Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.11.1 (protein kinase)
 81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The second messenger cAMP stimulates the expression of numerous genes via the protein kinase A-mediated phosphorylation of the cAMP response element-binding protein (CREB) at Ser-133. Ser-133 phosphorylation, in turn, appears to induce target gene expression by promoting interaction between CREB and CBP, a 265-kDa nuclear phospho-CREB-binding protein. It is unclear, however, whether Ser-133 phosphorylation per se is sufficient for CREB-CBP complex formation and for target gene induction in vivo. Here we examine CREB activity in Jurkat T cells after stimulation of the T-cell receptor (TCR), an event that leads to calcium entry and diacylglycerol production. Triggering of the TCR stimulated Ser-133 phosphorylation of CREB with high stoichiometry, but TCR activation did not promote CREB-CBP complex formation or target gene induction unless suboptimal doses of cAMP agonist were provided as a costimulus. Our results demonstrate that, in addition to mediating Ser-133 phosphorylation of CREB, protein kinase A regulates additional proteins that are required for recruitment of the transcriptional apparatus to cAMP-responsive genes.
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PMID:Multiple protein kinase A-regulated events are required for transcriptional induction by cAMP. 747 32

Cyclic AMP-regulated gene expression frequently involves a DNA element known as the cAMP-regulated enhancer (CRE). Many transcription factors bind to this element, including the protein CREB, which is activated as a result of phosphorylation by protein kinase A. This modification stimulates interaction with one or more of the general transcription factors or, alternatively, allows recruitment of a co-activator. Here we report that CREB phosphorylated by protein kinase A binds specifically to a nuclear protein of M(r) 265K which we term CBP (for CREB-binding protein). Fusion of a heterologous DNA-binding domain to the amino terminus of CBP enables the chimaeric protein to function as a protein kinase A-regulated transcriptional activator. We propose that CBP may participate in cAMP-regulated gene expression by interacting with the activated phosphorylated form of CREB.
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PMID:Phosphorylated CREB binds specifically to the nuclear protein CBP. 841 73

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.
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PMID:Phosphorylation of CREB at Ser-133 induces complex formation with CREB-binding protein via a direct mechanism. 855 98

CBP (CREB-binding protein) is a transcriptional coactivator of CREB (cAMP response element-binding) protein, which is directly phosphorylated by PKA (cAMP-dependent protein kinase A). CBP interacts with the activated phosphorylated form of CREB but not with the nonphosphorylated form. We report here that CBP is also a coactivator of the c-myb proto-oncogene product (c-Myb), which is a sequence-specific transcriptional activator. CBP directly binds to the region containing the transcriptional activation domain of c-Myb in a phosphorylation-independent manner in vitro. The domain of CBP that touches c-Myb is also required for binding to CREB. A c-Myb/CBP complex in vivo was demonstrated by a yeast two-hybrid assay. CBP stimulates the c-Myb-dependent transcriptional activation. Conversely, the expression of antisense RNA of CBP represses c-Myb-induced transcriptional activation. In addition, adenovirus EIA, which binds to CBP, inhibits c-Myb-induced transcriptional activation. Our data thus identify CBP as a coactivator of c-Myb. These results suggest that CBP functions as a coactivator for more transcriptional activators than were thought previously.
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PMID:CBP as a transcriptional coactivator of c-Myb. 859 84

