Growth state and cell cycle dependent phosphorylation of DNA topoisomerase II in Swiss 3T3 cells.

We have investigated the amount of DNA topoisomerase II and phosphorylation of the enzyme in Swiss 3T3 cells during the transition from cell quiescence to proliferation. A relatively high level of phosphorylation was observed with proliferating cells while no or a very low level of phosphorylation was observed with quiescent cells. Phosphoamino acid analysis of the phosphorylated topoisomerase II revealed that the phosphorylated aminoacyl residue was serine. When quiescent cells were stimulated to grow by the addition of serum, DNA synthesis began to increase at 9 h after serum addition, reaching a maximum at 15 h and then declining. The amount of topoisomerase II began to increase at 6 h and reached a maximum at 22-27 h, corresponding to the G2 phase. The phosphorylation of topoisomerase II measured by pulse-labeling gradually increased from 6 to 18 h and reached a maximum at 22 h when the amount of the enzyme was maximum. The level of phosphorylation measured by continuous-labeling increased gradually up to 12 h and markedly up to 28 h, and then declined. The increase in the rate of phosphorylation in the G2 phase was affected by inhibiting DNA synthesis, but the increase in the amount of the enzyme was not. Thus, it was suggested that the regulation of phosphorylation of topoisomerase II differs from that of the amount of the enzyme.     

A biochemical analysis of topoisomerase II alpha and beta kinase activity found in HIV-1 infected cells and virus

Human topoisomerase II plays a crucial role in DNA replication and repair. It exists in two isoforms: topoisomerase II alpha (alpha) and topoisomerase II beta (beta). The alpha isoform is localized predominantly in the nucleus, while the beta isoform exhibits a reticular pattern of distribution both in the cytosol and in the nucleus. We show that both isoforms of topoisomerase II are phosphorylated in HIV infected cells and also by purified viral lysate. An analysis of the phosphorylation of topoisomerase II isoforms showed that extracts of HIV infected cells at 8 and 32 h. post-infection (p.i.) contain maximal phosphorylated topoisomerase II alpha, whereas infected cell extracts at 4 and 64 h p.i. contain maximum levels of phosphorylated topoisomerase II beta. In concurrent to phosphorylated topoisomerase II isoforms, we have also observed increased topoisomerase II alpha kinase activity after 8h p.i and topoisomerase beta kinase activity at 4 and 64 h p.i. These findings suggest that both topoisomerase II alpha and beta kinase activities play an important role in early as well as late stages of HIV-1 replication. Further analysis of purified virus showed that HIV-1 virion contained topoisomerase II isoform-specific kinase activities, which were partially isolated. One of the kinase activities of higher hydrophobicity can phosphorylate both topoisomerase II alpha and beta, while lower hydrophobic kinase could predominantly phosphorylate topoisomerase II alpha. The phosphorylation status was correlated with catalytic activity of the enzyme. Western blot analysis using phosphoamino-specific antibodies shows that both the kinase activities catalyze the phosphorylation at serine residues of topoisomerase II alpha and beta. The catalytic inhibitions by serine kinase inhibitors further suggest that the alpha and beta kinase activities associated with virus are distinctly different.     

Phosphorylation of DNA topoisomerase II in vivo and in total homogenates of Drosophila Kc cells. The role of casein kinase II

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.     

Purification and characterization of type II DNA topoisomerase from mouse FM3A cells: phosphorylation of topoisomerase II and modification of its activity

Type II topoisomerase has been purified from mouse FM3A cells by using P4 phage knotted DNA as a substrate. Analysis of the purified enzyme by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed two bands of apparent molecular masses of 167 and 151 kDa. Partial digestion of the two bands with Staphylococcus aureus V8 protease indicated that the two polypeptides were structurally related. The enzyme required ATP and Mg2+ for activity. dATP could substitute for ATP, and ITP was slightly effective at 5-10 mM. The activity was sensitive to 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA), coumermycin, and ethidium bromide. A protein kinase activity was detected in the partially purified topoisomerase II fraction, and this protein kinase was further purified. The protein kinase phosphorylated the purified topoisomerase II, and the phosphorylation of topoisomerase II by the kinase increased the activity by 8.6-fold over that of the unmodified enzyme. The treatment of the purified topoisomerase II with alkaline phosphatase abolished the enzyme activity almost completely, and the treatment of the dephosphorylated topoisomerase II with the protein kinase restored the enzyme activity. The protein kinase activity was not stimulated by Ca2+ or cyclic nucleotides, and the aminoacyl residue phosphorylated by the kinase was serine. Enzymatic properties of the kinase were very similar to those of the kinase reported to be tightly associated with the Drosophila topoisomerase II [Sander, M., Nolan, J. M., & Hsieh, T.-S. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 6938-6942]. The immunoprecipitation of nuclear extracts prepared from 32P-labeled cells with anti-mouse topoisomerase II antiserum indicated that DNA topoisomerase II existed in mouse cells as a phosphoprotein.     

