Phosphorylation of DNA topoisomerase II by casein kinase II: modulation of eukaryotic topoisomerase II activity in vitro.

Abstract

The phosphorylation of Drosophila melanogaster DNA topoisomerase II by purified casein kinase II was characterized in vitro. Under the conditions used, the kinase incorporated a maximum of 2-3 molecules of phosphate per homodimer oftopoisomerase II. No autophosphorylation of the topoisomerase was observed. The only amino acid residue modified by casein kinase II was serine. Apparent Km and Vm.. values for the phosphorylation reaction were 0.4 ,.M topoisomerase II and 3.3 ,umol of phosphate incorporated per min per mg of kinase, respectively. Phosphorylation stimulated the DNA relaxation activity of topoisomerase II by 3-fold over that of the dephosphorylated enzyme, and the effects of modification could be reversed by treatment with alkaline phosphatase. Therefore, this study demonstrates that posttranslational enzymatic modifications can be used to modulate the interaction between topoisomerase II and DNA.The topology of DNA has a profound influence on how its genetic information is regenerated, rearranged, and expressed in vivo (1, 2). Consequently, the enzymes that affect the topological structure of nucleic acids play a crucial role in controlling the cellular functions of DNA (1-4). One class of enzymes, the type II topoisomerases, catalyzes changes in the topological state of nucleic acids by passing one intact DNA helix through a transient double-stranded break made in a second helix (5-7). These ubiquitous enzymes are essential for the viability of eukaryotic cells (8)(9)(10) and are involved in many aspects of DNA metabolism (1-4), including replication' (11)(12)(13)(14)(15)(16)(17), repair (18), transcription (13), and chromosome segregation (9,10).Despite the importance of type II topoisomerases to eukaryotic organisms, little information exists concerning the physiological regulation of these enzymes. Although their activity is stimulated >10-fold by cell proliferation (19-21, §), nothing is known about the events that control this increase in activity. Clearly, before the cellular functions of topoisomerase II can be completely described, the factors that regulate its activity must be well understood.Recently, the type I topoisomerase from Novikoff hepatoma cells was shown to exist in vivo as a phosphoprotein (22). Moreover, when purified, the enzyme was found to be sensitive to its state of phosphorylation, with higher levels of incorporation yielding increased rates of activity in vitro (23,24). This suggests that post-translational modification may play a role in the regulation of topoisomerases. With this in mind, the effects of phosphorylation on the activity of topoisomerase II have been examined. This paper describes the interaction between topoisomerase II and casein kinase II from Drosophila melanogaster. While topoisomerase II showed no autophosphorylation under the conditions used, kinase-mediated phosphorylation of the enzyme stimulated its DNA relaxation activity. Furthermore, the effects of phosphorylation could be reversed by treatment with alkaline phosphatase. This study, th...

Altered stability of etoposide-induced topoisomerase II-DNA complexes in resistant human leukaemia K562 cells

“…Topoisomerase II has been shown to be phosphorylated in intact cells at serine and threonine residues (Saijo et al, 1990;Kroll & Rowe, 1991;Cardenas et al, 1992) and in vitro serves as a substrate for casein kinase II, protein kinase C and p34cdc2 kinase (Ackerman et al, 1985;1988;Sahyoun et al, 1986;Cardenas et al, 1992;Devore et al, 1992;Corbett et al, 1993). In addition, the activity and degree of phosphorylation of topoisomerase II has been found to increase during cell cycle progression from GI to G2-M phase (Heck et al, 1989;Woessner et al, 1991;Saijo & Enomoto, 1992).…”

Immunocytochemical localization of casein kinase II during interphase and mitosis.

“…However, in Rana temporaria oocytes, the enzyme is found exclusively in the cytoplasm (Kandror et al, 1989) . The CKII activity purified from nuclei has similar characteristics to those used to define cytoplasmic CKII (Edelman et al ., 1987), including (a) cAMP independence ; (b) similar Kms (Ackerman et al ., 1985) RNA polymerase I and II (Stetler and Rose, 1982) Glycogen synthase (Picton et al ., 1982) DNA polymerase a (Podust et al ., 1990) Ornithine decarboxylase (Peng and Richards, 1988) Cytoskeletal Tubulin (Serrano et al ., 1987;Diaz-Nido et al ., proteins 1990x) Brain myosin heavy chain (Murakami et al ., 1990) MAP-lA, MAP-1B (Diaz-Nido et al ., 1988) Clathrin (Cantournet et al ., 1987) Transcription Serum response factor (Manak et al ., 1990) factors…”

Characterisation of VP-16-induced DNA cleavage in oestrogen-stimulated human breast cancer cells

“…However, the report that enzyme levels (as determined by immunoblotting) rise only in S-and G2-phase cells following stimulation -even though enhancement of VP-16-induced DNA cleavage is unaffected by inhibition of DNA synthesis using aphidicolin (Chow & Ross, 1987) suggests that enzyme activation within GI-phase cells may accompany cellular activation, a model supported by the data presented here. Indeed, in vitro data have already implicated phosphorylation (Ackerman et al, 1985), poly(ADP-ribosyl)ation (Darby et al, 1985) and calciummediated pathways (Osheroff & Zechiedrich, 1987) …”