Genetic Investigation of Lammer Protein Kinase Structure and Function in Drosophila

RABINOW博士的实验室正在描述一种被称为Doa的果蝇基因，该基因编码LAMMER激酶的一个成员，LAMMER激酶是最近发现的真核蛋白激酶家族。蛋白激酶将磷酸基团转移到其他蛋白质上，作为调节其活性的一种手段，并调节许多基本的细胞过程。在动物界，与Doa同源物在具有酶活性的蛋白质部分具有非常相似的氨基酸序列，这表明它们在细胞过程中具有同样相似的功能。隐性Doa突变在果蝇中几乎是致命的；然而，很少有成年人存活下来，但眼睛发育有缺陷。Doa突变体的其他缺陷包括正常胚胎中枢神经系统（CNS）发育失败和分割缺陷。DOA激酶广泛表达，在胚胎中枢神经系统中发现其表达水平特别高，而胚胎中枢神经系统是细胞快速分裂和分化发生的地方。DOA激酶有两种主要形式，大小不同。其中较大的（105 kD）只存在于细胞质中，而较小的（55 kD）也存在于细胞核中。105kd细胞质形态的结构是完全未知的。在目前的研究项目中，Rabinow博士的实验室将对DOA激酶进行进一步的结构和功能表征，以确定该激酶家族在细胞分化的启动和维持中的作用。第一个任务是确定105kd蛋白的结构。此外，将产生针对这两种蛋白同种异构体的抗体来检查它们的表达。将构建表达特异性改变形式的激酶的果蝇，以确定这两种蛋白亚型是否具有不同的功能，并提供更完整的Doa突变缺陷的遗传表征。这些数据将用于识别突变体中发生改变的一个或多个细胞过程。该项目的R结果将大大推进阐明LAMMER蛋白激酶家族的特定细胞功能及其发挥作用的发育信号通路的长期目标。

ABSTRACT 9724006 RABINOW Dr. Rabinow's laboratory is characterizing a Drosophila gene known as Doa, which encodes a member of the LAMMER kinases, a recently discovered eukaryotic family of protein kinases. Protein kinases transfer phosphate groups to other proteins as a means of regulating their activity, and regulate many essential cellular processes. Homologues to Doa throughout the animal kingdom have very similar amino-acid sequences in the part of the protein that has enzymatic activity, suggesting that they have equally similar function in cellular processes. Recessive Doa mutations in flies are almost lethal; however, rare adults do survive, but show defects in eye development. Other defects in Doa mutants include failure of a normal embryonic central nervous system (CNS) to develop and segmentation defects. The DOA kinase is widely expressed and is found at particularly high levels in the embryonic CNS, where rapid cell division and differentiation occurs. The DOA kinase comes in two major forms that differ in size. The larger of these (105 kD) is found only in the cell cytoplasm, whereas the smaller version (55 kD) is also found in the cell nucleus. The structure of the 105 kD cytoplasmic form is completely unknown. In the current research project, Dr. Rabinow's laboratory will carry out further structural and functional characterization of DOA kinase to define the role of this kinase family in the initiation and maintenance of cellular differentiation. The first task will be to determine the structure of the 105 kD protein. In addition, antibodies against the two protein isoforms will be generated to examine their expression. Drosophila expressing specifically altered forms of the kinase will be constructed to determine whether the two protein isoforms perform different functions, as well as to provide more complete genetic characterization of Doa mutant defects. These data will be used to identify one or more of the cellular processes that are altered in mutants. R esults from this project will substantially advance the long-term goal of elucidating the specific cellular functions of the LAMMER protein kinase family and the developmental signaling pathway(s) in which they function.