0 Mitotic Phosphorylation Stimulates DNA Relaxation Activity, otherFeat=[]-->, belongsTo=title 1 of Human Topoisomerase I*S, otherFeat=[]-->, belongsTo=parrnote 2 Received for publication, September 14, 2007, and in revised form, March 20, 2008 Published, JBC Papers in Press, April 11, 2008, DOI 10.1074/jbc.M802246200, otherFeat=[]-->, belongsTo=parrnote 3 Jennifer S. Hackbarth, Marina Galvez-Peralta?, Nga T. Dai?, David A. Loegering?, Kevin L. Peterson?,, otherFeat=[]-->, belongsTo=parrnote 4 Xue W. Meng?, Larry M. Karnitz?, and Scott H. Kaufmann?1, otherFeat=[]-->, belongsTo=title 5 From the Department of Biochemistry and Molecular Biology and ?Division of Oncology Research, Mayo Clinic, Mayo Graduate, otherFeat=[]-->, belongsTo=title 6 School, Rochester, Minnesota 55905, otherFeat=[]-->, belongsTo=title 7 Human DNA topoisomerase I (topo I) is an essential mamma-, otherFeat=[]-->, belongsTo=parrnote 8 lian enzyme that regulates DNA supercoiling during transcrip-, otherFeat=[]-->, belongsTo=parrnote 9 tion and replication. In addition, topo I is specifically targeted by, otherFeat=[]-->, belongsTo=parrnote 10 the anticancer compound camptothecin and its derivatives. Pre-, otherFeat=[]-->, belongsTo=parrnote 11 vious studies have indicated that topo I is a phosphoprotein and, otherFeat=[]-->, belongsTo=parrnote 12 that phosphorylation stimulates its DNA relaxation activity., otherFeat=[]-->, belongsTo=parrnote 13 The locations of most topo I phosphorylation sites have not been, otherFeat=[]-->, belongsTo=parrnote 14 identified, preventing a more detailed examination of this mod-, otherFeat=[]-->, belongsTo=parrnote 15 ification. To address this issue, mass spectrometry was used to, otherFeat=[]-->, belongsTo=parrnote 16 identify four topo I residues that are phosphorylated in intact, otherFeat=[]-->, belongsTo=parrnote 17 cells: Ser10, Ser21, Ser112, and Ser394. Immunoblotting using, otherFeat=[]-->, belongsTo=parrnote 18 anti-phosphoepitope antibodies demonstrated that these sites, otherFeat=[]-->, belongsTo=parrnote 19 are phosphorylated during mitosis. In vitro kinase assays dem-, otherFeat=[]-->, belongsTo=parrnote 20 onstrated that Ser10 can be phosphorylated by casein kinase II,, otherFeat=[]-->, belongsTo=parrnote 21 Ser21 can be phosphorylated by protein kinase C , and Ser112, otherFeat=[]-->, belongsTo=parrnote 22 and Ser394 can be phosphorylated by Cdk1. When wild type topo, otherFeat=[]-->, belongsTo=parrnote 23 I was pulled down from mitotic cells and dephosphorylated with, otherFeat=[]-->, belongsTo=parrnote 24 alkaline phosphatase, topo I activity decreased 2-fold. Likewise,, otherFeat=[]-->, belongsTo=parrnote 25 topo I polypeptide with all four phosphorylation sites mutated, otherFeat=[]-->, belongsTo=parrnote 26 to alanine exhibited 2-fold lower DNA relaxation activity than, otherFeat=[]-->, belongsTo=parrnote 27 wild type topo I after isolation from mitotic cells. Further muta-, otherFeat=[]-->, belongsTo=parrnote 28 tional analysis demonstrated that Ser21 phosphorylation was, otherFeat=[]-->, belongsTo=parrnote 29 responsible for this change. Consistent with these results, wild, otherFeat=[]-->, belongsTo=parrnote 30 type topo I (but not S21A topo I) exhibited increased sensitivity, otherFeat=[]-->, belongsTo=parrnote 31 to camptothecin-induced trapping on DNA during mitosis. Col-, otherFeat=[]-->, belongsTo=parrnote 32 lectively these results indicate that topo I is phosphorylated dur-, otherFeat=[]-->, belongsTo=parrnote 33 ing mitosis at multiple sites, one of which enhances DNA relax-, otherFeat=[]-->, belongsTo=parrnote 34 ation activity in vitro and interaction with DNA in cells., otherFeat=[]-->, belongsTo=parrnote 35 Human topo I2 is a type IB topoisomerase that relieves posi-, otherFeat=[]-->, belongsTo=parr 