FUNCTIONAL DOMAINS OF DROSOPHILA DLG PROTEIN

果蝇的DLG抑癌基因产生一种蛋白质DlgA， 定位于上皮细胞之间的分隔连接处。 该基因突变导致成像性视盘肿瘤性过度生长 在幼虫中，随后在蛹阶段死亡。DlgA是 Maguk蛋白质家族的原型，也就是这一主题 计划项目。我们的目标是了解DlgA在间隔中的作用 连接结构和在细胞增殖控制。域名(S) 负责将DlgA本地化到分隔结点 通过检查表位标记的部分的定位来鉴定 由表达构建体在体内产生的蛋白质。这些蛋白质可以 与DlgA的结合将通过亲和纯化或共- 免疫沉淀，然后用特异性抗体进行测试 候选结合蛋白，或通过对纯化的结合进行微测序 蛋白质。酵母双杂交系统也将用于搜索 DlgA相互作用的蛋白质。候选蛋白质将直接进行检测 DlgA绑定，任何绑定域都将与 奇什蒂医生。编码DlgA相互作用蛋白的基因将被克隆 以及从cDNA序列预测的蛋白质的结构。我们会 还在果蝇中使用遗传方法来选择DLG的修饰物 可以识别相互作用的蛋白质。在与奇什蒂博士的合作下， 将对DlgA的GUK结构域进行鸟苷酸激酶和GMP结合测试 活动，并与安德森博士合作，DlgA蛋白将 进行酪氨酸磷酸化检测。一种新发现的果蝇 基因编码一种名为CAMGUK的蛋白质，它与MAGUK同源，但也 包含一个N端扩展名，与 钙/钙调蛋白依赖的蛋白激酶(CAM激酶)。细胞间黏附分子激酶 以及CAMGUK的GUK结构域将在细菌或 动物细胞，并测试它们各自的催化活性，以及 其他CAMGUK结构域的功能将使用相同的方法进行检测 至于DlgA。将使用遗传方法来确定细胞的功能 CAMGUK在细胞内和发育过程中。我们还提议开始一项 新发现的一种新果蝇的分子遗传分析 Maguk，DLG-2，这与人类Hdlg有很强的同源性。这项工作将 有助于阐明DlgA和CAMGUK中结构域的功能以及 在MAGUKS中。

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.