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cAMP-dependent protein kinase, but not the cGMP-dependent enzyme, rapidly phosphorylates delta-CREB, and a synthetic delta-CREB peptide.

Phosphorylation of the cAMP response element binding protein (CREB) by the catalytic subunit of cAMP-dependent protein kinase (cAK) has been implicated in the cAMP-dependent stimulation of gene transcription. delta-CREB, a spliced variant of CREB, and CREBtide (KRREILSRRPSYR), a synthetic peptide based on the phosphorylation sequence in delta-CREB, were tested as substrates of cAK. Phosphorylation of delta-CREB (0.17 microM) was stoichiometric within 30 s when using a concentration of cAK which approximated the intracellular level (0.2 microM). The rate of phosphorylation of delta-CREB was comparable to the rates of the best physiological substrates of cAK tested. The rate of CREBtide phosphorylation was at least as great as that of delta-CREB, indicating that the peptide retained the determinants of delta-CREB which were responsible for substrate efficacy. The apparent Km of CREBtide phosphorylation by cAK was 3.9 microM, which is 10-fold lower than that of kemptide (Km = 39 microM), the synthetic peptide substrate most often employed for cAK measurement. The Vmax values were 12.4 mumol/(min.mg) for CREBtide and 9.8 mumol/(min.mg) for kemptide. The apparent Km of CREBtide phosphorylation by cGMP-dependent protein kinase (cGK) was 2.9 microM and the Vmax value was 3.2 mumol/(min.mg). Both delta-CREB and CREBtide were phosphorylated at a much slower rate by cGK as compared with cAK, implying that the high cAK/cGK specificity exhibited by delta-CREB was retained by the peptide. Taken together, the results indicated that delta-CREB and CREBtide are among the best substrates tested for cAK and suggested that phosphorylation of CREB by this enzyme could occur in intact cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1992

Beta-adrenergic stimulation of cFOS via protein kinase A is mediated by cAMP regulatory element binding protein (CREB)-dependent and tissue-specific CREB-independent mechanisms in corticotrope cells.

Catecholamines stimulate proopiomelanocortin (POMC) gene expression in corticotrope cells, but the molecular mechanisms of these effects are not known. While beta-adrenergic receptors stimulate the protein kinase A (PKA) system, the POMC promoter does not have classical cAMP-response elements (CREs). Therefore, we investigated the induction of the c-fos protooncogen, previously shown to increase POMC transcription in AtT20 cells. In this corticotrope-derived cell line, we show that activation of beta-receptors with isoprenaline (Iso) induces a transient rise in c-fos mRNA levels. Gel mobility shift assays with a labeled AP1 consensus sequence (TGACTCA) showed induction of specific binding activity after Iso treatment. Cotransfection experiments with dominant inhibitory PKA mutants and reporter genes containing c-fos promoter sequences showed that c-fos induction by Iso is entirely dependent on a functional PKA activity. Furthermore, we show that beta-receptor induction of c-fos in corticotrophs is mediated by at least two distinct cAMP-responsive sequences. cAMP regulatory element binding (CREB)-dependent induction is observed on the CRE located at -60 bp on the c-fos promoter. A region located in the vicinity of the dyad symetry element (-290) is also found to mediate tissue-specific cAMP induction. Transcriptional activation by this site, although sensitive to PKA antagonism, is not blocked by CREB mutants.

Animals↗
PubMed1990

CREB regulation of cellular cyclic AMP-responsive and adenovirus early promoters.

