Identification and Functional Analysis of Novel cAMP Response Element Binding Protein Splice Variants Lacking the Basic/Leucine Zipper Domain

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.

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

Biochemical Journal ◽

10.1042/bj20070796 ◽

2007 ◽

Vol 408 (1) ◽

pp. 69-77 ◽

Cited By ~ 24

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.

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Glutamine Rich and Basic Region/Leucine Zipper (bZIP) Domains Stabilize cAMP-response Element-binding Protein (CREB) Binding to Chromatin

Journal of Biological Chemistry ◽

10.1074/jbc.m414144200 ◽

2005 ◽

Vol 280 (15) ◽

pp. 15103-15110 ◽

Cited By ~ 26

Author(s):

Bernhard M. Mayr ◽

Ernesto Guzman ◽

Marc Montminy

Keyword(s):

Leucine Zipper ◽

Binding Protein ◽

Camp Response Element Binding ◽

Camp Response Element ◽

Response Element ◽

Element Binding Protein ◽

Basic Region

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Identification of a family of cAMP response element-binding protein coactivators by genome-scale functional analysis in mammalian cells

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1932773100 ◽

2003 ◽

Vol 100 (21) ◽

pp. 12147-12152 ◽

Cited By ~ 269

Author(s):

V. Iourgenko ◽

W. Zhang ◽

C. Mickanin ◽

I. Daly ◽

C. Jiang ◽

...

Keyword(s):

Functional Analysis ◽

Mammalian Cells ◽

Binding Protein ◽

Camp Response Element Binding ◽

Camp Response Element ◽

Response Element ◽

Element Binding Protein ◽

Genome Scale

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

Technology in Cancer Research & Treatment ◽

10.1177/1533033820962111 ◽

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.

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

Journal of Biological Chemistry ◽

10.1074/jbc.m010263200 ◽

2000 ◽

Vol 276 (15) ◽

pp. 11719-11728 ◽

Cited By ~ 48

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.

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Faculty Opinions recommendation of Does cAMP response element-binding protein have a pivotal role in hippocampal synaptic plasticity and hippocampus-dependent memory?

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1014814.194668 ◽

2003 ◽

Author(s):

Yadin Dudai

Keyword(s):

Synaptic Plasticity ◽

Binding Protein ◽

Camp Response Element Binding ◽

Pivotal Role ◽

Camp Response Element ◽

Response Element ◽

Hippocampal Synaptic Plasticity ◽

Element Binding Protein ◽

Hippocampus Dependent Memory

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Faculty Opinions recommendation of Nuclear inositol 1,4,5-trisphosphate receptors regulate local Ca2+ transients and modulate cAMP response element binding protein phosphorylation.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1027399.329527 ◽

2005 ◽

Author(s):

Thomas Cremer

Keyword(s):

Protein Phosphorylation ◽

Binding Protein ◽

Camp Response Element Binding ◽

Camp Response Element ◽

Response Element ◽

Inositol 1,4,5 Trisphosphate ◽

Element Binding Protein

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Scutellarin Mitigates Cancer-Induced Bone Pain by Suppressing CaMKII/CREB Pathway in Rat Models

Current Topics in Nutraceutical Research ◽

10.37290/ctnr2641-452x.17:249-253 ◽

2019 ◽

Vol 17 (3) ◽

pp. 249-253

Author(s):

Liu Chenglong ◽

Liu Haihua ◽

Zhang Fei ◽

Zheng Jie ◽

Wei Fang

Keyword(s):

Protein Kinase ◽

Bone Pain ◽

Binding Protein ◽

Camp Response Element Binding ◽

Camp Response Element ◽

Dependent Protein Kinase ◽

Response Element ◽

Protein Kinase Ii ◽

Element Binding Protein ◽

Dependent Protein

Cancer-induced bone pain is a severe and complex pain caused by metastases to bone in cancer patients. The aim of this study was to investigate the analgesic effect of scutellarin on cancer-induced bone pain in rat models by intrathecal injection of Walker 256 carcinoma cells. Mechanical allodynia was determined by paw withdrawal threshold in response to mechanical stimulus, and thermal hyperalgesia was indicated by paw withdrawal latency in response to noxious thermal stimulus. The paw withdrawal threshold and paw withdrawal latencies were significantly decreased after inoculation of tumor cells, whereas administration of scutellarin significantly attenuated tumor cell inoculation-induced mechanical and heat hyperalgesia. Tumor cell inoculation-induced tumor growth was also significantly abrogated by scutellarin. Ca2+/calmodulin-dependent protein kinase II is a multifunctional kinase with up-regulated activity in bone pain models. The activation of Ca2+/calmodulin-dependent protein kinase II triggers phosphorylation of cAMP-response element binding protein. Scutellarin significantly reduced the expression of phosphorylated-Ca2+/calmodulin-dependent protein kinase II and phosphorylated-cAMP-response element binding protein in cancer-induced bone pain rats. Collectively, our study demonstrated that scutellarin attenuated tumor cell inoculation-induced bone pain by down-regulating the expression of phosphorylated-Ca2+/calmodulin-dependent protein kinase II and phosphorylated-cAMP-response element binding protein. The suppressive effect of scutellarin on phosphorylated-Ca2+/calmodulin-dependent protein kinase II/phosphorylated-cAMP-response element binding protein activation may serve as a novel therapeutic strategy for CIBP management.

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