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PubMed1994

Differential activation of CREB by Ca2+/calmodulin-dependent protein kinases type II and type IV involves phosphorylation of a site that negatively regulates activity.

The cAMP response element-binding protein (CREB) has been shown to mediate transcriptional activation of genes in response to both cAMP and calcium influx signal transduction pathways. The roles of two multifunctional calcium/calmodulin-dependent protein kinases, CaMKIV and CaMKII, were examined in transient transfection studies that utilized either the full-length or the constitutively active forms of these kinases. The results indicate that CaMKIV is much more potent than CaMKII in activating CREB in three different cell lines. It was also found in these studies that Ser133 of CREB is essential for its activation by CaMKIV. Because both CaMKII and CaMKIV can phosphorylate CREB, we pursued further the mechanism by which CaMKII and CaMKIV differentially regulate CREB activity. Mutagenesis studies and phosphopeptide mapping analysis demonstrated that in vitro, CaMKIV phosphorylates CREB at Ser133 only, whereas CaMKII phosphorylates CREB at Ser133 and a second site, Ser142. Transient transfection studies revealed that phosphorylation of Ser142 by CaMKII blocks the activation of CREB that would otherwise occur when Ser133 is phosphorylated. When Ser142 was mutated to alanine, CREB was activated by CaMKII, as well as by CaMKIV. Furthermore, mutation of Ser142 to alanine enhanced the ability of Ca2+ influx to activate CREB, suggesting a physiological role for the phosphorylation of Ser142 in modulation of CREB activity. These data provide evidence for a new mechanism for regulation of CREB activity involving phosphorylation of a negative regulatory site in the transcriptional activation domain. The studies also provide new insights into possible interactions between the cAMP and Ca2+ signaling pathways in the regulation of transcription. In particular, changes in intracellular Ca2+ have the potential to either inhibit or augment the ability of cAMP to stimulate transcription, depending on the presence of specific forms of Ca2+/calmodulin-dependent protein kinases.

Amino Acid Sequence↗
PubMed2004

Ubiquitin-proteasome-mediated CREB repressor degradation during induction of long-term facilitation.

Abstract Long-term facilitation in Aplysia and other forms of long-term memory in invertebrates and vertebrates require the gene expression cascade induced by cAMP-responsive element binding protein (CREB). Normally, gene expression by CREB is inhibited by repressors. The molecular mechanisms by which the repression is relieved are not understood. Our results show that Aplysia CREB repressor is a substrate for degradation by the ubiquitin-proteasome pathway. Treatment with the facilitatory neurotransmitter 5-hydroxy tryptamine (5-HT) leads to CREB repressor degradation in vivo and the degradation can be blocked by a specific proteasome inhibitor. Our biochemical studies show that attachment of ubiquitin molecules marks the CREB repressor for degradation by the proteasome. Protein kinase C (PKC) stimulates ubiquitination and degradation of the CREB repressor. Our results suggest that proteolytic removal of the CREB repressor is a potential mechanism for controlling gene expression by CREB. Without stimulation, gene expression is suppressed by the CREB repressor. Upon stimulation with 5-HT, PKC is activated, causing enhancement in ubiquitination and degradation of the CREB repressor. Thus, regulation of proteolysis of the CREB repressor by PKC might be critical in determining whether or not CREB-mediated gene expression goes forward during induction of long-term facilitation.

Animals↗
PubMed1995

Chimeric proteins composed of Jun and CREB define domains required for interaction with the human T-cell leukemia virus type 1 Tax protein.