The cyclic AMP (cAMP) response element-binding protein (CREB) has been demonstrated to be a key mediator of cellular promoter response to cAMP. The binding site for this protein in many cellular cAMP inducible promoters (CRE) contains the palindrome sequence TGACGTCA, which contains two half-sites for CREB binding. A related promoter element, with the core sequence TGACG, has significant homology to an AP1-binding site and contains only one half-site for CREB binding. A group of factors known as activating transcription factors (ATF) have been found to bind to the latter and related sequences found upstream of early adenovirus promoters induced by E1A, and these factors are highly homologous to the CREB protein. We wished to characterize CREB, c-jun, and c-fos binding to these sites in the somatostatin gene (CRE) and in the adenovirus early region 3 promoter (E3/ATF). Oligonucleotides complementary to each of these sites were used in gel retardation assays with in vitro-translated CREB protein. These studies indicated that CREB bound primarily as a dimer to both a single and two half-sites, though there was increased affinity to the double compared with the single half-site. The c-jun and c-fos proteins also bound to both the somatostatin CRE- and E3/ATF-binding sites, but CREB did not bind to AP1 recognition sites nor was it capable of forming heterodimers with either c-jun or c-fos. Truncations of the CREB protein, which eliminated regions of the protein containing consensus sites for phosphorylation by protein kinase A, protein kinase C, and casein kinase II, bound to both the CRE and ATF sites, indicating that these consensus sites were not essential for DNA binding or dimer formation. Transfection of CREB and protein kinase A expression constructs into F9 cells with promoters containing either a single or two half-sites for CREB binding indicated that CREB was capable of similar levels of activation of these constructs. However, the fold activation by CREB was higher for constructs containing a single half-site compared with those containing two half-sites. These results demonstrate that multiple mechanisms may regulate CREB binding, including variations in the sequences in the promoter-binding site and the presence of related DNA-binding proteins.

Adenoviridae↗
PubMed1991

Developmental stage-specific expression of cyclic adenosine 3',5'-monophosphate response element-binding protein CREB during spermatogenesis involves alternative exon splicing.

Spermatogenesis is a temporally regulated developmental process by which the gonadotropin-responsive somatic Sertoli and Leydig cells act interdependently to direct the maturation of the germinal cells. The metabolism of Sertoli and Leydig cells is regulated by the pituitary gonadotropins FSH and LH, which, in turn, activate adenylate cyclase. Because the cAMP-second messenger pathway is activated by FSH and LH, we postulated that the cAMP-responsive element-binding protein (CREB) plays a physiological role in Sertoli and Leydig cells, respectively. Immunocytochemical analyses of rat testicular sections show a remarkably high expression of CREB in the haploid round spermatids and, to some extent, in pachytene spermatocytes and Sertoli cells. Although most of the CREB antigen is detected in the nuclei, some CREB antigen is also present in the cytoplasm. Remarkably, the cytoplasmic CREB results from the translation of a unique alternatively spliced transcript of the CREB gene that incorporates an exon containing multiple stop codons inserted immediately up-stream of the exons encoding the DNA-binding domain of CREB. Thus, the RNA containing the alternatively spliced exon encodes a truncated transcriptional transactivator protein lacking both the DNA-binding domain and nuclear translocation signal of CREB. Most of the CREB transcripts detected in the germinal cells contain the alternatively spliced exon, suggesting a function of the exon to modulate the synthesis of CREB. In the Sertoli cells we observed a striking cyclical (12-day periodicity) increase in the levels of CREB mRNA that coincides with the splicing out of the restrictive exon containing the stop codons. Because earlier studies established that FSH-stimulated cAMP levels in Sertoli cells are also cyclical, and the CREB gene promoter contains cAMP-responsive enhancers, we suggest that the alternative RNA splicing controls a positive autoregulation of CREB gene expression mediated by cAMP.

Animals↗
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↗
PubMed1992

The transcription factors c-JUN, JUN D and CREB, but not FOS and KROX-24, are differentially regulated in axotomized neurons following transection of rat sciatic nerve.

