MAMMALIAN DSDNA-DEPENDENT PROTEIN KINASES

我们的目标是确定一种新发现的 磷酸化系统，存在于各种各样的提取物中， 真核细胞，包括人HeLa细胞。 一个新的特点是， 磷酸化依赖于 双链DNA（dsDNA）。 大约有十几种HeLa蛋白质 在体外响应dsDNA磷酸化：一种dsDNA依赖性 磷酸化底物已被确定为90，000 MW热休克 蛋白，热休克蛋白90。 我们将以下列方式达到我们的目标。 1.的 dsDNA依赖性激酶将从Hela提取物和兔 网织红细胞裂解物。 2. dsDNA依赖性磷酸化的定位 通过蛋白质序列分析确定热休克蛋白90中的位点， 其中位点在体内被磷酸化。 3.其他dsDNA依赖性 将鉴定磷酸化底物。 4.特异性抗血清 将制备HeLa或兔dsDNA依赖性激酶并用于分析 酶在不同细胞类型中的数量和位置。 5.细胞 以各种方式处理，可能揭示dsDNA依赖性磷酸化 将分析体内活性。 6.将测定酵母菌 dsDNA依赖性磷酸化活性。 因为在大肠杆菌提取物中观察到dsDNA依赖性磷酸化， 来自不同组织和差异很大的生物体的细胞，我们 这可能是一个重要的控制机制。 据我们所 尽管已知，但尚未报道dsDNA的类似调节作用。 我们 这表明dsDNA依赖性磷酸化可以调节细胞 对应激或损伤的反应，或者可能是细胞周期的调节方面 控制和胚胎发育。 我们建议的研究应该建立 dsDNA依赖性磷酸化在 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.