Protein kinase C phosphorylates topoisomerase II : Topoisomerase activation and its possible role in phorbol ester - induced differentiation of HL - 60 cells ( gene expression / cell cycle / tumor promoter / calmodulin ) NAJi SAHYOUN
چکیده
DNA topoisomerase II from Drosophila was phosphorylated effectively by protein kinase C. With a Km of about 100 nM, the reaction was rapid, occurring at 40C as well as at 30'C and requiring as little as 0.6 ng of the protein kinase per 170 ng of topoisomerase. About 0.85 mol of phosphate could be incorporated per mol of topoisomerase II, with phosphoserine as the only phospho amino acid produced. The reaction was dependent on Ca2+ and phosphatidylserine and was stimulated by phorbol esters. Calmodulin-dependent protein kinase I, but not cyclic AMP-dependent protein kinase, was also able to phosphorylate the topoisomerase. Phosphorylation of topoisomerase U by protein kinase C resulted in appreciable activation of the topoisomerase, suggesting that it may represent a possible target for the regulation of nuclear events by protein kinase C. This possibility is supported by the finding that the phorbol ester-induced differentiation ofHL-60 cells was blocked by the topoisomerase II inhibitors novobiocin and 4'-(9-acridinylamino)methanesulfon-m-anisidide(mAMSA), but not by the inactive analog o-AMSA. Phorbol esters influence cellular functions at various levels (1, 2), presumably by activating protein kinase C (3, 4). Some cellular effects can be detected rapidly after the addition of phorbol esters. These effects include the regulation of ionic transport, release of bioactive substances, and receptor down-regulation (1, 2, 5). Other effects of phorbol esters target the genome, resulting in the regulation of DNA replication or in the modulation of gene expression. Examples of genes that appear to be induced by phorbol esters include ornithine decarboxylase in epidermal cells (6, 7), interleukin 2 and IL-2 receptor in mouse T cells and Tlymphoma cells (8-10), c-fos in U937 monocytes and in HL-60 cells (11), actin and vimentin in K562 erythroleukemia cells (12), and calcitonin in thyroid medullary carcinoma cells (13). Genes that seem to be repressed by phorbol esters include globin in Friend erythroleukemia cells (14), glycophorin in K562 erythroleukemia cells (15), and c-myc in thyroid medullary carcinoma cells (13). Moreover, phorbol esters increase the frequency of initiation ofDNA replication for bacteriophage X injected into Xenopus eggs (16). The effects of phorbol esters on gene transcription generally require more time to become manifest than the effects on ionic fluxes or receptors. Thus, the regulation of c-fos mRNA in U937 monocytes is maximal after 30 min (11) and that of glycophorin and actin genes becomes evident after about 1 hr (12, 15), whereas the effect on c-myc mRNA is observed 4 hr after exposure of the cells to the tumor promoter (13). The relative delay in these responses may reflect an indirect effect ofphorbol esters on gene transcription, requiring one or more intermediary steps or a cascade of reactions; alternatively, phorbol esters may alter gene transcription more directly by stimulating protein kinase C, which then may phosphorylate a polypeptide(s) involved in transcriptional regulation. In this case the delay in the appearance of the final mRNA product may be attributed to the nature of the reactions involved in gene activation, transcription, and RNA processing. Further, discrimination among events mediating the short-term and long-term effects of phorbol esters is a problem that pertains to the pleiotypic effects of growth factors in general. The present study was designed to investigate whether the phorbol ester receptor (3, 4), protein kinase C, can directly phosphorylate cellular components that are involved in DNA metabolism, which may lead to the modulation of DNA replication and/or transcription. Topoisomerases are prokaryotic and eukaryotic enzymes that produce DNA isomers by topological interconversions in reactions like "knotting/ unknotting," supercoiling/relaxation and catenation/decatenation of circular DNA (17-20). These processes basically entail cleavage and resealing of DNA with the production of enzyme-DNA covalent complexes as intermediates; type I topoisomerases produce single-strand DNA breaks, while double-strand breaks are produced by type II topoisomerases (17-20). Recent work on eukaryotic type II topoisomerases has implicated these enzymes in the regulation of DNA replication and in the transcription of specific genes. The evidence derives mainly from study of yeast topoisomerase mutants (21, 22), mapping of