36 tive and negative DNA supercoiling caused by transcription,, otherFeat=[]-->, belongsTo=parr 37 replication, and chromosome condensation (1). The 91-kDa,, otherFeat=[]-->, belongsTo=parr 38 765-amino acid polypeptide contains four domains: a poorly, otherFeat=[]-->, belongsTo=parr 39 conserved lysine-rich N-terminal domain that contains nuclear, otherFeat=[]-->, belongsTo=parr 40 and nucleolar localization signals, a linker region, and the core, otherFeat=[]-->, belongsTo=parr 41 and C-terminal domains that contain the residues important, otherFeat=[]-->, belongsTo=parr 42 for DNA interaction and relaxation of supercoils (2). A trans-, otherFeat=[]-->, belongsTo=parr 43 esterification reaction at the active site of topo I ligates Tyr723 of, otherFeat=[]-->, belongsTo=parr 44 the enzyme to the 3 phosphate of the DNA, thereby creating a, otherFeat=[]-->, belongsTo=parr 45 nick in the DNA backbone (3). This nick allows controlled rota-, otherFeat=[]-->, belongsTo=parr 46 tion of the DNA to relieve supercoils. Mutation of Tyr723 pre-, otherFeat=[]-->, belongsTo=parr 47 vents the transesterification reaction and abolishes all relax-, otherFeat=[]-->, belongsTo=parr 48 ation activity (4). The anticancer drug CPT and its derivatives, otherFeat=[]-->, belongsTo=parr 49 slow topo I-mediated DNA relaxation (5, 6) and inhibit the, otherFeat=[]-->, belongsTo=parr 50 religation reaction step of the enzyme (7, 8), trapping topo I on, otherFeat=[]-->, belongsTo=parr 51 DNA (9, 10) and causing cell death (11, 12)., otherFeat=[]-->, belongsTo=parr 52 A number of observations have raised the possibility that, otherFeat=[]-->, belongsTo=parr 53 phosphorylation can modulate the activity and CPT sensitivity, otherFeat=[]-->, belongsTo=parr 54 of topo I. Treatment with calf intestine alkaline phosphatase, otherFeat=[]-->, belongsTo=parr 55 decreases topo I enzymatic activity in vitro (13?15). Conversely, otherFeat=[]-->, belongsTo=parr 56 subsequent treatment with PKC or CKII, two kinases that, otherFeat=[]-->, belongsTo=parr 57 copurify with topo I and phosphorylate it in vitro (16 ?18), stim-, otherFeat=[]-->, belongsTo=parr 58 ulates topo I activity 2?3-fold (14, 15, 19) and enhances the, otherFeat=[]-->, belongsTo=parr 59 ability of CPT to trap covalent topo I-DNA cleavage complexes, otherFeat=[]-->, belongsTo=parr 60 (20), suggesting that phosphorylation by these kinases might, otherFeat=[]-->, belongsTo=parr 61 make topo I more sensitive to CPT., otherFeat=[]-->, belongsTo=parr 62 Despite its potential importance, many aspects of topo I, otherFeat=[]-->, belongsTo=parr 63 phosphorylation remain poorly understood. The number of, otherFeat=[]-->, belongsTo=parr 64 phosphorylation sites, for example, remains unclear because, otherFeat=[]-->, belongsTo=parr 65 the number of phosphopeptides detected after metabolic label-, otherFeat=[]-->, belongsTo=parr 66 ing and immunoprecipitation has varied from one (16) to as, otherFeat=[]-->, belongsTo=parr 67 many as five (21) or six (17). The effect of cell cycle progression, otherFeat=[]-->, belongsTo=parr 68 on phosphorylation has likewise been unclear. Initial studies, otherFeat=[]-->, belongsTo=parr 69 suggested that topo I is phosphorylated in interphase cells as, otherFeat=[]-->, belongsTo=parr 70 evidenced by its labeling in unsynchronized cell populations, otherFeat=[]-->, belongsTo=parr 71 and a rapid increase in phosphorylation after certain treat-, otherFeat=[]-->, belongsTo=parr 72 ments (13, 16, 17, 20, 21). In contrast, a more recent study found, otherFeat=[]-->, belongsTo=parr 73 that topo I in rodent cells quantitatively shifts to a slower, otherFeat=[]-->, belongsTo=parr 74 migrating, phosphorylated state exclusively during mitosis (22)., otherFeat=[]-->, belongsTo=parr 