In adult rats, expression of c-JUN, JUN B, JUN D, c-FOS, FOS B, KROX-24 and CREB proteins was investigated by immunocytochemistry in L4 and L5 dorsal root ganglia and lumbar spinal cord for up to 300 days following transection of the left sciatic nerve. In dorsal root ganglia, expressions of c-JUN and JUN D were increased 10 h and 15 h after sciatic nerve transection, respectively. c-JUN was still at an elevated level after 300 days predominantly in small diameter neurons, whereas JUN D had declined to control levels after 100 days. In contrast to the JUN proteins, expression of CREB showed a delayed onset after 10 days and reached a maximum between 70 and 150 days. In motoneurons, expression of c-JUN and JUN D was increased 15 h and 25 h after sciatic nerve transection, respectively. Expression of c-JUN remained increased after 150 days, whereas JUN D had declined to control levels after 70 days. In contrast, expression of CREB declined within 30 h in axotomized motoneurons and remained on a reduced level for up to 150 days. JUN B, c-FOS, FOS B and KROX-24 were not induced either following axotomy or following a repeated nerve crush. Sciatic nerve transection including the surgical procedure transynaptically provoked a transient expression of all JUN, FOS and KROX-24 proteins in neurons of spinal dorsal horn which disappeared after 5 days except the expression of JUN D which lasted for up to 20 days. In contrast, CREB immunoreactivity was not at all altered in neurons of spinal dorsal horn. In untreated animals, CREB and to a lesser extent JUN D showed an ubiquitous expression in neurons and glia cells of spinal cord, whereas expression of c-JUN and a weak expression of FOS B were restricted to motoneurons. In neurons of the dorsal root ganglia, a basal expression was found for c-JUN, JUN D and CREB and, at a low level, for FOS B and KROX-24. c-JUN and JUN D were colocalized with CREB in many cells such as interneurons, motoneurons, dorsal root ganglion cells and glial cells indicating the possibility for both the control of c-jun and jun D expression by CREB and the competition of JUN and CREB proteins for CRE consensus sequences.

Animals↗
PubMed1992

Multiple mRNA isoforms of the transcription activator protein CREB: generation by alternative splicing and specific expression in primary spermatocytes.

We have characterized cDNA clones representing mouse CREB (cyclic AMP responsive element binding protein) mRNA isoforms. These include CREB delta and CREB alpha, of which the rat and human homologues have been previously identified. Both encode proteins with CRE-binding activity and identical transactivation potential. The additional CREB mRNA isoforms potentially encode CREB related proteins. From the structural organization of the mouse CREB gene we conclude that the multiple transcripts are generated by alternative splicing. Furthermore we show that specific CREB mRNA isoforms are expressed at a high level in the adult testis. Expression of these isoforms is induced after commencement of spermatogenesis. In situ hybridization suggests that this expression occurs predominantly in the primary spermatocytes. Comparison of the CREB gene with the recently isolated CREM (cAMP responsive element modulator) cDNAs illustrates that the two genes have arisen by gene duplication and have diverged to encode transcriptional activators and repressors of the cAMP signal transduction pathway.

Amino Acid Sequence↗
PubMed1989

A cluster of phosphorylation sites on the cyclic AMP-regulated nuclear factor CREB predicted by its sequence.

Cyclic AMP regulates the expression of a number of genes through a conserved promoter element, the CRE1. Moreover, transcriptional induction by cAMP requires the activation of cAMP-dependent protein kinase (protein kinase A). We have previously characterized the cAMP response element binding protein (CREB) in PC12 cells and brain tissue as a nuclear factor, of relative molecular mass 43,000, whose transcriptional efficacy is regulated by protein kinase A phosphorylation. CREB stimulates transcription on binding to the CRE as a dimer. Experiments suggesting that the dimerization and transcriptional efficacy of CREB are each stimulated by phosphorylation at distinct sites prompted us to suggest that CREB is regulated by multiple kinases in vivo. We now report the isolation of a cDNA clone for rat CREB using amino-acid sequence information from purified CREB protein. Sequence analysis of this CREB cDNA predicts a cluster of protein kinase A, protein kinase C and casein kinase II consensus recognition sites near the N terminus of the protein. The proximity of these potential phosphorylation sites to one another indicates that they may interact either positively or negatively to regulate CREB bioactivity.

Activating Transcription Factor 2↗
PubMed1990

Two distinct forms of active transcription factor CREB (cAMP response element binding protein).

