scholarly journals Consensus and Variant cAMP-regulated Enhancers Have Distinct CREB-binding Properties

2000 ◽  
Vol 276 (15) ◽  
pp. 11719-11728 ◽  
Author(s):  
Johanna C. Craig ◽  
Maria A. Schumacher ◽  
Steven E. Mansoor ◽  
David L. Farrens ◽  
Richard G. Brennan ◽  
...  
Keyword(s):  
Dna Binding ◽  
Binding Affinity ◽  
Leucine Zipper ◽  
Camp Response Element Binding ◽  
Camp Response Element ◽  
Basic Leucine Zipper ◽  
Binding Characteristics ◽  
Element Binding Protein ◽  
Dna Binding Affinity

Recent determination of the cAMP response element-binding protein (CREB) basic leucine zipper (bZIP) consensus CRE crystal structure revealed key dimerization and DNA binding features that are conserved among members of the CREB/CREM/ATF-1 family of transcription factors. Dimerization appeared to be mediated by a Tyr307–Glu312interhelical hydrogen bond and a Glu319–Arg314electrostatic interaction. An unexpected hexahydrated Mg2+ion was centered above the CRE in the dimer cavity. In the present study, we related these features to CREB dimerization and DNA binding. A Y307F substitution reduced dimer stability and DNA binding affinity, whereas a Y307R mutation produced a stabilizing effect. Mutation of Glu319to Ala or Lys attenuated dimerization and DNA binding. Mg2+ions enhanced the binding affinity of wild-type CREB to the palindromic CRE by ∼20-fold but did not do so for divergent CREs. Similarly, mutation of Lys304, which mediates the CREB interaction with the hydrated Mg2+, blocked CREB binding to the palindromic but not the variant CRE sequences. The distinct binding characteristics of the K304A mutants to the consensus and variant CRE sequences indicate that CREB binding to these elements is differentially regulated by Mg2+ions. We suggest that CREB binds the consensus and variant CRE sequences through fundamentally distinct mechanisms.

scholarly journals Phosphorylation of CREB-Specific Coactivator CRTC2 at Ser238 Promotes Proliferation, Migration, and Invasion of Colorectal Cancer Cells

2020 ◽  
Vol 19 ◽  
pp. 153303382096211
Author(s):  
Yi Wang ◽  
Qian Liu ◽  
Hanshuo Zhang
Keyword(s):  
Colorectal Cancer ◽  
Leucine Zipper ◽  
Camp Response Element Binding ◽  
Serine Phosphorylation ◽  
Site Directed Mutagenesis ◽  
Cell Counting ◽  
Migration And Invasion ◽  
Camp Response Element ◽  
Basic Leucine Zipper ◽  
Element Binding Protein

cAMP response element binding protein (CREB)-regulated transcription coactivator 2 (CRTC2), a member of the novel CRTC family of transcriptional coactivators that activates basic leucine zipper transcription factors, including CREB, is overexpressed in many carcinomas, including colon cancer. Phosphorylation of CRTC2 protein at different residues is important for its subcellular localization and activity. However, the functions of some of the serine phosphorylation sites have not been elucidated. This study aimed to investigate the effects of phosphorylation of Ser127, Ser238, and Ser245 sites of CRTC2 in colorectal cancer (CRC) cells. Recombinant lentiviral particles with a CRTC2-targeting small hairpin RNA (shRNA) sequence were transfected into CRC cells to obtained shCRTC2 cell lines. Site-directed mutagenesis of Ser127, Ser238, and Ser245 cells were constructed by transfecting CRTC2 cDNA containing S127A, S238A, and S245A mutations into shCRTC2. Cell proliferation was measured by cell counting kit-8. Cell migration and invasion were examined by transwell assay. mRNA expression was assayed by qRT-PCR, and protein expression was determined by Western blot. Our results indicate that CRTC2 is overexpressed in CRC cells. Knockdown of CRTC2 inhibits the proliferation, migration, and invasion of CRC cells. When the phosphorylation of CRTC2 Ser238 decreases due to the lack of ERK2, the phosphorylation of Ser171 site increases. The proliferation, migration and invasion of CRC cells were inhibited, the nuclear aggregation of CRTC2 in the nucleus was reduced, and the interaction between CRTC2 and CREB was weaken. It is shown that the phosphorylation of CRTC2 Ser238 is important for CREB transcriptional activity. These findings may help in the identification of potentially new targets for CRC therapy.