DNA topoisomerase I phosphorylation in murine fibroblasts treated with 12-O-tetradecanoylphorbol-13-acetate and in vitro by protein kinase.

The phosphorylation of DNA topoisomerase I in quiescent murine 3T3-L1 fibroblasts treated with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) was characterized by in vivo labeling with [32P] orthophosphate and immunoprecipitation with a scleroderma anti-DNA topoisomerase I autoantibody. DNA topoisomerase I phosphorylation was stimulated 4-fold by 2 h of TPA treatment (TPA at 100 ng/ml maximally enhanced phosphorylation). Purified DNA topoisomerase I was phosphorylated in vitro in a Ca2+ and phospholipid-dependent fashion by types I, II, and III protein kinase C. The phosphorylation reaction was stimulated by TPA and had an apparent K(m) of 0.4 microM. DNA topoisomerase I was phosphorylated in vivo and in vitro predominantly at serine. The major tryptic phosphopeptides from DNA topoisomerase I in TPA-treated fibroblasts and phosphorylated by protein kinase C comigrated in thin-layer electrophoresis. The half-life of incorporated phosphate on DNA topoisomerase I was 40 min in both TPA-treated and control cells. These results suggest that phosphorylation is a mechanism for activating DNA topoisomerase I in fibroblasts treated with TPA and that protein kinase C functions in the phosphorylation.     

Specific phosphorylation of proteins in pore complex-laminae from the sponge Geodia cydonium by the homologous aggregation factor and phorbol ester. Role of protein kinase C in the phosphorylation of DNA topoisomerase II.

We have recently shown that the aggregation factor (AF) from the sponge Geodia cydonium stimulates DNA synthesis in quiescent, dissociated cells from the same organism; this event was correlated with the release of the two second messengers: inositol trisphosphate and diacylglycerol. Here we describe that after binding of the AF to the plasma membrane-bound aggregation receptor, a rapid and drastic increase in the incorporation of 32Pi into a series of proteins in the pore complex-lamina fraction occurs. Addition of the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate, to quiescent cells resulted in a similar stimulation of phosphorylation of nuclear proteins. Among them we have selected one protein with a polypeptide Mr of 170,000 (pp170) for detailed studies. By immunoblotting pp170 was identified as DNA topoisomerase II. In vitro studies with nuclei and purified, homogeneous protein kinase C together with the required activators of this enzyme also showed a phosphorylation of pp170. After phosphorylation, DNA topoisomerase II activity was found to be 2.5-fold that of the non-phosphorylated enzyme. From these data we conclude that protein kinase C is involved in AF induced transmembrane signalling, ultimately leading to an initiation of DNA synthesis.     

Topoisomerase activity in irradiated mammalian cells

The role of topoisomerase enzymes in the response of HeLa S3 cells to ionizing radiation was investigated. Exposure of cells to 100 Gy of X-radiation had no detectable effect either on the total cellular topoisomerase activity as measured by the relaxation of supercoiled plasmid DNA by cell sonicates or on the total cellular topoisomerase II activity as measured by plasmid DNA catenation. Total topoisomerase II activity remained constant for up to 90 min after cell irradiation. The effect of 2 drugs (caffeine and novobiocin) which inhibit topoisomerase II activity on the HeLa cell response to radiation was determined. Both drugs were found to inhibit topoisomerase II in vitro and to inhibit the recovery of nucleoid sedimentation in irradiated cells in vivo to the same extent. Topoisomerase II was inhibited by 50% by exposure to 10 mM caffeine and 0.79 mM novobiocin. At low concentrations neither drug affected the induction frequency, nor the rejoining rate, of DNA double-strand breaks. Caffeine (5 mM) inhibited the short-term recovery of cells from radiation while novobiocin (0.79 mM) had no detectable effect on the capacity of cells to recover from radiation exposure. The results indicate that topoisomerase II is not required for DNA double-strand break rejoining though it could be required for the recovery of DNA coiling in the irradiated cell. If topoisomerase II is involved at all in cell recovery from irradiation, this role does not apparently involve an ATP-dependent enzyme activity.     