Mammalian cells express two distinct forms of transcription factor CREB (cAMP response element binding protein) that are apparently the products of alternative splicing of the CREB gene transcript. The two proteins differ by a 14-amino acid serine-rich insertion present in one of the CREB isoforms. We show that both CREB isoforms are expressed in many cell types and mammalian species. Both encode proteins that bind specifically to a cAMP response element in vitro. As expected for proteins of this class, the CREB proteins bind DNA as dimers. Both proteins impart cAMP-regulated transcriptional activity to a heterologous DNA-binding domain, showing that cAMP directly modulates the transcriptional stimulatory activity of CREB. The presence of multiple CREB isoforms with identical DNA-binding specificities but differences in the presumed regulatory domain raises the possibility that CREB proteins may be able to integrate distinct regulatory signals at the level of gene transcription.

Amino Acid Sequence↗
PubMed1990

DNA-binding and dimerization domains of adenosine 3',5'- cyclic monophosphate-responsive protein CREB reside in the carboxyl-terminal 66 amino acids.

The expression of genes in response to cAMP is mediated by one or more trans-activator proteins, CREBs, that bind to cAMP-responsive enhancers (CREs) of the general motif 5'-TGACGTCA-3'. The carboxyl-terminal amino acid sequences of two isoforms of CREB, CREB-327 and CREB-341, deduced from the cDNAs consist of a positively charged (basic) region adjacent to a leucine zipper motif. Three peptides corresponding to the hypothetical DNA-binding and dimerization domains of CREB-327 were synthesized. A peptide that includes both the basic and leucine zipper domains binds to the CRE specifically. Moreover, this peptide readily forms CRE-binding heterodimers with full-length CREB both synthesized by in vitro cell-free translation and isolated from PC-12 cells, but did not heterodimerize with in vitro translated jun or fos. Two other peptides, either partially or totally lacking the basic region, but containing the intact leucine zipper domain, readily form dimers but do not bind to the CRE. We conclude that the carboxy-terminal basic and leucine zipper regions are necessary and sufficient for specific binding of CREB to the CRE as a homodimer. The leucine zipper domain is responsible for the dimerization, and the basic region confers binding specificity for the CRE. Heterodimerization of CREB-327 does not form heterodimers with jun or fos.

Amino Acid Sequence↗
PubMed1992

Direct interaction of CREB protein with 21 bp Tax-response elements of HTLV-ILTR.

The three 21-bp repeats (Tax-responsive elements) of the long terminal repeat (LTR) of the human T-cell leukemia virus (HTLV-I) mediates the response of the Tax protein. All three Tax-responsive elements (TREs) contain a TGACG motif, reminiscent of the CREB/ATF-binding site TGACGTCA. DNA-affinity chromatography with the 5'-TRE resulted in a previous study in proteins of about 32, 36 to 42, 50 and 110 kDa. Here we demonstrate that the 42 kDa protein is the cAMP-response element-binding (CREB) protein. This is shown by phosphorylation of the proteins eluted from the DNA-affinity column with protein kinase A (PKA) in vitro and subsequent indirect immunoprecipitation with a CREB-specific antiserum raised against an internal CREB-specific peptide. This method allows detection of phosphorylated proteins by autoradiography with high sensitivity and is superior to metabolic labeling. One of the phosphorylated proteins co-migrates with immuno-affinity-purified CREB protein--also phosphorylated in vitro--and competes with the peptide antigen, which proves the specificity of the reaction. The purified CREB protein leads to specific DNA-protein complexes in DNA mobility-shift analyses with all three TREs. Comparison of these TRE-CREB complexes with those formed by nuclear extracts from the HTLV-I-transformed T-cell line C81-66-45 indicates that additional cellular factors contribute to the complexes, especially to the middle TRE. This is also shown by using CREB-depleted instead of complete nuclear extracts for DNA mobility-shift assays. Antibodies against CREB but not Tax affect the mobility of the DNA-protein complex.

Base Sequence↗
PubMed1990

Both the basic region and the 'leucine zipper' domain of the cyclic AMP response element binding (CREB) protein are essential for transcriptional activation.

