Abstract
The phosphorylation of DNA topoisomerase II in Drosophila Kc tissue culture cells was characterized by in vivo labeling studies and in vitro studies that examined the modification of exogenous enzyme in total homogenates of these embryonic cells. Several lines of evidence identified casein kinase II as the kinase primarily responsible for phosphorylating DNA topoisomerase II. First, the only amino acyl residue modified in the enzyme was serine. Second, partial proteolytic maps of topoisomerase II which had been labeled with [32P]phosphate by Drosophila cells in vivo, by cell homogenates in vitro, or by purified casein kinase II were indistinguishable from one another. Third, phosphorylation in cell homogenates was inhibited by micrograms/ml concentrations of heparin, micromolar concentrations of nonradioactive GTP, or anti-Drosophila casein kinase II antiserum. Fourth, cell homogenates were able to employ [gamma-32P]GTP as a phosphate donor nearly as well as [gamma-32P]ATP. Although topoisomerase II was phosphorylated in homogenates under conditions that specifically stimulate protein kinase C, calcium/calmodulin-dependent protein kinase, or cAMP-dependent protein kinase, modification was always sensitive to anti-casein kinase II antiserum or heparin. Thus, under a variety of conditions, topoisomerase II appears to be phosphorylated primarily by casein kinase II in the Drosophila embryonic Kc cell system.
Highlights
From the $Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232 and the §Department of Biochemistry, University of Georgia,Athens, Georgia30602
Plot of [heparin]uersus percent phosphorylation, this range is somewhaltower than thatobserved for the homogenate-catalyzed activity, the heparin inhibition of casein kinase I1 can beovercomeby the presence of basic proteins, such as histones, which are present in high concentrations in the embryonic Kc cell line [49]
Ml heparin impaired the catalytic activity of the CAMP-dependent enzyme, as determined from experiments which employed either histone or Kemptide substrates
Summary
Despite the importance of topoisomerase I1 to theeukary- Drosophila melanogaster enzyme in totalKc cell homogenates otic cell, very little is understood about its physiological have been examined. S. aureus cells were removed by centrifugation and topoisomerase I1 samples were subjected to electrophoresis on 7%acrylamide gels by the procedure of Laemmli [51]. Reaction mixtures were prepared for the identification of phosphorylated amino acids and thegeneration of partial proteolytic maps by increasing the topoisomerase I1 concentration to 175 mM and the specific activity of the [Y-~'P]ATP t3o0 Ci/mmol. Phosphorylated topoisomerase I1was isolated as described under "Identificationof Phosphorylated AminoAcylResidues." Enzyme samples were dissolved in proteolysis buffer (125 mM Tris-C1, pH 6.8, 0.5%SDS, 10%glycerol, 0.001%bromphenol blue) containing bovine serum albumin (0.5 mg/ml) and digested by the method of Cleveland et al [55].
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