scholarly journals cAMP Response Element-Binding Protein Interacts With and Stimulates the Proteasomal Degradation of the Nuclear Receptor Coactivator GRIP1

Endocrinology ◽  
2013 ◽  
Vol 154 (4) ◽  
pp. 1513-1527 ◽  
Author(s):  
Tuyen Hoang ◽  
Ingvild S. Fenne ◽  
Andre Madsen ◽  
Olivera Bozickovic ◽  
Mona Johannessen ◽  
...  
Keyword(s):  
Nuclear Receptor ◽  
Leucine Zipper ◽  
Binding Protein ◽  
Proteasomal Degradation ◽  
Estrogen Receptor Α ◽  
Camp Response Element Binding ◽  
Camp Response Element ◽  
Basic Leucine Zipper ◽  
Response Element ◽  
Element Binding Protein

Abstract The glucocorticoid receptor interacting protein (GRIP1) belongs to the p160 steroid receptor coactivator family that plays essential roles in nuclear receptor-dependent transcriptional regulation. Previously, we reported that the cAMP-dependent protein kinase (PKA) induces ubiquitination leading to degradation of GRIP1. Here we show that the cAMP response element-binding protein (CREB) downregulates GRIP1 and is necessary for the PKA-stimulated degradation of GRIP1, which leads to changes in the expression of a subset of genes regulated by estrogen receptor-α in MCF-7 breast cancer cells. Our data of domain-mapping and ubiquitination analyses suggest that CREB promotes the proteasomal breakdown of ubiquitinated GRIP1 through 2 functionally independent protein domains containing amino acids 347 to 758 and 1121 to 1462. We provide evidence that CREB interacts directly with GRIP1 and that CREB Ser-133 phosphorylation or transcriptional activity is not required for GRIP1 interaction and degradation. The basic leucine zipper domain (bZIP) of CREB is important for the interaction with GRIP1, and deletion of this domain led to an inability to downregulate GRIP1. We propose that CREB mediates the PKA-stimulated degradation of GRIP1 through protein-protein interaction and stimulation of proteasomal degradation of ubiquitinated GRIP1.

scholarly journals Enhancement by lithium of cAMP-induced CRE/CREB-directed gene transcription conferred by TORC on the CREB basic leucine zipper domain

2007 ◽  
Vol 408 (1) ◽  
pp. 69-77 ◽  
Author(s):  
Ulrike Böer ◽  
Julia Eglins ◽  
Doris Krause ◽  
Susanne Schnell ◽  
Christof Schöfl ◽  
...  
Keyword(s):  
Dna Binding ◽  
Gene Transcription ◽  
Leucine Zipper ◽  
Luciferase Reporter ◽  
Lithium Salts ◽  
Transactivation Domain ◽  
Camp Response Element ◽  
Basic Leucine Zipper ◽  
Response Element ◽  
Leucine Zipper Domain

The molecular mechanism of the action of lithium salts in the treatment of bipolar disorder is not well understood. As their therapeutic action requires chronic treatment, adaptive neuronal processes are suggested to be involved. The molecular basis of this are changes in gene expression regulated by transcription factors such as CREB (cAMP-response-element-binding protein). CREB contains a transactivation domain, in which Ser119 is phosphorylated upon activation, and a bZip (basic leucine zipper domain). The bZip is involved in CREB dimerization and DNA-binding, but also contributes to CREB transactivation by recruiting the coactivator TORC (transducer of regulated CREB). In the present study, the effect of lithium on CRE (cAMP response element)/CREB-directed gene transcription was investigated. Electrically excitable cells were transfected with CRE/CREB-driven luciferase reporter genes. LiCl (6 mM or higher) induced an up to 4.7-fold increase in 8-bromo-cAMP-stimulated CRE/CREB-directed transcription. This increase was not due to enhanced Ser119 phosphorylation or DNA-binding of CREB. Also, the known targets inositol monophosphatase and GSK3β (glycogen-synthase-kinase 3β) were not involved as specific GSK3β inhibitors and inositol replenishment did not mimic and abolish respectively the effect of lithium. However, lithium no longer enhanced CREB activity when the CREB-bZip was deleted or the TORC-binding site inside the CREB-bZip was specifically mutated (CREB-R300A). Otherwise, TORC overexpression conferred lithium responsiveness on CREB-bZip or the CRE-containing truncated rat somatostatin promoter. This indicates that lithium enhances cAMP-induced CRE/CREB-directed transcription, conferred by TORC on the CREB-bZip. We thus support the hypothesis that lithium salts modulate CRE/CREB-dependent gene transcription and suggest the CREB coactivator TORC as a new molecular target of lithium.

