【作者】 刘永强；

【导师】 倪晓华；

【作者基本信息】 华东师范大学 ， 生物化学与分子生物学， 2006， 硕士

【摘要】 信号转导途径集中在转录水平对细胞进行调控，通过转录激活特异基因的表达对抗环境刺激因子的作用。可与基因调控区结合的特异转录因子的激活或协同表达能够调节这些信号途径的起始转录。对这些转录因子结构和功能的研究为探讨转录激活机制提供了新视点。cAMP反应元件结合蛋白（cAMP response element-binding proteins，CREB）是一个哺乳动物转录因子家族，通过cAMP反应元件（cAMP response element，CRE）介导cAMP和钙离子依赖性基因表达，参与细胞增殖和分化、胚胎发育、学习记忆和肿瘤发生等多种生理过程。这些蛋白的不正常表达，会引起组织的不正常发育和异常的细胞凋亡，甚至引起个体死亡。 CREB4是人CREB家族的一个新成员，人肿瘤MTC panel结果显示，CREB4在人肺癌LX-1，结肠腺癌CX-1，前列腺癌PC-3，结肠癌G1-112和胰腺癌G1-103中有表达，而在乳腺癌GI-101、卵巢癌组织GI-102和肺癌组织GI-117中未检测到特异性条带。提示CREB4在多种肿瘤组织的基因表达调控中起作用。全长CREB4蛋白定位于细胞质，而缺失C端假定跨膜结构域的CREB4^1-279蛋白突变体则转移至细胞核内，提示CREB4的C端跨膜结构域与其从细胞质到细胞核的转移有关。 构建表达CREB4^215-279-LexA和CREB^215-395-LexA的pLexA融合质粒分别转化含p8opLacZ报告质粒的酵母EGY48菌株，经酵母双杂交筛选人胎脑文库发现与CREB4^215-279相互作用的蛋白kayopherin α2和核孔复合物相关蛋白TPR。TPR为核孔复合物相关蛋白。karyopherin α2参与受体介导的Karyopherin α2β1途径，协助大分子蛋白从细胞质转运到细胞核，提示karyopherin α2可能参与CREB4的跨膜转运过程。 将CREB4，CREB4^1-279，karyopherin α2三种蛋白分别构建于真核表达载体pEGFP-C1和pDsRed-N1上共转染AD293细胞，荧光共定位进一步显示，CREB4和karyopherin α2共定位于细胞质中，CREB4^1-279和karyopherin α2共定位于细胞核中。CREB4和CREB4^1-279改变了karyopherin α2在细胞中的定位。结果提示CREB4与karyopherin α2结合，切掉C端之后，才转移到核内发挥转录作用。更多还原

【Abstract】 Signal-transduction pathways converge ultimately at the level of transcriptional activation to produce specific patterns of gene expression in response to environmental stimuli. The initiation of transcription mediated by these signaling pathways is regulated by the coordinate expression and/or activation of specific transcription factors that bind to the control regions of genes. Specific insights into the mechanisms underlying transcriptional activation have recently arisen from studies of the structure and functions of these transcription factors. The mammalian CREB/ATF family represents a large group of basic-region leucine zipper （bzip） transcription factors with rather diverse physiological functions. Defects in the apoptotic signaling pathway are often associated with uncontrolled cell proliferation, high mutation rate and malignant transformation. Transcriptional regulators ATF/CREB family have diverse functions in controlling cell proliferation and apoptosis. Aberrant expression of these proteins causes abnormal progression, cell apoptosis and death.CREB4 is a member of human CREB/ATF family. Subcellular location of CREB4 and CREB4^1-279 showed that a fusion protein of GFP and full-length CREB4 was localized in cytoplasm, whereas the fusion protein of GFP and a deletion mutant lacking the C-terminal putative transmembrane domain was translocated in nucleus, which suggested that C-terminal of CREB4 protein is associated with the process of translocation of CREB4 from cytoplasm to nucleus.Constructing CREB4^215-279 and CREB^2-5-395 fusion protein with the entire prokaryotic LexA protein respectively , Yeast two-hybrid system revealed two proteins, TPR and karyopherin α 2 ,may associate with CREB4（215-279）. TPR was a nuclear pore complex associated protein, karyopherin a 2 was concerned with the Karyopherin α 2 β 1 pathways mediated by receptors to assist the translocation of proteins from cytoplasm to nucleous, which suggusted that it may be associated with translocation of CREB4.Co-location showed that CREB4 and karyopherin α 2 were co-located in cytoplasm, whereas CREB4^1-279 and karyopherin α 2 were co-located in nucleus. These results suggested that CREB4 first combined with karyopherin α 2, followed by the C-terminal cleavage, finally translocation into nucleus to activate gene transcription.更多还原