The regulation of human T-cell leukemia virus type 1 (HTLV-1) long terminal repeat gene expression is dependent on three cis-acting elements known as 21-bp repeats and the transactivator protein Tax. Mutagenesis has demonstrated that sequences in each of the 21-bp repeats can be divided into three domains designated A, B, and C. Tax stimulates the binding of CREB to the B domain, which is essential for Tax activation of HTLV-1 gene expression. In this study, we demonstrate that Tax will stimulate the binding of CREB to the HTLV-1 21-bp repeats but does not stimulate CREB binding to the consensus cyclic AMP response element (CRE) element found in the somatostatin promoter. However, Tax stimulates CREB binding to a consensus CRE in the context of the 21-bp repeats, indicating the importance of these sequences in stimulating CREB binding. To determine the mechanism by which Tax stimulates CREB binding and determine potential interactions between Tax and CREB, we used the mammalian two-hybrid system in conjunction with in vitro binding and gel retardation assays. Two-hybrid analysis indicated that mutations in either the basic or leucine zipper region of CREB prevented interactions with Tax. Since several studies have demonstrated that Tax will also stimulate the binding of a variety of different basic region-leucine zipper proteins to their cognate binding sites, we assayed whether chimeric proteins composed of portions of CREB and another basic region-leucine zipper protein, Jun, could be used to map domains required for interactions with Tax. These studies were possible because we did not detect in vivo or in vitro interactions between Tax and Jun. The amino acid sequence of the CREB basic region and a portion of its leucine zipper were required for both in vivo and in vitro interactions with Tax and increased binding of CREB to the 21-bp repeats in response to Tax. These studies define the domains in CREB required for both in vivo and in vitro interactions by the HTLV-1 Tax protein.

Amino Acid Sequence↗
PubMed1999

Interaction of Gli2 with CREB protein on DNA elements in the long terminal repeat of human T-cell leukemia virus type 1 is responsible for transcriptional activation by tax protein.

The long terminal repeat (LTR) of human T-cell leukemia virus type 1 (HTLV-1) has two distinct DNA elements, one copy of TRE2S and three copies of a 21-bp sequence that respond to the viral trans-activator protein, Tax. Either multiple copies of the 21-bp sequence or a combination of one copy each of TRE2S and 21-bp sequence is required for efficient trans activation by Tax. In the trans activation of multiple copies of 21-bp sequence, CREB/ATF protein plays an essential role in forming a complex with Tax. To understand the role of TRE2S in trans activation of one copy of 21-bp sequence, we examined protein binding to the DNA elements by DNA affinity precipitation assay including Gli2 protein binding to TRE2S and CREB protein binding to 21-bp sequence. Binding of CREB to a DNA probe containing both elements, TRE2S-21bp probe, was dependent on Gli2 protein under restricted conditions and was enhanced in a dose-dependent fashion by the binding of Gli2 protein to the same probe. Mutation in either element abolished the efficient binding of CREB. A glutathione S-transferase fusion protein of a fragment of Gli2 was able to bind to CREB. Therefore, Gli2-CREB interaction on the DNA probe is proposed to stabilize CREB binding to DNA. Tax can bind to CREB protein on the DNA; therefore, stabilization of DNA binding of CREB results in more recruitment of Tax onto DNA. Conversely, Tax increased the DNA binding of CREB, although it had almost no effect on the binding of Gli2. These results suggest that Gli2 binds to the DNA element and interacts with CREB, resulting in more recruitment of Tax, which in turn stabilizes DNA binding of CREB. Similar cooperation of the protein binding to TRE2S-21bp probe was also observed in nuclear extract of an HTLV-1-infected T-cell line. Consistent with the Gli2-CREB interaction on the DNA elements, Tax-mediated trans activation was dependent on the size of the spacer between TRE2S and 21-bp sequence. The effective sizes of the spacer suggest that TRE2S in the LTR would cooperate with the second and third copies of the 21-bp sequence and contribute to trans activation of the viral gene transcription.

Cyclic AMP Response Element-Binding Protein↗
PubMed1991

Nuclear translocation and DNA recognition signals colocalized within the bZIP domain of cyclic adenosine 3',5'-monophosphate response element-binding protein CREB.

CREB is a cAMP-responsive nuclear DNA-binding protein that binds to cAMP response elements and stimulates gene transcription upon activation of the cAMP signalling pathway. The protein consists of an amino-terminal transcriptional transactivation domain and a carboxyl-terminal DNA-binding domain (bZIP domain) comprised of a basic region and a leucine zipper involved in DNA recognition and dimerization, respectively. Recently, we discovered a testis-specific transcript of CREB that contains an alternatively spliced exon encoding multiple stop codons. CREB encoded by this transcript is a truncated protein lacking the bZIP domain. We postulated that the antigen detected by CREB antiserum in the cytoplasm of germinal cells is the truncated CREB that must also lack its nuclear translocation signal (NTS). To test this hypothesis we prepared multiple expression plasmids encoding carboxyl-terminal deletions of CREB and transiently expressed them in COS-1 cells. By Western immunoblot analysis as well as immunocytochemistry of transfected cells, we show that CREB proteins truncated to amino acid 286 or shorter are sequestered in the cytoplasm, whereas a CREB of 295 amino acids is translocated into the nucleus. Chimeric CREBs containing a heterologous NTS fused to the first 248 or 261 amino acids of CREB are able to drive the translocation of the protein into the nucleus. Thus, the nine amino acids in the basic region involved in DNA recognition between positions 287 and 295 (RRKKKEYVK) of CREB contain the NTS. Further, mutation of the lysine at position 290 in CREB to an asparagine diminishes nuclear translocation of the protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1999

