FUNCTIONAL DOMAINS OF DROSOPHILA DLG PROTEIN

果蝇 DLG 蛋白的功能域

• 批准号: 6103031
• 负责人: PETER J BRYANT
• 金额: $ 21.93万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-07-01 至 2001-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6103031
• 关键词: Drosophilidae; SDS polyacrylamide gel electrophoresis; binding proteins; biological signal transduction; cell cycle; cell cycle proteins; cell differentiation; cell growth regulation; confocal scanning microscopy; epitope mapping; gene expression; guanosine monophosphate; immunoelectron microscopy; immunoprecipitation; molecular cloning; nucleic acid sequence; phosphorylation; polymerase chain reaction; protein kinase; protein structure function; southern blotting; tumor suppressor genes; western blottings

The dlg tumor suppressor gene of Drosophila gives rise to a protein, DlgA, that is localized at septate junctions between epithelial cells. Mutations in this gene lead to neoplastic overgrowth of the imaginal discs in the larva, followed by death during the pupal stage. DlgA is the prototype for the MAGUK family of proteins that are the subject of this Program Project. Our goal is to understand the role of DlgA in septate junction structure and in cell proliferation control. The domain(s) responsible for localization of DlgA to the septate junction will be identified by examining the localization of epitope-tagged parts of the protein produced in vivo from expression constructs. The proteins that bind to DlgA will be identified by affinity purification or co- immunoprecipitation, followed by tests with specific antibodies to candidate binding proteins, or by microsequencing of purified binding proteins. The yeast two-hybrid system will also be used to search for DlgA-interacting proteins. Candidate proteins will be tested directly for DlgA binding, and any binding domains will be mapped in collaboration with Dr. Chishti. The genes encoding DlgA-interacting proteins will be cloned and the structure of the proteins predicted from cDNA sequence. We will also use a genetic approach in Drosophila to select modifiers of dlg that may identify interacting proteins. In collaboration with Dr. Chishti, the GUK domain of DlgA will be tested for guanylate kinase and GMP-binding activities, and in collaboration with Dr. Anderson, the DlgA protein will be tested for tyrosine phosphorylation. A newly identified Drosophila gene encodes a protein, CAMGUK, that shows homology to MAGUKs but also contains an N-terminal extension with highly significant similarity to calcium/calmodulin-dependent protein kinase (CAM kinase). The CAM kinase as well as the GUK domains of CAMGUK will be produced in bacteria or animal cells and tested for their respective catalytic activities, and the functions of other CAMGUK domains will be assayed using the same methods as for DlgA. Genetic methods will be used to determine the function of CAMGUK in the cell and during development. We also propose to begin a molecular genetic analysis of a recently identified additional Drosophila MAGUK, Dlg-2, that shows strong homology to human Hdlg. The work will help to elucidate the functions of domains in DlgA and CAMGUK as well as in MAGUKs in general.

果蝇的 dlg 肿瘤抑制基因产生一种蛋白质 DlgA， 位于上皮细胞之间的隔膜连接处。 该基因的突变导致成虫盘肿瘤性过度生长 在幼虫期，随后在蛹期死亡。 DlgA 是 MAGUK 蛋白质家族的原型，这是本文的主题 计划项目。我们的目标是了解 DlgA 在隔膜中的作用 连接结构和细胞增殖控制。域名 负责将 DlgA 定位到隔膜连接处的是 通过检查表位标记部分的定位来识别 由表达构建体在体内产生的蛋白质。这些蛋白质 与 DlgA 的结合将通过亲和纯化或共-鉴定 免疫沉淀，然后用特异性抗体进行测试 候选结合蛋白，或通过纯化结合的微测序 蛋白质。酵母双杂交系统也将用于寻找 DlgA 相互作用蛋白。候选蛋白质将直接进行测试 DlgA 结合，任何结合域都将与 奇什蒂博士。编码 DlgA 相互作用蛋白的基因将被克隆 以及从 cDNA 序列预测的蛋白质结构。我们将 还在果蝇中使用遗传方法来选择 dlg 的修饰因子 可以识别相互作用的蛋白质。与 Chishti 博士合作， DlgA 的 GUK 结构域将进行鸟苷酸激酶和 GMP 结合测试 活动，并与安德森博士合作，DlgA 蛋白将 检测酪氨酸磷酸化。新发现的果蝇 基因编码一种蛋白质 CAMGUK，它与 MAGUK 具有同源性，但也 包含一个 N 末端延伸，与 钙/钙调蛋白依赖性蛋白激酶（CAM 激酶）。 CAM激酶 以及 CAMGUK 的 GUK 结构域将在细菌或 动物细胞并测试其各自的催化活性，以及 其他 CAMGUK 结构域的功能将使用相同的方法进行测定 至于DlgA。遗传方法将用于确定 CAMGUK 在细胞中和发育过程中。我们还建议开始 最近发现的另外一种果蝇的分子遗传学分析 MAGUK，Dlg-2，与人类 Hdlg 显示出很强的同源性。该工作将 有助于阐明 DlgA 和 CAMGUK 中结构域的功能以及 一般而言，在 MAGUK 中。