cAMP response element-binding protein phosphorylation and DNA binding activity are increased in the anterior pituitary gland following glucocorticoid depletion

1997 ◽  
Vol 19 (3) ◽  
pp. 291-297 ◽  
Author(s):  
D Whitehead ◽  
DA Carter
Keyword(s):  
Transcription Factors ◽  
Dna Binding ◽  
Camp Response Element Binding ◽  
Binding Activity ◽  
Binding Assays ◽  
Camp Response Element ◽  
Response Element ◽  
Creb Protein ◽  
Dna Binding Activity ◽  
Element Binding Protein

Activation of the hypothalamo-pituitary-adrenal (HPA) axis during stress is associated with increased expression of genes that code for regulatory hormones such as corticotrophin-releasing factor (CRF) and ACTH. The identity of the transcription factors that mediate these changes in gene expression is not known. In the present study we have investigated the expression of the cAMP response-element binding protein (CREB) in mouse pituitary, and its regulation during a pharmacological paradigm that simulates activation of the CRF-ACTH axis. Using Western blots and DNA binding assays we have shown that both CREB protein (43 kDa) and CRE binding exhibit a readily-detectable basal level of activity in the pituitary. Following treatment with the 11 beta-hydroxylase inhibitor metyrapone, CRE binding activity was increased at 1 and 2 h but levels of CREB protein were not found to be consistently elevated. However, using a Ser133 phosphopeptide-specific antibody, that detects the functionally important phosphorylated form of CREB (P-CREB), we have shown that levels of pituitary P-CREB are markedly elevated following metyrapone. The same antibody was also used in DNA binding assays, and in the presence of this antiserum CRE binding activity in samples extracted from metyrapone-treated animals was reduced to levels similar to controls. Parallel experiments have confirmed previous studies showing increases in c-Fos expression and AP-1 DNA binding activity following metyrapone treatment but we have shown that c-Fos-associated binding activity does not appear to contribute to the increase in activity detected using the CRE binding probe. Our evidence of functionally relevant changes in pituitary CREB activity following glucocorticoid depletion must be viewed in the context of numerous other novel pituitary transcription factors that are implicated in HPA regulation, but our use of mice as an experimental model has facilitated the use of novel mouse mutants that can be used to dissect the role of individual factors.

Identification of a novel dimer stabilization region in a plant bZIP transcription activator

1992 ◽  
Vol 12 (11) ◽  
pp. 4809-4816
Author(s):  
F Katagiri ◽  
K Seipel ◽  
N H Chua
Keyword(s):  
Dna Binding ◽  
Binding Affinity ◽  
Binding Proteins ◽  
Leucine Zipper ◽  
Dna Binding Proteins ◽  
Transcription Activator ◽  
Bzip Domain ◽  
Dna Binding Affinity ◽  
Necessary And Sufficient

We have carried out deletion analyses of a tobacco transcription activator, TGA1a, in order to define its functional domains. TGA1a belongs to the basic-region-leucine zipper (bZIP) class of DNA-binding proteins. Like other proteins of this class, it binds to its target DNA as a dimer, and its bZIP domain is necessary and sufficient for specific DNA binding. A mutant polypeptide containing the bZIP domain alone, however, shows a lower DNA-binding affinity than the full-length TGA1a. The C-terminal portion of TGA1a, which is essential for the higher DNA-binding affinity, contains a polypeptide region that can stabilize dimeric forms of the protein. This polypeptide region is designated the dimer stabilization (DS) region. Under our in vitro conditions, TGA1a derivatives with the DS region and those without the region do not form a detectable mixed dimer. This result indicates that in addition to the leucine zipper, the DS region can serve as another determinant of the dimerization specificity of TGA1a. In fact, the DS region, when fused to another bZIP protein, C/EBP, can inhibit dimer formation between the fusion protein and native C/EBP, whereas each of these can form homodimers. Such a portable determinant of dimerization specificity has potential application in studies of DNA-binding proteins as well as in biotechnology.

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