Second messengers like cAMP can activate the transcription of genes containing consesus cAMP response element (CRE). A 43 kd nuclear phosphoprotein previously identified as the cAMP response element binding (CREB) protein has been shown to bind as a dimer to CRE and activate gene transcription. The rat and human CREB protein contain the 'leucine zipper' motif. We have analyzed the role of both leucine zipper domain and the amino-terminal basic region by making site-specific mutations. Our results show that the first three leucines int he leucine zipper domain are essential for efficient dimer formation. Mutations of two consecutive leucines in the leucine zipper domain completely abolish the ability to form dimers. Mutant CREB protein unable to form homodimers is also unable to bind to DNA. In contrast, however, mutations, in the DNA binding region had no effect on dimer formation but were unable to bind to CRE sites or activate transcription. We propose that CREB protein functions by forming homodimers which bind to CRE and activate transcription. Furthermore, the CREB protein needs to be phosphorylated before activating transcription. Finally, we show that the CREB basic region mutant acts as a trans-dominant transcriptional suppressor of wild-type CREB function.

Animals↗
PubMed1990

Characterization of a bipartite activator domain in transcription factor CREB.

In this paper, we characterize a trans-activating region in CREB, termed alpha, that interacts cooperatively with the kinase A phosphorylation motif to stimulate transcription. The alpha region appears to be encoded by an alternate exon that is deleted in a CREB-related cDNA named delta CREB. Both proteins are expressed in eukaryotic cells, although the activity of CREB is 10-fold higher than that of delta CREB. Circular dichroism data on a synthetic "alpha peptide" combined with results from in vitro mutagenesis experiments support the hypothesis that the alpha region contains an amphipathic alpha helix whose structure is critical to CREB activity. We propose that phosphorylation by kinase A may stimulate CREB activity in part by modulating the structure of alpha and thus may stimulate its ability to interact with other proteins in the polymerase II complex.

Adrenal Gland Neoplasms↗
PubMed1992

The mouse CREB (cAMP responsive element binding protein) gene: structure, promoter analysis, and chromosomal localization.

In this paper we report the isolation and characterization of the mouse CREB gene. It is composed of 11 exons and 10 introns and spans a region of 70 kb. BR-A and BR-B, the two alpha-helical regions of the proposed basic DNA binding domain of CREB, are encoded separately on exons 10 and 11. The mouse CREB gene is expressed from a promoter that is situated in a CpG island. The promoter contains no TATA or CCAAT box homologies but has a number of putative binding sites for the acidic transcriptional activator Sp1 and a 9/11 match with the initiator region. Transcriptional start site mapping identified five major start sites spread over at least 41 nucleotides. Northern blot analysis indicated that expression of the CREB gene is almost ubiquitous with expression at differing levels of multiple transcripts. Testis expressed a predominant RNA species of approximately 1.6 kb. The CREB gene was found to be single copy in the mouse and well conserved through evolution. Finally Creb-1, the CREB locus, was mapped to the proximal region of mouse chromosome 1.

Amino Acid Sequence↗
PubMed1991

Somatotroph hypoplasia and dwarfism in transgenic mice expressing a non-phosphorylatable CREB mutant.

Most of the transcriptional effects of cyclic AMP are mediated by the cAMP response element binding protein (CREB). After activation of cAMP-dependent protein kinase A, the catalytic subunits of this enzyme apparently mediate the phosphorylation and activation of CREB. As cAMP serves as a mitogenic signal for anterior pituitary somatotrophic cells, we investigated whether CREB similarly regulates proliferation of these cells. We prepared transgenic mice expressing a transcriptionally inactive mutant of CREB (CREBM1), which cannot be phosphorylated, in cells of the anterior pituitary. If CREB activity is required for proliferation, the overexpressed mutant protein would effectively compete with wild-type CREB activity and thereby block the response to cAMP. As predicted, the CREBM1 transgenic mice exhibited a dwarf phenotype with atrophied pituitary glands markedly deficient in somatotroph but not other cell types. We conclude that transcriptional activation of CREB is necessary for the normal development of a highly restricted cell type, and that environmental cues, possibly provided by the hypothalamic growth hormone-releasing factor, are necessary for population of the pituitary by somatotrophic cells.

