CREB TRANSCRIPTION FACTOR IN LPS TREATED MACROPHAGES

LPS 处理的巨噬细胞中的 CREB ​​转录因子

• 批准号: 2884790
• 负责人: STEVEN LEE WEINSTEIN
• 金额: $ 9.78万
• 依托单位: SAN FRANCISCO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-08-15 至 2001-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2884790
• 关键词: biological signal transduction; cAMP response element binding protein; gel mobility shift assay; immunoprecipitation; leukocyte activation /transformation; lipopolysaccharides; macrophage; nucleic acid sequence; phosphorylation; protein kinase

DESCRIPTION (Adapted from the applicant's abstract): 	Macrophages have evolved the capacity to recognize conserved structural componentsof LPS to facilitate effective anti-microbial responses to Gram negative bacterial infections. The objective of this research is to understand how LPS-elicited gene regulation is mediated by intracellular events. Because many LPS-regulated genes contain cAMP responsive elements (CRE) in their promoters, the cAMP response element binding protein, CREB, is likely to be important in LPS signaling. The activity of CREB is thought to be governed by phosphorylation on serine-133, and LPS treatment of macrophages activates phosphorylation of S133. The proposed research tests the hypothesis that LPS regulates CREB phosphorylation on S133, thus altering the capacity of CREB to activate transcription of CRE-containing LPS-responsive genes (LRE). Three specific aims will test this hypothesis. 1) Assess the effect of CREB phosphorylation on i) association between CREB and CBP, ii) capacity of CREB to bind CRE sequences and iii) the capacity of CREB to induce transcription. Aim 2 will determine the signaling pathways that mediate phosphorylation of CREB and identify CREB kinase(s). Aim 3 will identify the CREB-dependent LPS responsive genes. The association between CREB and CBP will be examined by co-immunoprecipitation. CREB binding to CRE will be determined by EMSA. Antibodies to CREB or phosphoCREB will detect these proteins in supershift assays. CREB transcriptional activity will be determined using a transiently transfected GAL4-CREB chimera and GAL4-CAT reporter construct. Inhibitors will be used to block LPS activated signal pathways to assess the role of that pathway in phosphorylation of CREB. An in-gel kinase assay using a peptide containing S133 of CREB will identify CREB kinases in nuclear extracts. A dominant interfering allele of CREB will be used to block the wild type protein function to identify LPS responsive genes that are CREB dependent.

描述（改编自申请人的摘要）：宏观经济学具有 进化出识别LPS保守结构成分的能力， 促进对革兰氏阴性细菌的有效抗微生物应答 感染.本研究的目的是了解LPS是如何引起 基因调节由细胞内事件介导。因为许多LPS调节的 基因在其启动子中含有cAMP反应元件（CRE），cAMP 反应元件结合蛋白CREB可能在LPS中起重要作用 发信号。CREB的活性被认为是由磷酸化控制的。 丝氨酸-133，并且巨噬细胞的LPS处理激活S133的磷酸化。 这项研究验证了LPS调节CREB的假设 S133的磷酸化，从而改变CREB激活 含CRE的LPS应答基因（LRE）的转录。三个具体目标 将检验这个假设。1)评估CREB磷酸化对i） CREB和CBP之间的关联，ii）CREB结合CRE序列的能力 和iii）CREB诱导转录的能力。目标2将决定 介导CREB磷酸化并识别CREB的信号通路 激酶。目的3：寻找CREB依赖的LPS应答基因。 CREB和CBP之间的关联将通过以下方式进行检查： 免疫共沉淀。将通过EMSA测定CREB与CRE的结合。 CREB或磷酸化CREB的抗体将在超移位中检测这些蛋白质 测定。CREB转录活性将使用瞬时荧光法测定。 转染的GAL 4-CREB嵌合体和GAL 4-CAT报告基因构建体。抑制剂将 用于阻断LPS激活的信号通路，以评估其作用， CREB的磷酸化途径。使用肽的凝胶内激酶测定 含有CREB的S133将鉴定核提取物中的CREB激酶。一 CREB的显性干扰等位基因将用于阻断野生型蛋白 其功能是鉴定CREB依赖的LPS应答基因。