CREB-mediated transcriptional control.

cAMP-response-element-binding protein, CREB, is a 43-kDa leucine zipper transcription factor identified and cloned via the study of cAMP-regulated genes. In the last decade, numerous studies have contributed much to our understanding of CREB structure, function, and CREB-mediated transcription. CREB binds to the cAMP-response-element (CRE) as a homodimer formed via the leucine zipper motif present at its C-terminus; its transcriptional activity is regulated by phosphorylation at Ser133, located within the N-terminal transactivation domain. Active, Ser133-phosphorylated CREB effects transcription of CRE-dependent genes via interaction with the 265-kDa co-activator protein CREB-binding-protein, CBP, which bridges the CRE/CREB complex to components of the basal transcriptional apparatus. This mechanism of CREB activation is effected by diverse signals, including those regulating the intracellular levels of cAMP and Ca+2, growth factors, and cellular stress. Accordingly, CREB-mediated transcription regulates diverse cellular responses, including intermediary metabolism, neuronal signaling, cell proliferation, and apoptosis. In addition to the regulation of CREB by phosphorylation, the viral oncoproteins HBV pX and HTLVI Tax regulate CREB transcriptional efficacy by an alternative mechanism, by increasing its DNA-binding affinity for viral and/or cellular CRE sites. In this review I describe key experiments that have defined the mechanism of CREB activation, with primary emphasis on emerging evidence linking CREB to cellular growth and development.

Amino Acid Sequence↗
PubMed1995

Protein kinase C mediates activation of nuclear cAMP response element-binding protein (CREB) in B lymphocytes stimulated through surface Ig.

The cAMP response element-binding protein (CREB) is generally considered to be responsive to elevation of cAMP through the activity of protein kinase A (PKA). Although it is well known that cAMP-raising agents can strongly influence B cell stimulation, the regulation of CREB has been little studied. Recently, cross-linking of surface Ig (sIg) was shown to result in trans-activation of a cAMP response element (CRE)-dependent promoter to which bound B cell CREB. In this study, we explored the mechanism underlying this unexpected linkage between sIg and CREB. We found that sIg cross-linking results in phosphorylation of CREB at Ser133. Although this phosphorylation step is mediated by PKA in pheochromocytoma cells, it depends on protein kinase C (PKC) in B lymphocytes. This conclusion is based on abrogation of sIg-induced CREB Ser133 phosphorylation by long-term phorbol-ester treatment to deplete PKC, and mimicking of sIg-induced CREB phosphorylation and CRE-dependent gene expression by short-term PKC agonism. Furthermore, CD40 ligand (CD40L) and LPS, two PKC-independent forms of B cell stimulation, failed to induce phosphorylation of CREB Ser133. These results suggest that CREB responds to specific surface-receptor signals in B cells and that this response is mediated by PKC. Interestingly, forskolin failed to induce phosphorylation of CREB Ser133 in B cells, although it did so in PC12 pheochromocytoma cells. Taken together with PKC mediation of CREB Ser133 phosphorylation in B cells, these results suggest that the dominant mode of CREB regulation is cell-type specific.

Animals↗
PubMed1996

Alternative exon splicing controls a translational switch from activator to repressor isoforms of transcription factor CREB during spermatogenesis.

The cAMP/protein kinase A signaling pathway activates the cAMP-responsive transcription factor CREB. Here we describe a unique alternative RNA splicing event that occurs during the development of germ cells in the testis, resulting in a translational switch from an mRNA encoding activator CREB to an mRNA encoding novel inhibitor CREB isoforms (I-CREBs). Alternative splicing of an additional exon into the CREB mRNA in mid to late pachytene spermatocytes results in the premature termination of translation and consequent downstream reinitiation of translation producing I-CREBs. The I-CREBs down-regulate cAMP-activated gene expression by inhibiting activator CREB from binding to cAMP response elements. Further, the developmental stage-specific expression of I-CREBs in germ cells of the seminiferous tubules correlates with the cyclical down-regulation of activator CREB, suggesting that I-CREBs repress expression of the cAMP-inducible CREB gene as well as other genes transiently induced by cAMP during the 12-day cycle of spermatogenesis.

