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CHARACTERIZATION OF NEWLY-IDENTIFIED PROTEIN KINASES

新鉴定的蛋白质激酶的表征

基本信息

批准号：
3466480
负责人：
Steven K. Hanks
金额：
$ 11.33万
依托单位：
VANDERBILT UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
1987
资助国家：
美国
起止时间：
1987-07-01 至 1992-06-30
项目状态：
已结题

项目摘要

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.

这项建议的长远目标是致力于蛋白质丝氨酸系统鉴定与表征在生长的HeLa细胞群中表达的激酶。的透彻这类重要的调节酶的调查，增殖的人类细胞群应该提供新的见解我们对细胞控制机制的理解特别是应该有助于定义所涉及的事件链遗传物质的有序复制和分离是细胞分裂的基础这一信息，反过来，将提示了通过结合生长因子对酶的更全面的了解和代谢事件参与调节哺乳动物细胞繁殖将导致更有效的治疗方法，治疗人类癌症。编码蛋白丝氨酸激酶成员的cDNA克隆将通过筛选HeLa文库来鉴定家族，寡核苷酸探针，其针对内部高度保守区域催化领域。家庭关系将由 DNA序列分析结构和功能信息克隆编码的蛋白质丝氨酸激酶通过分析推导的氨基酸序列，序列以及通过异源真核表达系统. 将针对编码的蛋白质产生抗血清用于1）免疫沉淀研究，以允许酶促和进一步的结构表征，2）间接免疫荧光研究，以确定细胞内定位，和3）免疫印迹研究，以测量细胞和表达的组织特异性。初步研究清楚地表明，寡核苷酸同源性筛选方法的价值。一已经鉴定了cDNA克隆，其编码一种蛋白质，与细胞编码的蛋白丝氨酸激酶相当同源酵母中的周期调节基因。建议的一个主要重点是研究将进一步探讨结构和功能这些蛋白质之间的关系。