75 Finally the location of topo I phosphorylation sites has not been, otherFeat=[]-->, belongsTo=parr 76 resolved. Cardellini et al. (23) reported that a 17-amino acid, otherFeat=[]-->, belongsTo=parr 77 peptide from the N terminus of topo I could be phosphorylated, otherFeat=[]-->, belongsTo=parr 78 at Ser10 by CKII in vitro, but phosphorylation of this site was not, otherFeat=[]-->, belongsTo=parr 79 verified in the full-length polypeptide in vitro or in vivo. More, otherFeat=[]-->, belongsTo=parr 80 recently, Yu et al. (24) reported that topo I is phosphorylated on, otherFeat=[]-->, belongsTo=parr 81 Tyr268 by c-Abl in vitro, but again the phosphorylation of this, otherFeat=[]-->, belongsTo=parr 82 site in intact cells was not confirmed. The location of other, otherFeat=[]-->, belongsTo=parr 83 possible phosphorylation sites on topo I, the cellular conditions, otherFeat=[]-->, belongsTo=parr 84 * This work was supported, in whole or in part, by National Institutes of Health, otherFeat=[]-->, belongsTo=parrnote 85 Grant R01 CA73709. The costs of publication of this article were defrayed in, otherFeat=[]-->, belongsTo=parrnote 86 part by the payment of page charges. This article must therefore be hereby, otherFeat=[]-->, belongsTo=parrnote 87 marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to, otherFeat=[]-->, belongsTo=parrnote 88 indicate this fact., otherFeat=[]-->, belongsTo=parrnote 89 S The on-line version of this article (available at http://www.jbc.org) contains, otherFeat=[]-->, belongsTo=parrnote 90 supplemental Figs. S1?S4., otherFeat=[]-->, belongsTo=parrnote 91 1 To whom correspondence should be addressed: Division of Oncology, otherFeat=[]-->, belongsTo=parrnote 92 Research, Guggenheim 1342C, Mayo Clinic, 200 First St. S.W., Rochester,, otherFeat=[]-->, belongsTo=parrnote 93 MN 55905. Tel.: 507-284-8950; Fax: 507-284-3906; E-mail: Kaufmann., otherFeat=[]-->, belongsTo=parrnote 94 scott@mayo.edu., otherFeat=[]-->, belongsTo=parrnote 95 2 The abbreviations used are: topo, topoisomerase; Cdk, cyclin-dependent, otherFeat=[]-->, belongsTo=parrnote 96 kinase; CKII, casein kinase II; CPT, camptothecin; PBS, calcium- and magne-, otherFeat=[]-->, belongsTo=parrnote 97 sium-free Dulbecco's phosphate-buffered saline; PKC, protein kinase C;, otherFeat=[]-->, belongsTo=parrnote 98 PMSF, -phenylmethylsulfonyl fluoride., otherFeat=[]-->, belongsTo=parrnote 99 THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 283, NO. 24, pp. 16711?16722, June 13, 2008, otherFeat=[]-->, belongsTo=nota_cab_pie 100 ? 2008 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A., otherFeat=[]-->, belongsTo=nota_cab_pie 101 JUNE 13, 2008 ? VOLUME 283 ? NUMBER 24, otherFeat=['U']-->, belongsTo=nota_cab_pie 102 JOURNAL OF BIOLOGICAL CHEMISTRY 16711, otherFeat=[]-->, belongsTo=nota_cab_pie 103 at, otherFeat=[u'a']-->, belongsTo=nota_cab_pie 104 Centro, otherFeat=[]-->, belongsTo=nota_cab_pie 105 Nacional, otherFeat=[]-->, belongsTo=nota_cab_pie 106 de, otherFeat=[]-->, belongsTo=nota_cab_pie 107 Investigaciones, otherFeat=[]-->, belongsTo=nota_cab_pie 108 Oncol?gicas,, otherFeat=[]-->, belongsTo=nota_cab_pie 109 on, otherFeat=[]-->, belongsTo=nota_cab_pie 110 May, otherFeat=[]-->, belongsTo=nota_cab_pie 111 28,, otherFeat=[]-->, belongsTo=nota_cab_pie 112 2010, otherFeat=[]-->, belongsTo=nota_cab_pie 113 www.jbc.org, otherFeat=[]-->, belongsTo=nota_cab_pie 114 Downloaded, otherFeat=[]-->, belongsTo=nota_cab_pie 115 from, otherFeat=[]-->, belongsTo=nota_cab_pie 116 http://www.jbc.org/content/suppl/2008/04/16/M802246200.DC1.html, otherFeat=[]-->, belongsTo=nota_cab_pie 117 Supplemental Material can be found at:, otherFeat=[]-->, belongsTo=nota_cab_pie 118 that cause phosphorylation, and the effects of individual