Alternative Splicing↗
PubMed1998

Chronic ethanol exposure impairs phosphorylation of CREB and CRE-binding activity in rat striatum.

This study examined influences of ethanol exposure on phosphorylation of cAMP response element-binding protein (CREB) and CRE-binding activity in the striatum of rats. The phosphorylated form of CREB increased 180% during acute intoxication, compared to sham conditions. After chronic ethanol exposure, induction of CREB phosphorylation by an acute ethanol challenge was markedly attenuated (50%) compared with acute ethanol exposure in the pair-fed condition. Gel retardation assays with oligomers encoding the rat proenkephalin CRE-1 and CRE-2 were performed to determine the effects of ethanol on CRE-binding activity. Supershift experiments demonstrated that striatal nuclear protein contains CREB and CEBPPbeta (CCAAT/enhancer-binding protein) and that both transcription factors are involved in specifically binding to the DNA sequence. Rechallenging rats that had been chronically exposed to ethanol with an acute ethanol challenge reduced the CRE/nuclear protein complexes. However, supershift analyses did not show that chronic ethanol exposure altered the dimerization patterns of CREB and CEBPbeta within the complexes. Associated with these impairments in CREB binding and CREB phosphorylation was a significant reduction in two CREB-modulated factors, proenkephalin and c-fos expression. In summary, acute ethanol exposure activates the phosphorylation of CREB. Neuroadaptation to chronic ethanol exposure includes alterations in CREB physiology that may impair genes that are dependent upon CREB for transcriptional activation.

Alcoholic Intoxication↗
PubMed2000

Microphthalmia due to p53-mediated apoptosis of anterior lens epithelial cells in mice lacking the CREB-2 transcription factor.

CREB-2 (also called ATF4, TAXREB67, or C/ATF) is an evolutionarily conserved member of the CREB/ATF family of basic-leucine zipper transcription factors. CREB-2 is expressed ubiquitously in the adult mouse and can function as both a transcriptional activator and a repressor. However, little was understood about the normal function of CREB-2 in mammalian development or organ physiology. In this report we have used gene targeting to produce CREB-2-deficient (CREB-2-/-) mice. Adult CREB-2-/- mice displayed microphthalmia due to the complete absence of a lens. Early embryonic lens development including formation of the optic vesicle, primary lens fibers, and proliferating anterior epithelial cells occurred normally in these mice. However, beginning at ED 14.5 the CREB-2-deficient anterior epithelial lens cells underwent massive and synchronous apoptosis. This was followed by the complete resorption of the developing lens. Consistent with this defect in anterior epithelial cell survival, in situ hybridization studies showed that CREB-2 is expressed at high levels in wild-type anterior epithelial lens cells at ED 14.5. The defect in lens formation seen in the CREB-2-/- mice was not associated with qualitative defects in the expression of Pax-6, alphaA-crystallin, c-maf, or PDGF-R alpha. However, apoptosis of the anterior epithelial cells was mediated by a p53-dependent cell death pathway because ablation of the p53 gene rescued anterior epithelial cell death and allowed the formation of a lens in the absence of CREB-2. Taken together, these results identify CREB-2 as an important regulator of mammalian lens development.

Animals↗
PubMed2001

Membrane depolarization mediates phosphorylation and nuclear translocation of CREB in vascular smooth muscle cells.

Diverse signals have the potential to modulate gene transcription through the Ca2+ and cAMP response element binding protein (CREB) in vascular smooth muscle cells (VSMCs). A key step in the transmission of these signals is import into the nucleus. Here, we provide evidence that the Ran GTPase, which regulates nuclear import, exerts different regulation over PDGF-BB, Ca2+, and cAMP signaling to CREB in VSMCs. PDGF-BB, membrane depolarization, and forskolin increased levels of activated CREB (P-CREB) and c-fos in VSMCs and intact aorta. The calcium channel antagonist nimodipine reduced the level of P-CREB stimulated by membrane depolarization, but not by PDGF-BB or forskolin. Block of Ran-mediated nuclear import, by wheat germ agglutinin or an inactivating Ran mutant (T24N Ran), significantly reduced nuclear P-CREB in response to PDGF-BB or membrane depolarization, but enhanced levels of P-CREB in response to forskolin. Contrary to expectation, block of nuclear import led to the appearance of P-CREB in the cytoplasm after depolarization. Furthermore, blocking nuclear export with leptomycin B reduced P-CREB stimulation by both depolarization and PDGF-BB. These results suggest that translocation of CREB between the nucleus and the cytoplasm provides an important role in CREB activating pathways in VSMCs.