Overexpression of the atypical protein kinase C zeta reduces topoisomerase II catalytic activity,cleavable complexes formation,and drug-induced cytotoxicity in monocytic U937 leukemia cells

In this study, we evaluated the influence of protein kinase C zeta (PKC zeta) on topoisomerase II inhibitor-induced cytotoxicity in monocytic U937 cells. In U937-zeta J and U937-zeta B cells, enforced PKC zeta expression, conferred by stable transfection of PKC zeta cDNA, resulted in total inhibition of VP-16- and mitoxantrone-induced apoptosis and decreased drug-induced cytotoxicity, compared with U937-neo control cells. In PKC zeta-overexpressing cells, drug resistance correlated with decreased VP-16-induced DNA strand breaks and DNA protein cross-links measured by alkaline elution. Kinetoplast decatenation assay revealed that PKC zeta overexpression resulted in reduced global topoisomerase II activity. Moreover, in PKC zeta-overexpressing cells, we found that PKC zeta interacted with both alpha and beta isoforms of topoisomerase II, and these two enzymes were constitutively phosphorylated. However, when human recombinant PKC zeta (rH-PKC zeta) was incubated with purified topoisomerase II isoforms, rH-PKC zeta interacted with topoisomerase II beta but not with topoisomerase II alpha. PKC zeta/topoisomerase II beta interaction resulted in phosphorylation of this enzyme and in decrease of its catalytic activity. Finally, this report shows for the first time that topoisomerase II beta is a substrate for PKC zeta, and that PKC zeta may significantly influence topoisomerase II inhibitor-induced cytotoxicity by altering topoisomerase II beta activity through its kinase function.     

Heat sensitivity of Hela S3 Cell DNA topoisomerase II

The sensitivity of HeLa DNA topoisomerase II to 45°C heat shock was measured both in the intact cell and in vitro. In the intact cell, DNA topoisomerase II activity was estimated by measuring the formation and reversal of enzyme-DNA cleavable complexes by alkaline filter elution of cells exposed to the enzyme poison 4′-(9-acridinylamino)(methanesulfon-m-anisidide). In vitro enzymatic activity was estimated by measuring changes in the topological state of plasmid and kinetoplast DNA produced by sonicates of nuclei from previously heated cells. The capacity of the enzyme to form, or reverse, enzyme-DNA cleavable complexes was inactivated during 45°C heating with a reciprocal slope of 120 or 15 min, respectively. In vitro estimates of the activity of the enzyme from previously heated cells indicated that the enzyme was inactivated with a reciprocal slope of 99, 45, and 21 min after 45, 46 and 47°C heating, respectively. DNA topoisomerase I activity was inactivated with a reciprocal slope of 130 min at 45°C. The cumulative results indicate that during 45°C heat shock, thermal inactivation of neither DNA topoisomerase I nor II is rate limiting for either cell survival or for DNA replication. While DNA topoisomerase II is resistant in situ to heat inactivation, in vivo assays indicate that the enzyme's capacity to function in the intact cell may be compromised by hyperthermic changes in the enzyme's environment. © 1994 wiley-Liss, Inc.     

Mitotic specific phosphorylation of serine-1212 in human DNA topoisomerase IIalpha.

It is known that topoisomerase IIalpha is phosphorylated by several kinases. To elucidate the role of phosphorylation of topoisomerase IIalpha in the cell cycle, we have examined the cell cycle behavior of phosphorylated topoisomerase IIalpha in HeLa cells using antibodies against several phospho-oligopeptides of this enzyme. Here we demonstrate that serine1212 in topoisomerase IIalpha is phosphorylated only in the mitotic phase. Using an antibody against an oligopeptide containing phosphoserine-1212 in topoisomerase IIalpha (PS1212), subcellular localization of topoisomerase IIalpha phosphorylated at serine1212 was examined by indirect immunofluorescence staining, and compared with that of overall topoisomerase IIalpha. Serine1212-phosphorylated topoisomerase IIalpha was localized specifically on mitotic chromosomes, but not on interphase chromosomes; this result contrasts with overall topoisomerase IIalpha which was observed on chomosomes in both interphase and mitosis. Serine1212-phosphorylated topoisomerase lIalpha first appeared on chromosome arms in prophase, became concentrated on the centromeres in metaphase, and disappeared in early telophase. In addition, ICRF-193, a catalytic inhibitor of topoisomerase II, prevented accumulation of serine1212-phosphorylated topoisomerase IIalpha at the centromeres. These results indicate that serine1212 of topoisomerase IIalpha is phosphorylated specifically during mitosis, and suggest that the serine1212-phosphorylated topoisomerase IIalpha acts on resolving topological constraint progressively from the chromosome arm to the centromere during metaphase chromosome condensation.     