phos-, otherFeat=[]-->, belongsTo=parr 119 phorylation events on topo I remain largely unknown., otherFeat=[]-->, belongsTo=parr 120 In the present study, we utilized mass spectrometry to map, otherFeat=[]-->, belongsTo=parr 121 four phosphorylation sites on topo I, generated phospho-, otherFeat=[]-->, belongsTo=parr 122 epitope-specific antibodies, and used these antibodies to study, otherFeat=[]-->, belongsTo=parr 123 the conditions leading to topo I phosphorylation in vitro and in, otherFeat=[]-->, belongsTo=parr 124 intact cells. In addition, we performed site-directed mutagene-, otherFeat=[]-->, belongsTo=parr 125 sis of these sites to assess the impact on localization and activity., otherFeat=[]-->, belongsTo=parr 126 Results of this analysis suggest that topo I is phosphorylated, otherFeat=[]-->, belongsTo=parr 127 during mitosis in untreated cells and that one of these mitotic, otherFeat=[]-->, belongsTo=parr 128 phosphorylations modestly enhances topo I activity in vitro as, otherFeat=[]-->, belongsTo=parr 129 well as sensitivity to CPT-induced trapping on DNA in intact, otherFeat=[]-->, belongsTo=parr 130 cells., otherFeat=[]-->, belongsTo=parr 131 EXPERIMENTAL PROCEDURES, otherFeat=['U']-->, belongsTo=title 132 Materials--Reagents were obtained from the following sup-, otherFeat=[]-->, belongsTo=parr 133 pliers: CPT, paclitaxel, aphidicolin, hydroxyurea, Hoechst, otherFeat=[]-->, belongsTo=parr 134 33258, PMSF, phorbol myristate acetate, and chloroquine from, otherFeat=[]-->, belongsTo=parr 135 Sigma; nocodazole from Aldrich; and DMSO from Fisher. Anti-, otherFeat=[]-->, belongsTo=parr 136 bodies to the following antigens were obtained from the indi-, otherFeat=[]-->, belongsTo=parr 137 cated suppliers: nucleolin from Santa Cruz Biotechnology, Inc.,, otherFeat=[]-->, belongsTo=parr 138 TATA-binding protein from BD Transduction Laboratories,, otherFeat=[]-->, belongsTo=parr 139 and topors from Novus. Anti-S peptide antibody was raised and, otherFeat=[]-->, belongsTo=parr 140 characterized as described previously (25). Antibodies to other, otherFeat=[]-->, belongsTo=parr 141 antigens were kind gifts from the following investigators:, otherFeat=[]-->, belongsTo=parr 142 murine monoclonal anti-poly(ADP-ribose) polymerase from, otherFeat=[]-->, belongsTo=parr 143 Guy Poirier (Laval University, Ste. Foy, Quebec, Canada), human, otherFeat=[]-->, belongsTo=parr 144 anti-topo I autoantiserum from Naomi Rothfield (University of, otherFeat=[]-->, belongsTo=parr 145 Connecticut, Farmington, CT), and C21 murine monoclonal anti-, otherFeat=[]-->, belongsTo=parr 146 topo I from Y.-C. Cheng (Yale University Medical School, New, otherFeat=[]-->, belongsTo=parr 147 Haven, CT)., otherFeat=[]-->, belongsTo=parr 148 Cell Culture--A549 human lung cancer cells and K562, otherFeat=[]-->, belongsTo=parr 149 human leukemia cells were grown in RPMI 1640 medium con-, otherFeat=[]-->, belongsTo=parr 150 taining 10% heat-inactivated fetal bovine serum, 100 units/ml, otherFeat=[]-->, belongsTo=parr 151 penicillin G, 100 g/ml streptomycin, 2 mML-glutamine, otherFeat=[]-->, belongsTo=parrnote 152 (medium A). Cells were incubated at 37 ?C in a humidified incu-, otherFeat=[]-->, belongsTo=parr 153 bator with 5% CO2., otherFeat=[]-->, belongsTo=parr 154 In the indicated experiments, cells were treated with 100 nM, otherFeat=[]-->, belongsTo=parr 155 paclitaxel for 16 h, resulting in arrest of 80% of cells in G2/M, otherFeat=[]-->, belongsTo=parr 156 as assessed by flow cytometry and 75? 