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A Protein Kinase Activity Tightly Associated with Drosophila Type II

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A Protein Kinase Activity Tightly Associated with Drosophila Type II Proc. Nati. Acad. Sci. USA Vol. 81, pp. 6938-6942, November 1984 Biochemistry A protein kinase activity tightly associated with Drosophila type II DNA topoisomerase (eukaryotic topoisomerase/protein phosphorylation/Drosophila melanogaster) MIRIAM SANDER, JAMES M. NOLAN, AND TAO-SHIH HSIEH Department of Biochemistry, Duke University Medical Center, Durham, NC 27710 Communicated by Robert L. Hill, July 19, 1984 ABSTRACT A protein kinase activity has been identified tial gene. The mechanism by which the eukaryotic topoiso- that is tightly associated with the purified Drosophila type II merase activities are regulated is not yet clear. Since varia- DNA topoisomerase. The kinase and topoisomerase activities tions in chromatin structure are associated with the are not separated when the enzyme is subjected to analytical processes of transcription, replication, and cell division, it is chromatography (phosphoceilulose, single-strand DNA aga- likely that topoisomerase-mediated alteration in chromatin rose, and Sephacryl S-300) and analytical glycerol gradient structure will be regulated in a complex manner. sedimentation. These two activities are also inactivated to the Recently, a type I topoisomerase isolated from Novikoff same extent by either heat or N-ethylmaleimide treatment. hepatoma cells has been shown to be a phosphoprotein (15). The evidence, however, does not rule out the possibility that In our studies on Drosophila type II topoisomerase we have the kinase activity resides in a polypeptide other than the to- established that the enzyme isolated from the Kco Drosophi- poisomerase polypeptide. The topoisomerase-associated pro- la cell culture line is present in a phosphorylated form (un- tein kinase activity is not stimulated by Ca21 or cyclic nucleo- published data). A substantial amount of evidence exists in- tides. It shows a broad substrate range, including the DNA dicating the importance of protein phosphorylation as a topoisomerase itself, casein, phosvitin, and histones. Phos- mechanism by which enzyme activity is regulated in higher phoamino acid analysis identified phosphoserine and phos- cells (16, 17). Thus, it will be of interest to determine if the phothreonine in polypeptides modified by the topoisomerase- functions of eukaryotic topoisomerase polypeptides might associated protein kinase. No similar activity has been identi- be regulated by phosphorylation. fied previously in Drosophila melanogaster. The purification (18) and physical characterization (19) of Drosophila type II topoisomerase was presented previously. Topoisomerase activities, first identified in Escherichia coli We report here the identification of a cyclic nucleotide- and cell extracts (1), have been found in many organisms and are Ca2'-independent protein kinase activity that catalyzes the considered to be ubiquitous in nature (for reviews, see refs. phosphorylation of the Drosophila type II topoisomerase 2-4). These enzymes are of two types: type I activities make and that remains tightly associated with the topoisomerase transient single-strand breaks in DNA and can change the throughout its purification. Other substrates of the activity linking number of DNA molecules by 1 unit at a time, where- are Drosophila histones H1, H3, H4, and H2a, Drosophila as type II topoisomerases make transient double-strand protein D1, casein, and phosvitin. breaks, changing DNA linking number by units of 2. w Pro- tein and DNA gyrase, two bacterial enzymes, are the proto- MATERIALS AND METHODS types of type I and type II topoisomerases, respectively. Enzymes and DNAs. Drosophila topoisomerase II was pu- In the bacterial system, some functional roles of the topo- rified to homogeneity according to the previously described isomerases are well defined. DNA gyrase plays a role in procedure (18). In some cases ion-exchange chromatography maintaining chromosomal supercoiling and functions in both on Bio-Rex 70 substituted for the phosphocellulose column the initiation and elongation phases of DNA replication (5- step. P4 knotted DNA was prepared according to Liu et al. 7). Recently, Steck and Drlica (8) presented evidence that (20). Single-strand fi phage DNA was the generous gift of DNA gyrase is also involved in facilitating separation of Robert Webster. daughter chromosomes. Variation in the level of superhelical Protein Substrates for Kinase Assay. Phosvitin and casein tension of the bacterial chromosome causes pleiotropic ef- were from Sigma. Samples were dephosphorylated at alka- fects (2, 9, 10) and the maintenance of a given level of chro- line pH and high temperature according to Reimann et al. mosomal supercoiling requires the coordinate activities of (21). They were neutralized and stored at -20°C at a protein type I and type II bacterial topoisomerases (11, 12). Only concentration of 50 mg/ml. Calf thymus histones were from recent work has addressed the question of how topoisomer- Sigma. Drosophila core histones, histone H1, and