Actins↗
PubMed2001

Distribution and partial characterization of CREB-like immunoreactivity in the medicinal leech Hirudo.

The presence and distribution of immunoreactivity to the cyclic AMP response element binding protein (CREB) were determined in the central nervous system (CNS) and in peripheral tissues of the medicinal leech Hirudo. Western blots revealed several CREB-immunoreactive (CREB-IR) bands including one whose molecular weight (43-44 kDa) was similar to mammalian CREB. The 43-44 kDa CREB-like protein was detected in nuclear extracts of the ventral nerve cord and was not observed following preincubation of the primary antiserum with the epitope sequence. CREB-like immunoreactivity was detected in extracts from each of six regions of the leech CNS, and in extracts from leech body wall musculature, crop, intestine, jaw musculature, pharynx, and salivary tissues. Whole mounts of leech ganglia revealed specific CREB-IR in a restricted population of neurons distributed throughout the leech CNS. Apparent homologues to a pair of CREB-IR dorsolateral neurons were observed in most ganglia along the ventral nerve cord. Several CREB-IR neurons exhibited segmental specificity. A number of neurons stained with an antiserum to the cyclic AMP response element modulator (CREM). These neurons showed no overlap in location with CREB-IR neurons, and this staining was not eliminated with a preabsorption control. Possible roles for a CREB-like protein in the leech are discussed.

Animals↗
PubMed2006

A nitric oxide signaling pathway controls CREB-mediated gene expression in neurons.

Prevailing views of neurotrophin action hold that the transcription factor CREB is constitutively bound to target genes with transcriptional activation occurring via CREB phosphorylation. However, we report that within several CRE-containing genes, CREB is not constitutively bound. Upon exposure of neurons to brain-derived neurotrophic factor (BDNF), CREB becomes rapidly bound to DNA coincident with phosphorylation at its transcriptional regulatory site, Ser133. This inducible CREB-DNA binding is independent of CREB Ser133 phosphorylation and is not affected by inhibition of the ERK or PI3K signaling pathways. Instead, BDNF regulates CREB binding by initiating a nitric oxide-dependent signaling pathway that leads to S-nitrosylation of nuclear proteins that associate with CREB target genes. Pharmacological manipulation of neurons in vitro and analysis of mice lacking neuronal nitric oxide synthase (nNOS) suggest that NO mediates BDNF and activity-dependent expression of CREB target genes. Thus, in conjunction with CREB phosphorylation, the NO pathway controls CREB-DNA binding and CRE-mediated gene expression.

Animals↗
PubMed1998

Inducible cAMP early repressor ICER down-regulation of CREB gene expression in Sertoli cells.

The cAMP response element binding protein (CREB) and the cAMP-responsive element modulator (CREM) are cyclically expressed in the seminiferous tubules during spermatogenesis. In the somatic Sertoli cells, which are the major supporters of germ cell development in the seminiferous tubules, the expression of CREB is cyclical and appears to be regulated by the levels of cAMP produced in response to the pituitary derived follicle-stimulating hormone FSH. Cyclic AMP response elements (CREs) located in the promoter of the CREB gene were shown earlier to be implicated in an autopositive feedback loop that up-regulates the expression of CREB. Here we show that in Sertoli cells FSH-mediated induction of the CREM repressor isoform, ICER (inducible cAMP early repressor) is correlated with the inhibition and delay of CREB gene expression in the seminiferous tubules. ICER binds to the two CREs located in the promoter of the CREB gene and in transient transfection assays of Sertoli cells, ICER expression vectors down-regulate transcription of a reporter gene driven by the CREB gene promoter. In addition, analyses of ICER and CREB gene expression in isolated segments of rat seminiferous tubules reveals stage-specific and cycle-dependent expression of ICER. The periods of enhanced expression of ICER correspond to the stages of spermatogenesis with the lowest levels of CREB expression. We suggest that the expression of ICER in Sertoli cells may contribute to the periodic repression of CREB gene expression during the repeated 12-day cycles of spermatogenesis, and may be required to reset the levels of activator CREB prior to the initiation of each new cycle of spermatogenesis.