The transcription factor CREB (cAMP responsive element binding protein) is activated by protein kinase A (PKA) phosphorylation of a single serine residue. To investigate possible mechanisms of CREB regulation by phosphorylation, we initiated a structural and biophysical characterization of the full-length, wild-type CREB protein, an altered CREB protein (CREB/SER) in which the three cysteine residues in the DNA-binding domain were replaced with serine residues and a truncated protein (ACT265) which encompasses the entire activation domain of CREB. Circular dichroism (CD) reveals that CREB and CREB/SER have identical secondary structures and contain approximately 20% alpha-helix, 9% beta-strand, 34% beta-turn, and 37% random coil structures. PKA phosphorylation does not alter the CD spectra, and therefore the secondary structure, of CREB or of CREB bound to DNA. Protease cleavage patterns indicate that PKA phosphorylation does not induce a global conformational change in CREB. Furthermore, PKA phosphorylation does not change the DNA binding affinity of CREB for either canonical or non-canonical CRE sequences as measured by a fluorescence anisotropy DNA binding assay. Since PKA phosphorylation of CREB results in its specific binding to the transcriptional co-activators CREB-binding protein and p300, we suggest that the PKA activation of CREB occurs by the production of specific, complementary interactions with these proteins, rather than through the previously proposed mechanisms of a phosphorylation-dependent conformational change or increased DNA binding affinity.
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PMID:Analysis of the structural properties of cAMP-responsive element-binding protein (CREB) and phosphorylated CREB. 866 19

Cyclic adenosine monophosphate (cAMP) stimulates transcription of somatostatin and other target genes with burst-attenuation kinetics. The kinetics of protein kinase (PK-A)-dependent cAMP response element binding protein (CREB) phosphorylation closely parallel the changes in transcription of cAMP-responsive genes by run-on assay. Nuclear translocation of PK-A, visualized by microinjection of fluorescently labeled PK-A holoenzyme, appears to represent the rate-limiting step in CREB phosphorylation and transcriptional activation. We and others have recently characterized a CREB-binding protein (CBP), which specifically recognizes sequences within the Ser133 phosphorylated form of CREB. CBP does not regulate the DNA binding, dimerization, or nuclear targeting properties of CREB, but binds selectively to the kinase-inducible 60 amino acid trans-activation domain (KID) of CREB, critical for PK-A-inducible transcription. We developed an antiserum directed against amino acid 634-648 within the CREB-binding domain of CBP. We detected a 265-kd polypeptide by Western blot as predicted from the cDNA, which coincided with the predominant phospho-CREB-binding activity in Hela nuclear extracts by "Far Western" blot assay. An identical phospho-CREB-binding activity was also found in NIH-3T3 cells. This phospho-CREB-binding protein appeared to be specific for Ser133-phosphorylated CREB, because no such band was detected with CREB labeled to the same specific activity at a nonregulatory phosphoacceptor site (Ser156) by casein kinase II (CKII). Following microinjection into nuclei of NIH-3T3 cells, a cAMP response element (CRE)-lacZ reporter was markedly induced by treatment with 8-Br cAMP plus isobutyl methyl xanthine (IBMX). Coinjection of CBP antiserum with the CRE-lacZ plasmid inhibited cAMP-dependent activity in a dose-dependent manner, but control immunoglobulin G (lgG) had no effect on this response. We can now begin reconstituting PK-A-dependent transcription in vitro, using well-characterized proteins such as CREB, TAF 110, and CBP. The assembly of such factors on cAMP-regulated promoters like somatostain may enable responsiveness to a variety of hormonal stimuli that employ cAMP as their second messenger.
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PMID:Regulation of somatostatin gene transcription by cyclic adenosine monophosphate. 876 68

CREB-binding protein (CBP) functions as a coactivator molecule for a number of transcription factors including CREB, c-Fos, c-Jun, c-Myb, and several nuclear receptors. Although binding sites for these factors within CBP have been identified, the regions of CBP responsible for transcriptional activation are unknown. In this report, we show that the N-terminal half of CBP is sufficient for activation of CREB-mediated transcription and that this region contains a strong transcriptional activation domain (TAD). Both deletion of this TAD or sequestering of factors that the TAD binds using a squelching assay were found to greatly decrease the ability of CBP to activate CREB-mediated transcription. In vivo studies by others have shown that p300/CBP associates with TBP; using an in vitro approach, we show the N-terminal TAD binds TBP. We also examined the ability of the C terminus of CBP to activate transcription using GAL-CBP chimeras. With this approach, we identified two C-terminal TADs located adjacent to the c-Fos binding site. In previous studies, cAMP-dependent protein kinase A (PKA) increased the transcriptional activity of a GAL full-length CBP chimera in F9 cells, and of the C terminus in PC-12 cells. Here, we demonstrate that PKA also increased the ability of the N-terminal TADs of CBP to activate transcription in PC-12 but not F9 or COS-7 cells, suggesting that this PKA-responsiveness is cell type-specific.
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PMID:CREB-binding protein activates transcription through multiple domains. 891 Apr 28