topoisomerase-induced DNA cleavage sites (23, 24), and use of topoisomerase II inhibitors such as novobiocin (25, 26). Further, by means of immunochemical and immunocytochemical methods, topoisomerase II has been visualized in association with the nuclear matrix ofDrosophila embryonic cells (27) and with the "scaffold" of mitotic MSB-1 lymphoblastoid chicken cells (28). This localization may facilitate the participation of topoisomerase II in transcriptional regulation. Regulation of topoisomerase activity itself may, therefore, result in alterations of gene expression. Phosphorylation of topoisomerases by protein kinases may represent such a regulatory mechanism. Thus, topoisomerase I from Novikoff ascites cells appears to be activated by serine/threonine phosphorylation (29), whereas calfthymus topoisomerase I is inhibited following tyrosine phosphorylation by pp6Osrc (30). Moreover, purified preparations of Drosophila topoisomerase II contain an endogenous protein kinase, and the topoisomerase II occurs as a phosphoprotein (31). This topoisomerase is also subject to serine phosphorylation by Abbreviations: PBt2, 4,&phorbol 12,13-dibutyrate; m/o-AMSA, 4'(9-acridinylamino)methanesulfon-m/o-anisidide. 1603 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 1604 Biochemistry: Sahyoun et al. casein kinase II, leading to a 3-fold increase in enzyme activity (32). We have examined the phosphorylation of Drosophila topoisomerase II by protein kinase C. The topoisomerase proved to be a high-affinity substrate which underwent very rapid phosphorylation even when subnanogram amounts of protein kinase C were used. Topoisomerase phosphorylation was accompanied by activation of the unknotting and relaxation enzyme activities. These results provide a putative target for the action of tumor promoters that may be relevant to the mechanism of transcriptional regulation for one or more genes. Consistent with this possibility is the observation that the topoisomerase II inhibitors novobiocin (33) and 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA) (34) prevented the phorbol ester-induced differentiation of HL-60 cells. MATERIALS AND METHODS [y32P]ATP (30-40 Ci/mmol; 1 Ci = 37 GBq) was purchased from New England Nuclear; 4p-phorbol 12,13-dibutyrate (PBt2), 4a-phorbol, and polyethylene glycol (PEG) 20,000 were obtained from Sigma; phenyl-Sepharose was supplied by Pharmacia; m-AMSA and o-AMSA were a generous gift from L. Elwell (Burroughs Wellcome). Protein kinase C was purified to homogeneity by a rapid, high-yield method (35). In brief, 5 g of brain tissue was obtained from male Sprague-Dawley rats (150-250 g) and homogenized in a buffer containing 1 mM CaCl2 and 50 ,ug of leupeptin/ml. Protein kinase C was released from the particulate fraction in the presence of EGTA, bound to insideout vesicles from human erythrocytes in the presence of 1 A Proc. Natl. Acad. Sci. USA 83 (1986) mM CaCl2, and then specifically released with EGTA. Minor contaminants were eliminated by phenyl-Sepharose chromatography, yielding about 150 ,ug ofpure enzyme. Calmodulindependent protein kinase type II was purified from rat brain either in the cytoskeletal or in the solubilized form (36). Cyclic AMP-dependent protein kinase from bovine heart (Sigma) was further purified (unpublished results) by HPLC, employing sequentially a Mono Q anion exchanger followed by TSK-400 and TSK-200 gel-permeation columns in tandem (Pharmacia). Topoisomerase II was purified to homogeneity from Drosophila melanogaster embryos as detailed previously (37). Phage P4 DNA and plasmid pBR322 DNA were prepared as described (38, 39). Phosphorylation assays were performed at 30°C unless indicated otherwise; assay mixtures (50 p4) contained 50 mM Tris HCl (pH 7.7), 7 mM MgCl2, 50 ,uM ATP, 2.5 ,Ci of [-32P]ATP, and various activators of protein kinase C or other protein kinases as indicated. Phosphorylated polypeptides were analyzed by NaDodSO4/PAGE in 7.5% or 10% acrylamide Laemmli gels (40); gels were stained with Coomassie blue, destained, dried, and exposed to Kodak direct-contact DEF-5 film. 32P-incorporation into proteins was estimated by densitometric scanning and by direct measurement of radioactivity in dried gel slices. Phospho amino acid analysis (41) was performed on topoisomerase II after separating it from [y-32P]ATP by gel-filtration chromatography on Sephadex G-50. Protein concentration was determined by the Coomassie blue G-250 method (42). Topoisomerase II activity was measured by unknotting of knotted bacteriophage P4 capsid DNA (38), or by relaxing negatively supercoiled plasmid pBR322 (43) in the presence of 1.25 mM ATP, and the products were analyzed by