80% of cells in metaphase, otherFeat=[]-->, belongsTo=parr 157 as assessed by fluorescence microscopy after Hoechst 33258, otherFeat=[]-->, belongsTo=parr 158 staining. Longer exposures to paclitaxel were avoided because, otherFeat=[]-->, belongsTo=parr 159 of adaptation of the mitotic checkpoint and exit of these cells, otherFeat=[]-->, belongsTo=parr 160 into a multinucleated, tetraploid G1 state (26)., otherFeat=[]-->, belongsTo=parr 161 Plasmid Construction--The yeast shuttle vector hGal1 con-, otherFeat=[]-->, belongsTo=parr 162 taining cDNA encoding catalytically inactive (Y723F) human, otherFeat=[]-->, belongsTo=parr 163 topo I was a kind gift from M.-A. Bjornsti (St. Jude Children's, otherFeat=[]-->, belongsTo=parr 164 Hospital, Memphis, TN). The cDNA was subjected to PCR, otherFeat=[]-->, belongsTo=parr 165 to add the S peptide and linker sequence (in brackets), otherFeat=[]-->, belongsTo=parr 166 [GAGAGAGAGGAP]MKETAAAKFERQHMDS or MKETA-, otherFeat=[]-->, belongsTo=parr 167 AAKFERQHMDS[GAGAGAGAGGAP] to either the C-termi-, otherFeat=[]-->, belongsTo=parr 168 nal (Topo I-S) or N-terminal (S-Topo I) end of the topo I open, otherFeat=[]-->, belongsTo=parr 169 reading frame, respectively. After ligation of Topo I-S or, otherFeat=[]-->, belongsTo=parr 170 S-Topo I into pcDNA 3.1 (Invitrogen), the entire insert was, otherFeat=[]-->, belongsTo=parr 171 sequenced. Topo I-S was used for generating a stably trans-, otherFeat=[]-->, belongsTo=parr 172 fected K562 cell line, which was then used for mass spectrom-, otherFeat=[]-->, belongsTo=parr 173 etry and in vitro kinase assays as described below. S-Topo I was, otherFeat=[]-->, belongsTo=parr 174 used for all other experiments., otherFeat=[]-->, belongsTo=parr 175 Transient and Stable Transfection--10 ?15 106 K562 cells, otherFeat=[]-->, belongsTo=parr 176 were washed in sterile PBS and resuspended in cytomix buffer, otherFeat=[]-->, belongsTo=parr 177 (120 mM KCl, 150 M CaCl2,2mM EGTA, 5 mM MgCl2,25mM, otherFeat=[]-->, belongsTo=parr 178 HEPES, 10 mM K2HPO4/KH2PO4 (pH 7.6)) containing 40 gof, otherFeat=[]-->, belongsTo=parr 179 plasmid. Cells were electroporated at 320 V for 10 ms in a BTX, otherFeat=[]-->, belongsTo=parr 180 ECM 830 square wave electroporator, incubated for 15 min at, otherFeat=[]-->, belongsTo=parr 181 room temperature, and diluted in medium A without antibiot-, otherFeat=[]-->, belongsTo=parr 182 ics. After 24 h, cells were utilized for the assays described below., otherFeat=[]-->, belongsTo=parr 183 Alternatively 48 h after electroporation, cells transfected with, otherFeat=[]-->, belongsTo=parr 184 Topo I-S (Y723F) were treated with 800 g/ml Geneticin to, otherFeat=[]-->, belongsTo=parr 185 select stable transfectants. Once Geneticin-resistant cells grew,, otherFeat=[]-->, belongsTo=parr 186 clones were isolated by limiting dilution and assayed for Topo, otherFeat=[]-->, belongsTo=parr 187 I-S expression by immunoblotting using anti-S peptide anti-, otherFeat=[]-->, belongsTo=parr 188 body. A resulting stable line with high expression was main-, otherFeat=[]-->, belongsTo=parr 189 tained in medium A containing 400 g/ml Geneticin., otherFeat=[]-->, belongsTo=parr 190 Immunoprecipitation and Pulldown--Endogenous topo I, otherFeat=[]-->, belongsTo=parr 191 was isolated from K562 cells using human anti-topo I autoan-, otherFeat=[]-->, belongsTo=parr 192 tibody and protein A-Sepharose beads. All steps were per-, otherFeat=[]-->, belongsTo=parr 193 formed at 4 ?C. Cells were washed twice in ice-cold PBS, incu-, otherFeat=[]-->, belongsTo=parr 194 bated in lysis buffer with protease and phosphatase inhibitors, otherFeat=[]-->, belongsTo=parr 195 (1% (w/v) Triton X-100, 400 mM NaCl, 50 mM HEPES (pH 7.5),, otherFeat=[]-->, belongsTo=parr 196 10% (w/v) glycerol, 5 mM MgSO4,1mM EDTA, 1 mM sodium, otherFeat=[]-->, belongsTo=parrnote 197 orthovanadate, 10 mM sodium pyrophosphate, 100 mM NaF, 1, otherFeat=[]-->, belongsTo=parr 198 mM PMSF, 10 g/ml leupeptin, 100 units/ml Trasylol, 1% (w/v), otherFeat=[]-->, belongsTo=parr 199 