Chromogranin A (CgA), a member of the granin/secretogranin family of acidic glycoproteins that play multiple roles in the process of regulated secretion of peptide hormones and neurotransmitters, is specifically expressed in endocrine and neuroendocrine cells. We previously cloned and characterized the human (h) CgA gene and showed that nucleotides -55 to +32 relative to the transcriptional start site that contain a consensus cAMP element (CRE) and TATA-box motif were sufficient for neuroendocrine cell-specific expression. Here, we examined the role of the well conserved CRE in basal and cAMP-stimulated transcription in neuroendocrine cells. Transient transfection studies with hCgA gene promoter/chloroamphenical acetyl transferase (CAT) reporter constructs were conducted in a panel of neuroendocrine cell lines as well as in nonendocrine cell lines. Deletion or mutation of the CRE resulted in loss of neuroendocrine cell specific transcriptional activity. Mutation of a well conserved region (the TG-box) located between the CRE and the TATA box had no effect or resulted in only a modest decrease in activity. Mutation of the CRE in 5'-extended (-2300 to +32 and -700 to +32) constructs resulted in a 50-75% decrease in basal activity in neuroendocrine cells. This emphasized the importance of the CRE in basal transcription and also suggested that other elements between -700 and -55 may act independently of the CRE to contribute to full basal activity in some neuroendocrine cells. Dibutyryl cAMP stimulated transcriptional activity in neuroendocrine cells, and this was abolished by mutation of the CRE. In the presence of a PKA inhibitor, dibutyryl cAMP-induced activity was completely abolished and basal activity was decreased by up to 85%. Similar protein-DNA complexes were formed in gel retardation assays with a CgA-CRE oligonucleotide and nuclear extracts from both neuroendocrine and nonendocrine cells. A predominant complex that was supershifted by addition of a CREB antibody was identical in all cell types. By immunoblot analysis, levels of total CREB protein and phosphorylated (Ser 133) CREB did not differ between neuroendocrine and nonendocrine cells. Phosphorylated CREB was increased by forskolin treatment, an effect that was blocked by a PKA-inhibitor. Expression of the transcriptional cointegrator, CREB-binding protein (CBP), assessed by both RT-PCR and Western blot analysis, did not differ between neuroendocrine and nonendocrine cells. In summary, the CRE in the hCgA gene proximal promoter is critical for both basal and cAMP-induced expression in neuroendocrine cells via a PKA-mediated pathway. However, the neuroendocrine specificity of hCgA gene transcription mediated by the CRE is not a function of levels of total CREB or phosphorylated CREB or its cointegrator CBP. Specificity may be achieved by a PKA-responsive CRE-binding protein other than CREB expressed specifically in neuroendocrine cells, expression of a repressor molecule that binds CREB in nonendocrine cells, or may lie downstream of a CRE-binding protein, e.g. in the activity or amount of cointegrators other than CBP, which are required to couple transactivators to the basal transcriptional machinery.
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PMID:Analysis of molecular mechanisms controlling neuroendocrine cell specific transcription of the chromogranin A gene. 949 53