Animals↗
PubMed2001

Phosphorylation of CREB in thoracolumbar spinal neurons and dorsal root ganglia after renal artery occlusion in rat.

These studies have demonstrated that ipsilateral renal artery occlusion (RAO) in rat results in the phosphorylation of cyclic AMP (cAMP) response element binding protein (p-CREB) in the thoracolumbar (T8-L2) spinal cord and associated dorsal root ganglia (DRG). p-CREB-immunoreactivity (IR) was expressed bilaterally in the thoracolumbar spinal cord, whereas expression in the DRG was ipsilateral relative to RAO. p-CREB-IR was primarily expressed in four distinct regions of the spinal cord: medial or lateral dorsal horn (MDH or LDH), dorsal commissural nucleus (DCN) and the region of the intermediolateral cell column (IML). After RAO, p-CREB-IR was greatest in the T13-L2 spinal segments. Within the T13-L1 spinal segments, p-CREB-IR was greatest in the MDH, LDH and DCN and expression in each of these regions was comparable within a segment. Following RAO, there was a significant (p < or = 0.001) increase in the percentage (86-98%) of p-CREB-IR spinal neurons expressing choline acetyltransferase (ChAT)-IR (a marker of preganglionic neurons) in the IML of the T10, T12 and L1 spinal segments examined. After ipsilateral RAO, expression of p-CREB-IR was increased in the ipsilateral, T8-L2 DRG with the greatest number of p-CREB-IR dorsal root ganglion cells being located in the L1 dorsal root ganglion. Retrograde tracing with Fluorogold (FG) to label renal afferent cells in the DRG revealed a significant (p < or = 0.01) increase in the percentage (75-86%) of renal afferent cells expressing p-CREB-IR after ipsilateral RAO. These studies demonstrate that p-CREB-IR is a useful tool for examining the distribution of spinal neurons and DRG involved in reflexes of renal origin. In addition, expression of p-CREB-IR may be coupled to late response genes that may exert long-term changes in neuronal function after RAO.

Animals↗
PubMed1998

Phosphorylation of the cAMP response element binding protein CREB by cAMP-dependent protein kinase A and glycogen synthase kinase-3 alters DNA-binding affinity, conformation, and increases net charge.

The cAMP response element binding protein CREB activates the transcription of genes in response to phosphorylation by cAMP-dependent protein kinase A (PKA) and other protein kinases. Phosphorylated CREB activates transcription by recruiting transcriptional co-activators such as the CREB binding protein. Here, we describe experiments that analyze the effects of phosphorylation on the DNA binding affinity of CREB and the structural characteristics of the CREB/DNA complex in solution. Analysis of deletion mutants of CREB indicate that amino acid sequences within the transactivation domain promote high-affinity binding of CREB to fluorescently labeled oligonucleotides containing cAMP response elements. In vitro experiments indicate that phosphorylation is processive between PKA as the initial kinase and glycogen synthase kinase-3 (GSK-3) but not casein kinase II as the secondary kinase. Fluorescent electrophoretic mobility shift assays show that phosphorylation by PKA results in a 3-5-fold increase in the binding affinity of CREB to both the symmetrical somatostatin CRE (SMS-CRE) and the asymmetric somatostatin upstream element (SMS-UE). Processive phosphorylation of CREB by GSK-3 attenuates the enhanced DNA binding in response to PKA thus acts as an inhibitor of PKA-induced binding. Ferguson plot analyses demonstrate that phosphorylation of CREB by PKA and GSK-3 result in an increase in the spherical size and the net positive surface charge of the CREB/DNA complex. Moreover, these analyses uncovered the unexpected finding that CREB associates as a tetramer both in the presence and absence of DNA. These findings suggest a model by which phosphorylation of CREB alters the secondary structure and charge characteristics of the CREB/DNA complex resulting in an alteration in binding affinity.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed2005

IGF-II induces CREB phosphorylation and cell survival in human lung cancer cells.