thiodiglycol, 20 nM microcystin) for 15 min, and sedimented at, otherFeat=[]-->, belongsTo=parr 200 12,000 g for 15 min. Topo I antibody was added to the result-, otherFeat=[]-->, belongsTo=parr 201 ing supernatant, which was rotated end over end overnight., otherFeat=[]-->, belongsTo=parr 202 Samples were supplemented with protein A beads and rotated, otherFeat=[]-->, belongsTo=parr 203 for an additional 4 h. Beads were spun down at 12,000 g for 1, otherFeat=[]-->, belongsTo=parr 204 min and washed four times with RIPA buffer containing phos-, otherFeat=[]-->, belongsTo=parr 205 phatase inhibitors (1% (w/v) Triton X-100, 1% (w/v) sodium, otherFeat=[]-->, belongsTo=parr 206 deoxycholate, 0.1% (w/v) SDS, 150 mM NaCl, 10 mM sodium, otherFeat=[]-->, belongsTo=parrnote 207 phosphate (pH 7.2), 2 mM EDTA, 1 mM sodium orthovanadate,, otherFeat=[]-->, belongsTo=parr 208 100 units/ml Trasylol, 50 mM NaF). To elute protein for SDS-, otherFeat=[]-->, belongsTo=parr 209 PAGE, beads were resuspended in sample buffer (4 M urea, 2%, otherFeat=[]-->, belongsTo=parr 210 (w/v) SDS, 62.5 mM Tris-HCl (pH 6.8), 1 mM EDTA, 5% (v/v), otherFeat=[]-->, belongsTo=parr 211 2-mercaptoethanol, 0.1% (w/v) bromphenol blue) and heated, otherFeat=[]-->, belongsTo=parr 212 to 65 ?C for 20 min., otherFeat=[]-->, belongsTo=parr 213 S-Topo I or Topo I-S was isolated using a similar procedure., otherFeat=[]-->, belongsTo=parr 214 In brief, lysates were prepared as described above and sedi-, otherFeat=[]-->, belongsTo=parr 215 mented at 12,000 g for 15 min. S protein beads (Novagen), otherFeat=[]-->, belongsTo=parr 216 were added to the resulting supernatant, which was rotated end, otherFeat=[]-->, belongsTo=parr 217 over end overnight. Beads were then sedimented and washed, otherFeat=[]-->, belongsTo=parr 218 four times with RIPA buffer containing phosphatase inhibitors, otherFeat=[]-->, belongsTo=parr 219 prior to SDS-PAGE or incubation with kinases. Alternatively, otherFeat=[]-->, belongsTo=parr 220 immobilized topo I was washed with 0.1% Nonidet P-40 in PBS, otherFeat=[]-->, belongsTo=parr 221 rather than RIPA buffer and assayed for topo I activity., otherFeat=[]-->, belongsTo=parr 222 Metabolic Labeling--2.5 107 K562 cells were washed once, otherFeat=[]-->, belongsTo=parr 223 and resuspended in 10 ml of phosphate- and serum-free RPMI, otherFeat=[]-->, belongsTo=parr 224 1640 medium (BIOSOURCE). After cells were incubated for 30, otherFeat=[]-->, belongsTo=parr 225 min at 37 ?C, 2.5 mCi of [32P]orthophosphate (Amersham Bio-, otherFeat=[]-->, belongsTo=parr 226 sciences) and dialyzed fetal calf serum (10% (v/v) final concen-, otherFeat=[]-->, belongsTo=parr 227 tration) were added for 4 h. Topo I was recovered from radio-, otherFeat=[]-->, belongsTo=parr 228 labeled cells by immunoprecipitation or pulldown with S, otherFeat=[]-->, belongsTo=parr 229 protein-agarose as described above, subjected to SDS-PAGE,, otherFeat=[]-->, belongsTo=parr 230 and visualized by autoradiography., otherFeat=[]-->, belongsTo=parr 231 Two-dimensional Tryptic Mapping--5.0 107 K562 cells, otherFeat=[]-->, belongsTo=parr 232 stably transfected with Topo I-S (Y723F) were treated for, otherFeat=[]-->, belongsTo=parr 233 Mitotic Phosphorylation of Human Topoisomerase I, otherFeat=[]-->, belongsTo=nota_cab_pie 234 16712 JOURNAL OF BIOLOGICAL CHEMISTRY, otherFeat=['U']-->, belongsTo=nota_cab_pie 235 VOLUME 283 ? NUMBER 24 ? JUNE 13, 2008, otherFeat=['U']-->, belongsTo=nota_cab_pie 236 at, otherFeat=[u'a']-->, belongsTo=nota_cab_pie 237 Centro, otherFeat=[]-->, belongsTo=nota_cab_pie 238 Nacional, otherFeat=[]-->, belongsTo=nota_cab_pie 239 de, otherFeat=[]-->, belongsTo=nota_cab_pie 240 Investigaciones, otherFeat=[]-->, belongsTo=nota_cab_pie 241 Oncol?gicas,, otherFeat=[]-->, belongsTo=nota_cab_pie 