The involvement of serine/threonine protein phosphatases in signaling pathways that control the expression of the cyclooxygenase-2 (COX-2) gene in human chondrocytes was examined. Okadaic acid (OKA), an inhibitor of protein phosphatases 1 (PP-1) and 2A (PP-2A), induced a delayed, time-dependent increase in the rate of COX-2 gene transcription (runoff assay) resulting in increased steady-state mRNA levels and enzyme synthesis. The latter response was dose dependent over a narrow range of 1-30 nmol/L with declining expression and synthesis of COX-2 at higher concentrations due to cell toxicity. The delayed increase in COX-2 mRNA expression was accompanied by the induction of the proto-oncogenes c-jun, junB, junD, and c-fos (but not FosB or Fra-1). Increased phosphorylation of CREB-1/ATF-1 transcription factors was observed beginning at 4 h and reached a zenith at 8 h. Gel-shift analysis confirmed the up-regulation of AP-1 and CRE nuclear binding proteins, though there was little or no OKA-induced nuclear protein binding to SP-1, AP-2, NF-kappaB or NF-IL-6 regulatory elements. OKA-induced nuclear protein binding to 32P-CRE oligonucleotides was abrogated by a pharmacological inhibitor of protein kinase A (PKA), KT-5720; the latter compound also inhibited OKA-induced COX-2 enzyme synthesis. Calphostin C (CalC), an inhibitor of PKC isoenzymes, had little effect in this regard. Inhibition of 12P-CRE binding was also observed in the presence of an antibody to CREB-binding protein (265-kDa CBP), an integrator and coactivator of cAMP-responsive genes. The binding to 32P-CRE was unaffected in the presence of excess radioinert AP-1 and COX-2 NF-IL-6 oligonucleotides, although a COX-2 CRE-oligo competed very efficiently. 32P-AP-1 consensus sequence binding was unaffected by incubation of chondrocytes with KT-5720 or CalC, but was dramatically diminished by excess radioinert AP-1 and CRE-COX-2 oligos. Supershift analysis in the presence of antibodies to c-Jun, c-Fos, JunD, and JunB suggested that AP-1 complexes were composed of c-Fos, JunB, and possibly c-Jun. OKA has no effect on total cellular PKC activity but caused a delayed time-dependent increase in total PKA activity and synthesis. OKA suppressed the activity of the MAP kinases, ERK1/2 in a time-dependent fashion, suggesting that the Raf-1/MEKK1/MEK1/ERK1,2 cascade was compromised by OKA treatment. By contrast, OKA caused a dramatic increase in SAPK/JNK expression and activity, indicative of an activation of MEKK1/JNKK/SAPK/JNK pathway. OKA stimulated a dose-dependent activation of CAT activity using transfected promoter-CAT constructs harboring the regulatory elements AP-1 (c-jun promoter) and CRE (CRE-tkCAT). We conclude that in primary phenotypically stable human chondrocytes, COX-2 gene expression may be controlled by critical phosphatases that interact with phosphorylation dependent (e.g., MAP kinases:AP-1, PKA:CREB/ATF) signaling pathways. AP-1 and CREB/ATF families of transcription factors may be important substrates for PP-1/PP-2A in human chondrocytes.
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PMID:Transcriptional induction of cyclooxygenase-2 gene by okadaic acid inhibition of phosphatase activity in human chondrocytes: co-stimulation of AP-1 and CRE nuclear binding proteins. 962 Jan 67

Calcium is the principal second messenger in the control of gene expression by electrical activity in neurons. Recruitment of the coactivator CREB-binding protein, CBP, by the prototypical calcium-responsive transcription factor, CREB and stimulation of CBP activity by nuclear calcium signals is one mechanism through which calcium influx into excitable cells activates gene expression. Here we show that another CBP-interacting transcription factor, c-Jun, can mediate transcriptional activation upon activation of L-type voltage-gated calcium channels. Calcium-activated transcription mediated by c-Jun functions in the absence of stimulation of the c-Jun N-terminal protein kinase (JNK/SAPK1) signalling pathway and does not require c-Jun amino acid residues Ser63 and Ser73, the two major phosphorylation sites that regulate c-Jun activity in response to stress signals. Similar to CREB-mediated transcription, activation of c-Jun-mediated transcription by calcium signals requires calcium/ calmodulin-dependent protein kinases and is dependent on CBP function. These results identify c-Jun as a calcium-regulated transcriptional activator and suggest that control of coactivator function (i.e. recruitment of CBP and stimulation of CBP activity) is a general mechanism for gene regulation by calcium signals.
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PMID:c-Jun functions as a calcium-regulated transcriptional activator in the absence of JNK/SAPK1 activation. 1006 99


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