREB-1 sequences 3' to the exon was shifted such that the encoded proteins terminate after the transactivation domain, but before the target serine for cAMP-dependent protein kinase. The beta exon-containing CREB-1 mRNAs were more abundant in tissues that respond poorly to cAMP, suggesting that the generation of beta CREB-1 mRNAs may contribute to the down-regulation of CREB-1 activity and cAMP responsiveness.

Amino Acid Sequence↗