Amino Acid Sequence↗
PubMed1992

3',5'-cyclic adenosine monophosphate-regulated enhancer binding (CREB) activity is required for normal growth and differentiated phenotype in the FRTL5 thyroid follicular cell line.

The thyroid follicular cell requires elevated levels of cAMP for normal growth and optimal expression of the differentiated phenotype. The recent discovery of cAMP-regulated enhancer binding (CREB) proteins prompted us to analyze the possible role of these transcription factors in controlling thyroid cell growth and differentiated phenotype using the FRTL5 thyroid cell line as a model system. FRTL5 cells were stably transfected with an expression vector containing either the gene for wild type CREB (WTCREB) or a dominant negative mutant form of CREB, termed KCREB, which dimerizes with and inactivates endogenous CREB. Transfected clones were found to express the transfected KCREB and WTCREB mRNAs at higher levels than the endogenous CREB mRNA. Transient expression of a somatostatin-chloramphenicol acetyltransferase fusion gene in these clones demonstrated a 60% reduction of cAMP-regulated enhancer-dependent transcriptional activity in the KCREB transfected clones and wild type levels of activity in the WTCREB transfected clones. Parameters of growth (DNA synthesis and growth rate) and differentiation (iodide uptake and thyroglobulin mRNA levels) were then analyzed in the transfected clones. Transfection of WTCREB had no effect on any of the parameters examined in comparison to untransfected cells, presumably because CREB is already constitutively expressed at maximal levels in normal FRTL5 cells. However, cells expressing KCREB showed an 18-40% reduction in TSH-stimulated thymidine incorporation, a 31% increase in the length of the cell cycle, and a 4-fold reduction in TSH-stimulated iodide uptake in comparison with wild type cells or cells tranfected with wild type CREB.(ABSTRACT TRUNCATED AT 250 WORDS)

3T3 Cells↗
PubMed1992

Phosphorylation of CREB affects its binding to high and low affinity sites: implications for cAMP induced gene transcription.

Cyclic AMP treatment of hepatoma cells leads to increased protein binding at the cyclic AMP response element (CRE) of the tyrosine aminotransferase (TAT) gene in vivo, as revealed by genomic footprinting, whereas no increase is observed at the CRE of the phosphoenolpyruvate carboxykinase (PEPCK) gene. Several criteria establish that the 43 kDa CREB protein is interacting with both of these sites. Two classes of CRE with different affinity for CREB are described. One class, including the TATCRE, is characterized by asymmetric and weak binding sites (CGTCA), whereas the second class containing symmetrical TGACGTCA sites shows a much higher binding affinity for CREB. Both classes show an increase in binding after phosphorylation of CREB by protein kinase A (PKA). An in vivo phosphorylation-dependent change in binding of CREB increases the occupancy of weak binding sites used for transactivation, such as the TATCRE, while high affinity sites may have constitutive binding of transcriptionally active and inactive CREB dimers, as demonstrated by in vivo footprinting at the PEPCK CRE. Thus, lower basal level and higher relative stimulation of transcription by cyclic AMP through low affinity CREs should result, allowing finely tuned control of gene activation.

Amino Acid Sequence↗
PubMed1989

Cyclic AMP stimulates somatostatin gene transcription by phosphorylation of CREB at serine 133.

In this paper, we demonstrate that phosphorylation of CREB at Ser-133 is induced 6-fold in vivo, following treatment of PC12 cells with forskolin. By contrast, no such induction was observed in the kinase A-deficient PC12 line A126-1B2 (A126). Using F9 teratocarcinoma cells, which are unresponsive to cAMP, we initiated a series of transient expression experiments to establish a causal link between phosphorylation of CREB and trans-activation of cAMP-responsive genes. Inactivating the kinase A phosphorylation site by in vitro mutagenesis of the cloned CREB cDNA at Ser-133 completely abolished CREB transcriptional activity. As CREB mutants containing acidic residues in place of the Ser-133 phosphoacceptor were also transcriptionally inactive, these results suggest that phosphorylation of CREB may stimulate transcription by a mechanism other than by simply providing negative charge.

Adrenal Gland Neoplasms↗