A diazirine-based photoaffinity etoposide probe for labeling topoisomerase II

Etoposide is a widely used anticancer drug that targets topoisomerase II, an essential nuclear enzyme. However, despite the fact that it has been in use and studied for more than 30 years the specific site on the enzyme to which it binds is unknown. In order to identify the etoposide binding site(s) on topoisomerase II, a diazirine-based photoaffinity etoposide analog probe has been synthesized and its photoreactivity and biological activities have been characterized. Upon UV irradiation, the diazirine probe rapidly produced a highly reactive carbene species that formed covalent adducts containing stable carbon-based bonds indicating that it should also be able to form stable covalent adducts with amino acid residues on topoisomerase II. The human leukemia K562 cell growth and topoisomerase II inhibitory properties of the diazirine probe suggest that it targets topoisomerase II in a manner similar to etoposide. The diazirine probe was also shown to act as a topoisomerase II poison through its ability to cause topoisomerase IIα-mediated double-strand cleavage of DNA. Additionally, the diazirine probe significantly increased protein–DNA covalent complex formation upon photoirradiation of diazirine probe-treated K562 cells, as compared to etoposide-treated cells. This result suggests that the photoactivated probe forms a covalent adduct with topoisomerase IIα. In conclusion, the present characterization of the chemical, biochemical, and biological properties of the newly synthesized diazirine-based photoaffinity etoposide analog indicates that use of a proteomics mass spectrometry approach will be a tractable strategy for future identification of the etoposide binding site(s) on topoisomerase II through covalent labeling of amino acid residues.     

Golgi galactosyltransferase contains serine-linked phosphate

In HeLa and HepG2 cells the Golgi complex enzyme galactosyltransferase became phosphorylated following incubation with 32Pi-Analysis on sodium dodecyl sulphate/polyacrylamide gel electrophoresis revealed incorporation of 32P into the mature 54-kDa form. This phosphorylation was independent of protein synthesis. Serine was identified as the sole phosphorylated amino acid; no radioactive phosphate was detected on N-linked oligosaccharide. The phosphate-labelled galactosyltransferase has the same turnover as [35S]methionine-labelled polypeptides (t1/2 = 20 h). Soluble enzyme, released by the cells, contained very little phosphate relative to that which remained cell-associated. Charge heterogeneity arising from phosphorylation contributes in part to the heterodispersed appearance of the enzyme on two-dimensional gels, as the degree of radioactive phosphate differs among the different iso-enzymes.     

Topoisomerase II: its functions and phosphorylation

The gene encoding topoisomerase II in yeast is unique and essential, required for both mitotic and meiotic proliferation. The use of temperature-sensitive mutants in topoisomerase II have demonstrated roles in the relaxation of tortional stress, reduction of recombination rates, and in the separation of sister chromatids after replication. In vertebrate cells, topoisomerase II was shown to be the most abundant component of the metaphase chromosomal scaffold, and has been shown to play a role in chromosome condensationin vitro. The cell cycle control of chromosome condensation may well require phosphorylation of topoisomerase II, since the enzyme is more highly phosphorylated in metaphase than in G1. Recent studies have identified casein kinase II as the major enzyme phosphorylating topoisomerase II in intact yeast cells. The target sites of CKII are exclusively in the C-terminal 400 amino acids of topoisomerase II, the region that is most divergent among the eukaryotic type II enzymes and which is absent in the bacterial gyrase homologues.Abbreviations topoII topoisomerase II - CKII Casein Kinase II - SV40 Simian Virus 40     

Topoisomerase I phosphorylation in vitro and in rapidly growing Novikoff hepatoma cells.