242 on, otherFeat=[]-->, belongsTo=nota_cab_pie 243 May, otherFeat=[]-->, belongsTo=nota_cab_pie 244 28,, otherFeat=[]-->, belongsTo=nota_cab_pie 245 2010, otherFeat=[]-->, belongsTo=nota_cab_pie 246 www.jbc.org, otherFeat=[]-->, belongsTo=nota_cab_pie 247 Downloaded, otherFeat=[]-->, belongsTo=nota_cab_pie 248 from, otherFeat=[]-->, belongsTo=nota_cab_pie 249 http://www.jbc.org/content/suppl/2008/04/16/M802246200.DC1.html, otherFeat=[]-->, belongsTo=nota_cab_pie 250 Supplemental Material can be found at:, otherFeat=[]-->, belongsTo=nota_cab_pie 251 16 h with 100 nM paclitaxel and then radiolabeled with, otherFeat=[]-->, belongsTo=parr 252 [32P]orthophosphate as described above. Topo I pulled down, otherFeat=[]-->, belongsTo=parr 253 with S protein-agarose was identified by SDS-PAGE followed, otherFeat=[]-->, belongsTo=parr 254 by autoradiography. The radiolabeled topo I band was excised, otherFeat=[]-->, belongsTo=parr 255 and subjected to reduction, alkylation, and digestion with 2 g, otherFeat=[]-->, belongsTo=parr 256 of trypsin as described by Meisenhelder et al. (27). The resulting, otherFeat=[]-->, belongsTo=parr 257 sample was spotted onto a 20 20-cm, 100- m cellulose thin, otherFeat=[]-->, belongsTo=parr 258 layer chromatography plate (EM Science), subjected to electro-, otherFeat=[]-->, belongsTo=parr 259 phoresis at 1000 V for 30 min in pH 1.9 buffer, and exposed to, otherFeat=[]-->, belongsTo=parr 260 ascending chromatography overnight in buffer consisting of, otherFeat=[]-->, belongsTo=parr 261 37.5% (v/v) n-butyl alcohol, 25% (v/v) pyridine, 7.5% (v/v) acetic, otherFeat=[]-->, belongsTo=parr 262 acid. Radiolabeled spots were detected using a Storm 840 Phos-, otherFeat=[]-->, belongsTo=parr 263 phorImager (GE Healthcare)., otherFeat=[]-->, belongsTo=parr 264 Mass Spectrometry--Topo I-S was isolated from 1 107 sta-, otherFeat=[]-->, belongsTo=parr 265 bly transfected K562 cells, subjected to SDS-PAGE, and stained, otherFeat=[]-->, belongsTo=parr 266 with Coomassie Blue. The topo I band was excised, digested, otherFeat=[]-->, belongsTo=parr 267 using either trypsin or Arg-C, and analyzed by quadrupole, otherFeat=[]-->, belongsTo=parr 268 time-of-flight tandem mass spectrometry at the Taplin Biolog-, otherFeat=[]-->, belongsTo=parr 269 ical Mass Spectrometry Facility (Harvard Medical School,, otherFeat=[]-->, belongsTo=parr 270 Boston, MA)., otherFeat=[]-->, belongsTo=parr 271 Generation of Phosphospecific Antibodies--Phosphory-, otherFeat=[]-->, belongsTo=parr 272 lated and nonphosphorylated peptides surrounding Ser10, otherFeat=[]-->, belongsTo=parr 273 (CDHLHNDSQIEADFR), Ser21 (CADFRLNDSHKHKDKH),, otherFeat=[]-->, belongsTo=parrnote 274 Ser112 (CEKENGFSSPPQIKDE), and Ser394 (CSKDAKVPSP-, otherFeat=[]-->, belongsTo=parr 275 PPGHKW) were synthesized. Phosphorylated peptides were, otherFeat=[]-->, belongsTo=parr 276 conjugated to keyhole limpet hemocyanin through their, otherFeat=[]-->, belongsTo=parr 277 N-terminal cysteines and utilized to immunize rabbits., otherFeat=[]-->, belongsTo=parr 278 Bleeds were screened by immunoblotting using whole cell, otherFeat=[]-->, belongsTo=parr 279 lysates of K562 cells and dot blots of both the phosphorylated, otherFeat=[]-->, belongsTo=parr 280 and nonphosphorylated peptides coupled to bovine serum, otherFeat=[]-->, belongsTo=parr 281 albumin at concentrations from 1 g to 100 pg. Bleeds show-, otherFeat=[]-->, belongsTo=parr 282 ing evidence of specificity for phosphorylated peptides were, otherFeat=[]-->, belongsTo=parr 283 affinity-purified by sequential passage over SulfoLink col-, otherFeat=[]-->, belongsTo=parr 284 umns (Pierce) derivatized with nonphosphorylated and, otherFeat=[]-->, belongsTo=parr 285 phosphorylated peptides. Antibodies were eluted in 