We have previously shown that lung tumors arising in MMTV-IGF-II transgenic mice displayed elevated levels of phosphorylated cAMP response element binding protein (CREB). To investigate the role that insulin-like growth factor II (IGF-II) and CREB play in human lung tumorigenesis, A549 and NCI-H358 cells were examined. In these cell lines, IGF-II administration enhances human tumor cell survival and CREB phosphorylation. Further, the effects of IGF-II on cell survival and CREB phosphorylation appeared to be mediated, at least in part, by activation of the Erk pathways, as inhibition of these signaling pathways reduced tumor cell survival and CREB phosphorylation. Specifically, Erk5 appeared as the predominant mediator of CREB phosporylation. To further verify the importance of CREB in human lung tumorigenesis, A549 and NCI-H358 cells were stably transfected with a vector containing a dominant negative CREB construct (KCREB). KCREB transfection significantly inhibited the soft agar growth of both human tumor cell lines. In contrast, overexpression of wild-type CREB in the normal human bronchial epithelial cell line, HBE135, enhanced soft agar growth. Therefore, our results indicate that CREB and its associated proteins play a significant role in lung adenocarcinoma and IGF-II induces CREB phosphorylation, at least in part, via the Erk5 signaling pathway.

Adenocarcinoma, Bronchiolo-Alveolar↗
PubMed1998

Stimuli which entrain the circadian clock of the neonatal Syrian hamster in vivo regulate the phosphorylation of the transcription factor CREB in the suprachiasmatic nucleus in vitro.

Photic resetting of the adult mammalian circadian clock in vivo is associated with phosphorylation of the Ser133 residue of the calcium/cyclic AMP response-element binding-protein (CREB) in the retinorecipient region of the suprachiasmatic nucleus (SCN). Western blotting and immunocytochemistry were used to investigate whether agonists known to reset the clock of neonatal hamsters in vivo are also able to influence the phosphorylation of CREB in the suprachiasmatic hypothalamus in vitro. Antisera raised against synthetic CREB peptide sequences were used to differentiate between total CREB and the Ser133 phosphorylated form of CREB (pCREB). Western blot analysis of proteins isolated from suprachiasmatic tissue of 1-day-old Syrian hamsters revealed bands at approximately 45 kDa corresponding to total CREB and pCREB. Treatment of the tissue with a mixture of glutamatergic agonists [N-methyl-D-aspartate (NMDA), amino-methyl proprionic acid (AMPA) and kainate, all at 1 microM], or native glutamate (1 microM) had no effect on the total CREB signal, but increased the pCREB signal, indicative of agonist-stimulated phosphorylation of CREB on Ser133. A similar effect was seen following treatment of the suprachiasmatic blocks with either dopamine (1 microM) or forskolin (1 microM). Simultaneous treatment with melatonin (1 microM) significantly attenuated stimulation by forskolin. The effect of the agonists on nuclear pCREB-immunoreactivity (-ir) was investigated in primary cultures which contained a mixture of cell types characteristic of the suprachiasmatic nuclei in vivo. Basal expression of nuclear total CREB-ir was high, whereas expression of pCREB-ir was low. Treatment with glutamate (1 microM) or dopamine (1 microM) had no effect on total CREB-ir, but increased pCREB-ir in approximately 50 and 30% of cells, respectively, whereas forskolin (1 microM) increased pCREB-ir in almost all cells (> 90%). The effects of all three agonists were rapid (< 15 min), and dose and time dependent. Melatonin reversed the effects of forskolin in mixed cultures, but not in pure astrocyte cultures. Dual-immunocytochemistry (ICC) revealed that glutamate (1 microM) increased nuclear pCREB-ir in cells immunoreactive for microtubule-associated protein II (MAP II-ir), but not other cells, indicating an effect predominantly on neurons. This occurred equally in gamma-amino butyric acid (GABA)-ir and non-GABA-ir neurons. Dopamine (1 microM) was more selective, increasing pCREB-ir only in GABA-ir neurons, whereas forskolin increased pCREB-ir in all cells. The specific stimulation of pCREB-ir in GABA-ir neurons by dopamine was reversed by melatonin, but melatonin had no effect on the increase in pCREB-ir induced in GABA-ir neurons by glutamate. These results demonstrate that agonists known to entrain the circadian clock in vivo modulate phosphorylation of CREB in GABA-ir neurons derived from the neonatal suprachiasmatic nuclei.

Animals↗