Changes in phosphorylation modulate the activity of topoisomerase I in vitro. Specifically, enzymatic activity is stimulated by phosphorylation with a purified protein kinase (casein kinase type II). The purpose of this study was to compare the sites that are phosphorylated in vitro by casein kinase type II with the site(s) phosphorylated in vivo in rapidly growing Novikoff hepatoma cells. Topoisomerase I labeled in vitro was characterized by three major tryptic phosphopeptides (I-III). Separation of these peptides by a C18-reverse phase h.p.l.c. column resulted in their elution at fractions 18 (I), 27 (II) and 44 (III) with 17%, 22.5% and 33% acetonitrile, respectively. In contrast, only one major phosphopeptide was identified by h.p.l.c. in topoisomerase I labeled in vivo. This phosphopeptide eluted at fraction 18 corresponding to the elution properties of phosphopeptide I labeled in vitro. It also co-migrated with tryptic phosphopeptide I when subjected to high-voltage electrophoresis on thin-layer cellulose plates. Preliminary experiments suggest that phosphorylation occurs at a serine residue six amino acids from the N-terminus of the peptide. These data indicate that topoisomerase I is phosphorylated in vivo and in vitro within the same tryptic peptide and suggest that topoisomerase I is phosphorylated in vivo by casein kinase II.     

Isolation and characterization of a human cDNA clone encoding a novel DNA topoisomerase II homologue from HeLa cells

We have isolated and sequenced 3 human DNA topoisomerase II (topo II) partial cDNA clones from a HeLa carcinoma cell cDNA library. Two clones were identical to an internal fragment of HeLa topo II cDNA. The third clone, CAA5, had a different and novel sequence which shared significant nucleotide (62%) and predicted peptide (70%) homologies with a region of the HeLa topo II cDNA. Our results suggest that HeLa cells express at least two homologous forms of DNA topoisomerase II. The new HeLa topo II homologue is discussed in relation to topo II isoenzymes recently described in a Burkitt lymphoma and other cell lines.     

Biosynthesis and modification of Golgi mannosidase II in HeLa and 3T3 cells

The biosynthesis and post-translational modification of mannosidase II, an enzyme required in the maturation of asparagine-linked oligosaccharides in the Golgi complex, has been investigated. Antibody raised against this enzyme purified from rat liver Golgi membranes was used to immunoprecipitate mannosidase II from rat liver, 3T3 cells, or HeLa cells. Mannosidase II immunoprecipitated from rat liver Golgi membranes, when analyzed by polyacrylamide gel electrophoresis, migrated with an apparent molecular weight of approximately 124,000. In contrast, the enzyme purified from rat liver Golgi membranes was shown to contain both the 124,000-dalton component and a 110,000-dalton polypeptide believed to result from degradation of intact mannosidase II during purification. Mannosidase II from 3T3 and HeLa cells migrated on polyacrylamide gels with apparent molecular weights of approximately 124,000 and 134,000-136,000, respectively. When immunoprecipitated from radiolabeled cultures, mannosidase II from both cell types was similar in the following respects: (a) the initial synthesis product had an apparent molecular weight of approximately 124,000; (b) in cultures treated with tunicamycin the initial synthesis product had an apparent molecular weight of approximately 117,000; (c) endoglycosidase H digestion of the initial synthesis product gave an apparent molecular weight similar to the tunicamycin-induced polypeptide; (d) the mature enzyme was mostly (HeLa) or entirely (3T3) resistant to digestion by endoglycosidase H. Loss of [35S]methionine from intracellular mannosidase II occurred with a half-life of approximately 20 h; there was no appreciable accumulation of labeled immuno-reactive material in the medium. HeLa mannosidase II, but not the 3T3 enzyme, was additionally modified 1-3 h after synthesis, the initial synthesis product being converted to a doublet with an apparent molecular weight of approximately 134,000-136,000. Evidence is presented that this mobility shift may result from O-glycosylation. Mannosidase II from both cell types could be labeled with [32P]phosphate or [35S]sulfate. The latter is apparently attached to oligosaccharide as indicated by inhibition of labeling by tunicamycin; the former was shown with the HeLa enzyme to be present as serine phosphate moieties. In addition, [3H]palmitate could be incorporated into the enzyme in 3T3 cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Investigation of the biological mode of action of clerocidin using whole cell assays.

Clerocidin, a diterpenoid natural product, has been shown in vitro to inhibit DNA religation following cleavage by topoisomerase II. Herein, we characterize the efficacy and specificity of clerocidin in HeLa cells. Our results suggest that clerocidin recognizes topoisomerase II as its main intracellular target and binds to this enzyme prior to formation of the 'cleavable complex' with DNA. These pharmacological features attest to the promising chemotherapeutic potential of this natural product.