FUNCTIONAL DOMAINS OF DROSOPHILA DLG PROTEIN

果蝇的dlg肿瘤抑制基因产生一种蛋白质，DlgA， 其位于上皮细胞之间的分隔连接处。 该基因的突变会导致胚盘的肿瘤性过度生长 在幼虫期，然后在蛹期死亡。DlgA是 MAGUK蛋白质家族的原型是本研究的主题， 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定位到分隔连接处的是 通过检查表位标记的部分的定位来鉴定， 从表达构建体体内产生的蛋白质。的蛋白质 将通过亲和纯化或共沉淀鉴定与DlgA结合的抗体。 免疫沉淀，然后用特异性抗体进行测试， 候选结合蛋白，或通过纯化的结合蛋白的微测序， proteins.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检测， DlgA绑定，任何绑定域都将与 博士奇什提将克隆编码DlgA相互作用蛋白的基因 并根据cDNA序列预测了蛋白质的结构。我们将 我们还在果蝇中使用遗传学方法来选择dlg的修饰物， 可以识别相互作用的蛋白质。与Chishti博士合作， 将测试DlgA的GUK结构域的鸟苷酸激酶和GMP结合 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。新发现的果蝇 该基因编码一种蛋白质CAMGUK，该蛋白质与MAGUKs具有同源性，但也 含有一个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结构域将在细菌中产生，或 动物细胞并测试它们各自的催化活性， 其他CAMGUK结构域的功能将使用相同的方法进行测定 至于DlgA。遗传学方法将用于确定 CAMGUK在细胞和发育过程中。我们还提议开始一个 最近发现的另一种果蝇的分子遗传学分析 MAGUK，Dlg-2，其显示与人Hdlg的强同源性。这项工作将 有助于阐明DlgA和CAMGUK中结构域的功能， 一般来说，在MAGUKS。

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.