CHARACTERIZATION OF NEWLY-IDENTIFIED PROTEIN KINASES

这项提议的长期目标是努力实现 蛋白质丝氨酸的系统鉴定和性质研究 激酶在不断增长的HeLa细胞群中表达。一次彻底的 这类重要的调节酶在一种 不断增长的人类细胞数量应该提供新的见解 我们对细胞控制机制的总体理解 具体地说，应该有助于定义所涉及的事件链 在遗传物质的有序复制和分离中 这是细胞分裂的基础。这一信息，反过来，将 提示结合蛋白诱导的信号转导途径 增长因素。对酶的更全面的了解 和代谢事件参与哺乳动物的调节 细胞复制将带来更有效的治疗方法 人类癌症的治疗。 编码蛋白-丝氨酸激酶成员的cDNA克隆 将通过筛选HeLa文库来鉴定家庭 寡核苷酸探针针对的是 催化域。家庭关系将通过以下方式确认 DNA序列分析。结构和功能信息 关于克隆编码的可能的蛋白质-丝氨酸激酶 将通过分析推导出的氨基酸获得 序列以及通过异源真核表达 系统。将产生针对编码蛋白的抗血清。 用于1)免疫沉淀研究，以允许酶和 进一步的结构表征，2)间接 免疫荧光研究确定细胞内 定位，以及3)免疫印迹研究，以测量细胞和 表达的组织特异性。 初步研究已经清楚地证明了这种方法的实用性和 寡核苷酸同源性筛选方法的价值。一个 已鉴定出编码一种蛋白的cDNA克隆 与细胞编码的蛋白质-丝氨酸激酶有相当大的同源性 酵母中的循环调节基因。拟议中的一个主要主题是 研究将进一步探索结构和功能 这些蛋白质之间的关系。

The long term objective of this proposal is to work toward the systematic identification and characterization of protein-serine kinases expressed in a growing HeLa cell population. A thorough survey of this imporant class of regulatory enzymes in a proliferating human cell population should provide new insight into our understanding of cellular control mechanisms in general and specifically should help to define the chain of events involved in the ordered duplication and segregation of the genetic material which underlies cell division. This informalion, in turn, will suggest pathways for transduction of signals induced by binding of growth factors. A more complete understanding of the enzymatic and metabolic events involved in the regulation of mammalian cell reproduction will lead to more effective therapies for the treatment of human cancers. cDNA clones encoding members of the protein-serine kinase family will be identified by screening a HeLa library with oligonucleotide probes directed at highly conserved regions within the catalytic domain. Familial relationships will be confirmed by DNA sequence analysis. Structural and functional information about the putative protein-serine kinases encoded by the clones will be acquired through an analysis of deduced amino acid sequences as well as through heterologous eukaryotic expression systems. Antisera will be produced against the encoded proteins for use in 1) immunoprecipitation studies to allow enzymatic and further structural characterization, 2) indirect immunofluorescence studies to determine intracellular localization, and 3) immunoblotting studies to measure cell and tissue specificity of expression. Preliminary studies have clearly demonstrated the utility and value of the oligonucleotide homology screening approach. A cDNA clone has been identified which encodes a protein with considerable homology to protein-serine kinases encoded by cell cycle regulating genes in yeast. A major thrust of the proposed studies will be to further explore the structural and functional relationships between these proteins.