DOMAINS INVOLVED IN REGULATION OF RAF ACTIVITY

涉及英国皇家空军活动监管的领域

• 批准号: 3853480
• 负责人: G FANNING-HEIDECKER
• 金额: --
• 依托单位: DIVISION OF CANCER EPIDEMIOLOGY AND GENETICS
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3853480
• 关键词: X ray crystallography; Xenopus; bacterial proteins; biological signal transduction; enzyme structure; enzyme substrate; glucocorticoids; growth factor receptors; human tissue; immunofluorescence technique; insect virus; membrane proteins; mitogens; molecular oncology; monoclonal antibody; mutant; neoplasm /cancer genetics; oncogenes; oncoproteins; point mutation; protein kinase; protein signal sequence; protein structure function; protein transport; serine; stress proteins; temperature sensitive mutant; threonine; transfection; viral carcinogenesis

The protein products of the raf gene family are cytoplasmic serine/threonine protein kinases. They are active in the transmission of mitogenic signals from membrane-associated tyrosine kinase growth factor receptors to the nucleus. The Raf protein itself is phosphorylated, and its kinase is activated by several of the tyrosine kinases and other serine/threonine kinases. A series of point and deletion mutations have been used to identify the site of tyrosine phosphorylation and two of the major serine phosphorylation sites in the Raf kinase. They are Y230, S42, and S497/499. Several approaches to generate systems which produce activated Raf protein or the dominant negative raf mutant K375W conditionally have led to success. We have identified two point mutations responsible for the temperature-sensitive phenotype of the v-mil gene on an MH2 mutant stock. Both involve highly conserved residues found on all protein kinases. Incorporation of these mutations, either singly or in combination, into an activated raf background has conferred the temperature-sensitive phenotype. To study the effect of inactivating mutations, it is necessary to eliminate the endogenous Raf protein. To this end, we have generated vectors which express the antisense of the first 250 nucleotides of the raf mRNA. Constitutive expression of this antisense significantly lowers the number of colonies obtained after transfection compared to constructs expressing the sense sequence. Similar experiments have been done using other parts of the message with comparable results. Constructs which express these antisense RNAs, or the dominant negative raf mutant S375K under the control of either the heat-shock promoter or the glucocorticoid inducible MMTV-LTR, allow us to study their effects in a conditional system. Another approach to study the effect of wild-type and mutant Raf-1 on the signaling cascade is to analyze germinal vesicle breakdown in Xenopus oocytes following microinjection of the appropriate c-raf-1 mRNAs. To this end, we have obtained and sequenced a c-raf-l- homologous cDNA from Xenopus and have incorporated it, and several mutants, into an RNA expression vector.

raf基因家族的蛋白质产物是细胞质的 丝氨酸/苏氨酸蛋白激酶。 他们在传输中很活跃 来自膜相关酪氨酸激酶生长的促有丝分裂信号 细胞核的因子受体。 Raf蛋白本身 磷酸化，其激酶被几种酪氨酸激酶激活， 激酶和其它丝氨酸/苏氨酸激酶。 一系列的点和 缺失突变已被用于鉴定酪氨酸的位点 磷酸化和两个主要的丝氨酸磷酸化位点， Raf激酶。 它们是Y230、S42和S497/499。 的几项解决方案 产生产生活化Raf蛋白或显性Raf蛋白的系统， 阴性raf突变体K375 W有条件地导致成功。 我们有 确定了两个点突变，负责温度敏感性 图10示出了V-mil基因在MH 2突变体原种上的表型。 两者都涉及到高度 所有蛋白激酶上的保守残基。 纳入此等 单独或组合突变成活化的raf 背景技术已经赋予了温度敏感表型。 研究 失活突变的影响，有必要消除 内源性Raf蛋白。 为此，我们生成了 表达raf mRNA的前250个核苷酸的反义。 这种反义核酸的组成型表达显著降低了 转染后获得的集落与表达 sense序列。 类似的实验已经用其他部分做过了 具有可比结果的消息。 表达这些的结构 反义RNA，或显性负性raf突变体S375 K下， 控制热休克启动子或糖皮质激素诱导的 MMTV-LTR允许我们在条件系统中研究它们的作用。 另一种研究野生型和突变型Raf-1对细胞凋亡的影响的方法是， 信号级联是分析非洲爪蟾中的生殖囊泡破裂 显微注射适当的c-raf-1 mRNA后的卵母细胞。 到 为此，我们获得了一个c-raf-l同源cDNA，并进行了测序。 非洲爪蟾已经将它和几个突变体整合到RNA中， 表达载体