protein D1 ase activities might be regulated in the bacterial cell: Menzel were purified from Drosophila early embryos, according to and Gellert (10) provided evidence for the existence of a the methods of Alfageme et al. (22, 23). mechanism by which synthesis of gyrase is regulated by the Topoisomerase Assay. Drosophila type II topoisomerase level of DNA supercoiling. was assayed for unknotting activity as described (19). The Identification of functional roles for the eukaryotic en- purified topoisomerase had a specific activity of 5 x 105 zymes has been hampered by the lack of specific inhibitors units/mg, in which 1 unit of activity is defined as the amount and the greater difficulties in applying genetic techniques in of enzyme required to unknot 0.3 ,g of P4 knotted DNA eukaryotic organisms. Recently, mutant yeast strains with under the standard assay conditions. altered topoisomerase I activity have been reported (13). Kinase Assay. Acid precipitation. Assay solution con- Goto and Wang (14) report the cloning of the yeast topoiso- tained 20 mM Hepes (pH 7.4), 10 mM MgCl2, 1.0 mM dithio- merase II gene and its identification as a single-copy essen- threitol, and 1.0 A.M [y-32P]ATP (0.1-0.25 Ci/mmol; 1 Ci = 37 GBq). Protein substrate was 1 mg of casein per ml or as The publication costs of this article were defrayed in part by page charge indicated. The reaction was initiated by addition of enzyme payment. This article must therefore be hereby marked "advertisement" (1-1.5 ,ul) to 20 ul of assay, and incubation proceeded at 30°C in accordance with 18 U.S.C. §1734 solely to indicate this fact. for 10-20 min. The reaction was terminated by addition of 1 6938 Downloaded by guest on September 30, 2021 Biochemistry: Sander et aL Proc. NatL Acad Sci USA 81 (1984) 6939 ml of 10% trichloroacetic acid/0. 1 M sodium pyrophosphate. 1 2 3 4 5 6 7 Precipitates were then collected on Whatman glass fiber fil- ters (2.4 cm) by using an aspirator manifold. Each filter was washed with 25 ml of cold 10o trichloroacetic acid/0.1 M _-ropoTP( sodium pyrophosphate, rinsed with ethanol, dried, and as- DI sayed for radioactivity. In all cases, nonspecifically retained radioactivity was determined (no enzyme added), and this TOPO I - value was subtracted from all other samples. Filtration ofthe assay mixture prior to addition of substrate or enzyme was found to reduce background levels. NaDodSO4/polyacrylamide gel analysis. Assay solution and incubation was as above, except [y-32P]ATP was at a PHOSVITIIN specific activity of 1.0-2.5 Ci/mmol. The reaction was ter- minated by the addition of 80 Aul of phosphate-buffered saline _ ~~~~~H3 (0.137 M NaCl/2.68 mM KCI/1.47 mM KH2PO4/8.09 mM ' A SEIN H2 Na2HPO4) containing 2.5 mM Na3EDTA. Samples were then brought to 10% trichloroacetic acid. Precipitates were collected by centrifugation, washed once with acetone, and resuspended in gel loading buffer. NaDodSO4/polyacryla- mide gel analysis was carried out as described by Laemmli (24). Where histone substrates were used (acid precipitation or gel analysis), the trichloroacetic acid concentration for precipitation was 20%. Densitometry. Photographs of ethidium-stained agarose gels and autoradiographs of dried NaDodSO4/polyacryla- FIG. 1. Substrate specificity of topoisomerase-associated ki- mide gels were scanned by using a Zeineh soft laser scanning nase. Kinase reactions were carried out in the presence of 0.50 ug densitometer equipped with an integrator. In some cases, the (lane 1) or 0.25 yLg (lanes 2 and 4-7) of Drosophila type II topoiso- relative area under appropriate peaks was determined by merase. In lane 3 no enzyme was added. Protein substrates were as weighing tracing paper cut to the shape of the peak. For to- follows: lane 4, phosvitin, 1 mg/ml; lane 5, casein, 1 mg/ml; lane 6, poisomerase, some activity curves represent the average of Drosophila core histone, 0.5 mg/ml; lane 7, Drosophila histone H1 and protein D1, 0.5 mg/mil. NaDodSO4/polyacrylamide gels for more than one determination. analysis of the phosphoproteins were 8% in lanes 1-3, 10%o in lanes 5 Phosphoamino Acid Analysis. Phosphorylated proteins and 6, and 15% in lanes 6 and 7. Autoradiographs of the dried gels were purified by NaDodSO4/polyacrylamide gels, electro- are shown. The 32P-labeled topoisomerase polypeptide is indicated. eluted, and precipitated with acid or precipitated directly All indicated polypeptides were identified by Coomassie blue stain- from the kinase reaction. Precipitates were resuspended in ing. H20, made 6 M in HCl, and hydrolyzed 11/2-4 hr under vacu- um. Hydrolysates were dried in a Savant Speed Vac concen- species migrating with the topoisomerase subunit (Mr = trator and resuspended in a solution of 2 mg of phosphoser- 170,000) is a phosphorylated subunit of the species that is ine, phosphothreonine, and phosphotyrosine per ml. Paper active as a kinase in our enzyme preparation. electrophoresis on Whatman 3MM paper was carried out at As an initial step in characterizing this kinase activity, pH 3.5 (H20/acetic acid/pyridine, 945:50:5) at 1000 V for 3 phosphate transfer to the following protein substrates was hr, with cooling.
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