CREB信号通路在神经系统中的调控及功能

cAMP应答元件结合蛋白(cAMP response element binding protein,CREB)在神经元生成、突触可塑性及学习记忆等方面都具有重要的调节作用,这使得与CREB信号通路相关的分子成为较受关注的神经系统疾病干预的药物靶点.本文概述了CREB的基本构成、相关信号通路、其目的基因表达调控及其在阿尔茨海默病(Alzheimer’s disease,AD)中的作用.     

Involvement of the cAMP response element binding protein, CREB, and cyclin D1 in LPA-induced proliferation of P19 embryonic carcinoma cells

Lysophosphatidic acid (LPA) is a lipid growth factor that induces proliferation of fibroblasts by activating the cAMP response element binding protein (CREB). Here, we further investigated whether LPA induces proliferation of P19 cells, a line of pluripotent embryonic carcinoma cells. 5′-Bromo-2-deoxyuridine incorporation and cell viability assays showed that LPA stimulated proliferation of P19 cells. Immunoblot experiments with P19 cells revealed that the mitogen activated protein kinases, including p-ERK, p38, pAKT, glycogen synthase kinase 3β, and CREB were phosphorylated by treatment with 10 μM LPA. LPA-induced phosphorylation of CREB was efficiently blocked by U0126 and H89, inhibitors of the MAP kinases ERK1/2 and mitogen- and stress-activated protein kinase 1, respectively. Involvement of cyclin D1 in LPA-induced P19 cell proliferation was verified by immunoblot analysis in combination with pharmacological inhibitor treatment. Furthermore, LPA up-regulated CRE-harboring cyclin D1 promoter activity, suggesting that CREB and cyclin D1 play significant roles in LPA-induced proliferation of P19 embryonic carcinoma cells.     

急、慢性乙醇处理后大鼠伏核内cAMP反应元件结合蛋白磷酸化的变化

采用免疫组织化学的方法,检测急性、慢性乙醇作用及戒断后大鼠伏核内cAMP反应元件结合蛋白(cAMP response element binding protein,CREB)磷酸化的变化。结果显示,急性腹腔注射乙醇后15min,伏核内磷酸化CREB(Phospho-CREB,p-CREB)蛋白明显增加,30min后达高峰,至1和6h后仍明显高于对照组。而慢性饮乙醇溶液显著降低大鼠伏核内P—CREB蛋白含量,在撤除乙醇后24、72h时,伏核内p—CREB蛋白含量仍明显较低,戒断后7d,恢复到正常水平。结果表明,急性乙醇处理增加伏核内CREB磷酸化作用,而慢性乙醇作用则降低伏核内CREB磷酸化作用,这可能是乙醇依赖的分子机制之一。     

New insights into the autoinhibition mechanism of glycogen synthase kinase-3beta

It has been suggested that phosphorylation at serine 9 near the N-terminus of glycogen synthase kinase-3β (GSK-3β) mimics the prephosphorylation of its substrate and, therefore, the N-terminus functions as a pseudosubstrate. The molecular basis for the pseudosubstrate's binding to the catalytic core and autoinhibition has not been fully defined. Here, we combined biochemical and computational analyses to identify the potential residues within the N-terminus and the catalytic core engaged in autoinhibition of GSK-3β. Bioinformatic analysis found Arg4, Arg6, and Ser9 in the pseudosubstrate sequence to be extremely conserved through evolution. Mutations at Arg4 and Arg6 to alanine enhanced GSK-3β kinase activity and impaired its ability to autophosphorylate at Ser9. In addition, and unlike wild-type GSK-3β, these mutants were unable to undergo autoinhibition by phosphorylated Ser9. We further show that Gln89 and Asn95, located within the catalytic core, interact with the pseudosubstrate. Mutation at these sites prevented inhibition by phosphorylated Ser9. Furthermore, the respective mutants were not inhibited by a phosphorylated pseudosubstrate peptide inhibitor. Finally, computational docking of the pseudosubstrate into the catalytic active site of the kinase suggested specific interactions between Arg6 and Asn95 and of Arg4 to Asp181 (apart from the interaction of phosphorylated serine 9 with the “phosphate binding pocket”). Altogether, our study supports a model of GSK-3-pseudosubstrate autoregulation that involves phosphorylated Ser9, Arg4, and Arg6 within the N-terminus and identified the specific contact sites within the catalytic core.     

Bacterial expression and characterization of the CREB bZip module: circular dichroism and 2D 1H-NMR studies.

