MAMMALIAN DSDNA-DEPENDENT PROTEIN KINASES

我们的目标是确定一种新发现的 一种存在于多种植物提取物中的磷酸化系统 真核细胞，包括人的HeLa细胞。这是一个新的特点 系统是磷酸化依赖于是否存在 双链DNA(DsDNA)。大约有十几种HeLa蛋白成为 体外磷酸化对dsDNA的反应：一种dsDNA依赖的 磷酸化底物已被确定为90,000兆瓦的热休克 蛋白质，热休克蛋白90。 我们将通过以下方式实现我们的目标。1. 双链DNA依赖激酶(S)将从Hela提取液和兔中纯化 网织红细胞裂解物。2.依赖dsDNA的磷酸化的定位 将通过蛋白质序列分析确定HSP90中的位点并进行比较 这些位点在体内被磷酸化。3.其他依赖dsDNA的dsDNA 将确定磷酸化底物。4.特异性抗血清 将制备HeLa或兔dsDNA依赖激酶并用于分析 酶在不同细胞类型中的数量和位置。5.单元格 以各种方式处理，可能揭示dsDNA依赖的磷酸化 将分析体内的活性。6.将对酵母菌进行检测 DsDNA依赖的磷酸化活性。 因为已经观察到dsDNA依赖的磷酸化在黄连提取物中 来自不同组织和广泛分布的生物体的细胞，我们 相信这可能是一种重要的控制机制。尽我们最大努力 据了解，目前还没有关于dsDNA的类似调控作用的报道。我们 提示dsDNA依赖的磷酸化可能调节细胞 对应激或损伤的反应，或可能调节细胞周期的各个方面 控制和胚胎发育。我们建议的研究应该确定 DsDNA依赖的磷酸化在血管紧张素转换酶中的作用和重要性 真核细胞。

Our objective is to determine the function of a newly discovered phosphorylation system that is present in extracts from a wide variety of eukaryotic cells including, human HeLa cells. A novel feature of this system is that phosphorylation is dependent upon the presence of double-stranded DNA (dsDNA). About a dozen HeLa proteins become phosphorylated in vitro in response to dsDNA: one dsDNA-dependent phosphorylation substrate has been identified as the 90,000 MW heat shock protein, hsp90. We will approach our objective in the following way. 1. The dsDNA-dependent kinase(s) will be purified from Hela extracts and rabbit reticulocyte lysates. 2. The location of dsDNA-dependent phosphorylation sites in hsp90 will be determined by protein sequence analysis and compared with the sites phosphorylated in vivo. 3. Additional dsDNA-dependent phosphorylation substrates will be identified. 4. Antisera specific to HeLa or rabbit dsDNA-dependent kinase will be prepared and used to analyze the amount and location of the enzyme in different cell types. 5. Cells treated in various ways that might reveal dsDNA-dependent phosphorylation activity in vivo will be analyzed. 6. Yeast will be assayed for dsDNA-dependent phosphorylation activity. Because dsDNA-dependent phosphorylation has been observed in extracts of cells from different tissues and from widely divergent organisms, we believe it may be an important control mechanism. To the best of our knowledge, no similar regulatory role for dsDNA has been reported. We suggest that dsDNA-dependent phosphorylation may modulate cellular responses to stress or damage, or may regulated aspects of cell cycle control and embryonic development. Our proposed studies should establish the function and the importance of dsDNA-dependent phosphorylation in eukaryotic cells.