MAMMALIAN DSDNA-DEPENDENT PROTEIN KINASES

我们的目标是确定一个新发现的功能

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.