In this paper we describe the expression and purification from bacteria of the recombinant basic leucine zipper (bZip) domain of the cAMP response element binding protein, CREB327. The bZip peptide, CREB259-327, purified to near homogeneity, maintains the sequence-specific CRE site recognition demonstrated by in vitro competition assays. Alkylation of the three cysteine residues of CREB259-327 was employed to prevent aggregation of the peptide due to cysteine oxidation. The Kd of the purified native and modified CREB259-327 for the CRE site was determined by gel retardation assays to be on the order of 10(-7) M. We employed CD spectroscopy to study the folding properties of the native and modified CREB259-327. The CD analyses of the native/modified CREB259-327 peptide demonstrated a 20% increase in the alpha-helical content upon binding to the cAMP response-element. Only a 5% increase in the alpha-helical content of CREB259-327 is observed upon binding to the AP-1 site. This observation contrasts with CREB from the GCN4 protein (Weiss, M.A., et al., 1990, Nature 347, 575-578). In addition, the two-dimensional (2D) 1H-NMR studies of the bZip CREB peptide further support the distinct features of the CREB protein, in comparison to GCN4. Analysis by CD and 2D NMR of the dimerization domain of CREB suggests that the distinct DNA binding characteristics of CREB reside in the basic portion of the bZip module.     

CREB与其相关的非编码RNA在肿瘤发生发展中的作用

cAMP反应元件结合蛋白（cAMP responsive element binding protein, CREB）是亮氨酸拉链家族转录因子。新近研究发现,其在肿瘤组织中的表达显著高于癌旁,被认为是体内的原癌基因之一。非编码RNA（non-coding RNA, ncRNA）是生物体内不能翻译成蛋白质的RNA,主要包括微小RNA（microRNA, miRNA）和长链非编码RNA（long non-coding RNA, lncRNA）等,其异常表达与肿瘤的发生发展密切相关,是目前肿瘤研究的热点。研究表明,CREB与ncRNA之间存在互动效应,并且二者之间的相互作用影响肿瘤的发生发展,然而miRNA和lncRNA的作用机制却不相同。肿瘤细胞内高表达的CREB在影响下游靶基因表达时能够正调控miRNA,而对lncRNA则有促进和抑制两方面的作用。反之,肿瘤细胞中一些低表达的miRNA能促进CREB的表达;有趣的是,高表达的lncRNA能够促进CREB的表达和诱导其活性增强。在影响下游靶基因表达时miRNA仅仅发挥抑制作用,而lncRNA则分别具有促进和抑制作用。本文结合我们的系列报道和最新的研究结果,对ncRNA与CREB的互动效应及其与肿瘤的发生发展之间的关系作一综述。     

Constitutively active GPR6 is located in the intracellular compartments

We used multiple imaging assays to test the hypothesis that GPR6, a constitutively active Gs-coupled receptor, is present on the cell surface. A pHluorin tag at the N-terminus of rat GPR6 expressed in human embryonic kidney 293 (HEK293) cells was not accessible to protons, chymotrypsin or anti-green fluorescent protein antibody, demonstrating that GPR6 is primarily located in intracellular compartments. Similar intracellular localization of pHluorin-tagged GPR6 was found in striatal neurons, where endogenous GPR6 is expressed. Confirmation of Gs-mediated constitutive activity in HEK293 cells and striatal neurons led us to conclude that GPR6 can signal from intracellular compartments.     

Injury-induced activation of the MAPK/CREB pathway triggers apoptosis-induced compensatory proliferation in hydra head regeneration

After bisection, Hydra polyps regenerate their head from the lower half thanks to a head-organizer activity that is rapidly established at the tip. Head regeneration is also highly plastic as both the wild-type and the epithelial Hydra (that lack the interstitial cell lineage) can regenerate their head. In the wild-type context, we previously showed that after mid-gastric bisection, a large subset of the interstitial cells undergo apoptosis, inducing compensatory proliferation of the surrounding progenitors. This asymmetric process is necessary and sufficient to launch head regeneration. The apoptotic cells transiently release Wnt3, which promotes the formation of a proliferative zone by activating the beta-catenin pathway in the adjacent cycling cells. However the injury-induced signaling that triggers apoptosis is unknown. We previously reported an asymmetric immediate activation of the mitogen-activated protein kinase/ribosomal S6 kinase/cAMP response element binding protein (MAPK/RSK/CREB) pathway in head-regenerating tips after mid-gastric bisection. We show here that pharmacological inhibition of the MAPK/ERK pathway or RNAi knockdown of the RSK, CREB, CREB binding protein (CBP) genes prevents apoptosis, compensatory proliferation and blocks head regeneration. As the activation of the MAPK pathway upon injury plays an essential role in regenerating bilaterian species, these results suggest that the MAPK-dependent activation of apoptosis-induced compensatory proliferation represents an evolutionary-conserved mechanism to launch a regenerative process.