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      cAMP response element binding protein (CREB) activates transcription via two distinct genetic elements of the human glucose-6-phosphatase gene

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      1 , , 1 , 1
      BMC Molecular Biology
      BioMed Central

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          Abstract

          Background

          The enzyme glucose-6-phosphatase catalyzes the dephosphorylation of glucose-6-phosphatase to glucose, the final step in the gluconeogenic and glycogenolytic pathways. Expression of the glucose-6-phosphatase gene is induced by glucocorticoids and elevated levels of intracellular cAMP. The effect of cAMP in regulating glucose-6-phosphatase gene transcription was corroborated by the identification of two genetic motifs CRE1 and CRE2 in the human and murine glucose-6-phosphatase gene promoter that resemble cAMP response elements (CRE).

          Results

          The cAMP response element is a point of convergence for many extracellular and intracellular signals, including cAMP, calcium, and neurotrophins. The major CRE binding protein CREB, a member of the basic region leucine zipper (bZIP) family of transcription factors, requires phosphorylation to become a biologically active transcriptional activator. Since unphosphorylated CREB is transcriptionally silent simple overexpression studies cannot be performed to test the biological role of CRE-like sequences of the glucose-6-phosphatase gene. The use of a constitutively active CREB2/CREB fusion protein allowed us to uncouple the investigation of target genes of CREB from the variety of signaling pathways that lead to an activation of CREB. Here, we show that this constitutively active CREB2/CREB fusion protein strikingly enhanced reporter gene transcription mediated by either CRE1 or CRE2 derived from the glucose-6-phosphatase gene. Likewise, reporter gene transcription was enhanced following expression of the catalytic subunit of cAMP-dependent protein kinase (PKA) in the nucleus of transfected cells. In contrast, activating transcription factor 2 (ATF2), known to compete with CREB for binding to the canonical CRE sequence 5'-TGACGTCA-3', did not transactivate reporter genes containing CRE1, CRE2, or both CREs derived from the glucose-6-phosphatase gene.

          Conclusions

          Using a constitutively active CREB2/CREB fusion protein and a mutant of the PKA catalytic subunit that is targeted to the nucleus, we have shown that the glucose-6-phosphatase gene has two distinct genetic elements that function as bona fide CRE. This study further shows that the expression vectors encoding C2/CREB and catalytic subunit of PKA are valuable tools for the study of CREB-mediated gene transcription and the biological functions of CREB.

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          Most cited references37

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          Mediation by a CREB family transcription factor of NGF-dependent survival of sympathetic neurons.

          Nerve growth factor (NGF) and other neurotrophins support survival of neurons through processes that are incompletely understood. The transcription factor CREB is a critical mediator of NGF-dependent gene expression, but whether CREB family transcription factors regulate expression of genes that contribute to NGF-dependent survival of sympathetic neurons is unknown. CREB-mediated gene expression was both necessary for NGF-dependent survival and sufficient on its own to promote survival of sympathetic neurons. Moreover, expression of Bcl-2 was activated by NGF and other neurotrophins by a CREB-dependent transcriptional mechanism. Overexpression of Bcl-2 reduced the death-promoting effects of CREB inhibition. Together, these data support a model in which neurotrophins promote survival of neurons, in part through a mechanism involving CREB family transcription factor-dependent expression of genes encoding prosurvival factors.
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            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.
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              Differential signaling of cyclic AMP: opposing effects of exchange protein directly activated by cyclic AMP and cAMP-dependent protein kinase on protein kinase B activation.

              The recent discovery of Epac, a novel cAMP receptor protein, opens up a new dimension in studying cAMP-mediated cell signaling. It is conceivable that many of the cAMP functions previously attributed to cAMP-dependent protein kinase (PKA) are in fact also Epac-dependent. The finding of an additional intracellular cAMP receptor provides an opportunity to further dissect the divergent roles that cAMP exerts in different cell types. In this study, we probed cross-talk between cAMP signaling and the phosphatidylinositol 3-kinase/PKB pathways. Specifically, we examined the modulatory effects of cAMP on PKB activity by monitoring the specific roles that Epac and PKA play individually in regulating PKB activity. Our study suggests a complex regulatory scheme in which Epac and PKA mediate the opposing effects of cAMP on PKB regulation. Activation of Epac leads to a phosphatidylinositol 3-kinase-dependent PKB activation, while stimulation of PKA inhibits PKB activity. Furthermore, activation of PKB by Epac requires the proper subcellular targeting of Epac. The opposing effects of Epac and PKA on PKB activation provide a potential mechanism for the cell type-specific differential effects of cAMP. It is proposed that the net outcome of cAMP signaling is dependent upon the dynamic abundance and distribution of intracellular Epac and PKA.
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                Author and article information

                Journal
                BMC Mol Biol
                BMC Molecular Biology
                BioMed Central (London )
                1471-2199
                2005
                19 January 2005
                : 6
                : 2
                Affiliations
                [1 ]Department of Medical Biochemistry and Molecular Biology, Building 44, University of Saarland Medical Center, D-66421 Homburg, Germany
                Article
                1471-2199-6-2
                10.1186/1471-2199-6-2
                548273
                15659240
                656ad3fb-c9c8-4e6b-8501-6e5ec5df0d95
                Copyright © 2005 Thiel et al; licensee BioMed Central Ltd.

                This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

                History
                : 25 May 2004
                : 19 January 2005
                Categories
                Research Article

                Molecular biology
                Molecular biology

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