100 mM, otherFeat=[]-->, belongsTo=parr 286 glycine (pH 2.5), neutralized with 1 M Tris-HCl (pH 9.5),, otherFeat=[]-->, belongsTo=parr 287 assayed by dot blot and whole cell lysate immunoblotting,, otherFeat=[]-->, belongsTo=parr 288 diluted 1:1 in glycerol, and stored at 20 ?C. The specificity, otherFeat=[]-->, belongsTo=parr 289 of the antibodies was tested by a peptide competition assay, otherFeat=[]-->, belongsTo=parr 290 in which blots of mitotic K562 lysates were probed with each, otherFeat=[]-->, belongsTo=parr 291 phosphospecific antibody in the absence or presence of 1, otherFeat=[]-->, belongsTo=parr 292 g/ml synthetic phosphopeptide as indicated., otherFeat=[]-->, belongsTo=parr 293 Immunoblotting--Western blotting of whole cell lysates,, otherFeat=[]-->, belongsTo=parr 294 immunoprecipitates, or pulldown samples was performed on, otherFeat=[]-->, belongsTo=parr 295 nitrocellulose membranes as described previously (28). Either, otherFeat=[]-->, belongsTo=parr 296 ECL (Amersham Biosciences) or SuperSignal (Pierce) enhanced, otherFeat=[]-->, belongsTo=parr 297 chemiluminescence reagents were used depending on the, otherFeat=[]-->, belongsTo=parr 298 strength of the antibody., otherFeat=[]-->, belongsTo=parr 299 Cell Cycle Analysis of Topo I Phosphorylation--K562 cells, otherFeat=[]-->, belongsTo=parr 300 were treated for 16 h with 10 M aphidicolin, 2 mM hydroxyu-, otherFeat=[]-->, belongsTo=parrnote 301 rea, 100 nM paclitaxel, 150 nM nocodazole, or diluent (0.1%, otherFeat=[]-->, belongsTo=parr 302 DMSO). At the end of the incubation, cells were harvested,, otherFeat=[]-->, belongsTo=parr 303 subjected to SDS-PAGE, and analyzed by immunoblotting, otherFeat=[]-->, belongsTo=parr 304 using phosphoepitope-specific anti-topo I antibodies. Dupli-, otherFeat=[]-->, belongsTo=parr 305 cate aliquots were fixed in 50% ethanol, treated with RNase A,, otherFeat=[]-->, belongsTo=parr 306 stained with propidium iodide, and subjected to flow cytometry, otherFeat=[]-->, belongsTo=parr 307 as described previously (29)., otherFeat=[]-->, belongsTo=parr 308 To isolate mitotic cells without the use of pharmacological, otherFeat=[]-->, belongsTo=parr 309 agents, mitotic shake-off (30) was performed. T175 flasks of log, otherFeat=[]-->, belongsTo=parr 310 phase A549 cells were vigorously agitated for l min. After the, otherFeat=[]-->, belongsTo=parr 311 loosened cells and medium were removed, fresh medium was, otherFeat=[]-->, belongsTo=parr 312 added to the flasks. The process was repeated every 30 min for, otherFeat=[]-->, belongsTo=parr 313 9 h. The initial aliquot of dislodged cells was discarded. There-, otherFeat=[]-->, belongsTo=parr 314 after cells were sedimented at 100 g, resuspended in ice-cold, otherFeat=[]-->, belongsTo=parr 315 RPMI 1640 medium, 10 mM HEPES (pH 7.4), pooled, and, otherFeat=[]-->, belongsTo=parr 316 stored on ice. The isolated mitotic cells and remaining control, otherFeat=[]-->, belongsTo=parr 317 adherent cells were stained with Hoechst 33258 to determine, otherFeat=[]-->, belongsTo=parr 318 mitotic index and were subjected to immunoblotting., otherFeat=[]-->, belongsTo=parr 319 In Vitro Kinase Assay--Topo I-S pulled down from stably, otherFeat=[]-->, belongsTo=parr 320 transfected K562 cells using S protein beads or commercially, otherFeat=[]-->, belongsTo=parr 321 available purified topo I (Topogen, Columbus, OH) was mixed, otherFeat=[]-->, belongsTo=parr 322 with 200 M ATP and 10 units of purified CKII or Cdk1 (New, otherFeat=[]-->, belongsTo=parr 323 England Biolabs) in the CKII or Cdk1 reaction buffers provided, otherFeat=[]-->, belongsTo=parr 324 by the supplier. Alternatively the beads were incubated with, otherFeat=[]-->, belongsTo=parr 325 PKC reaction buffer (20 mM HEPES-NaOH (pH 7.4), 0.03%, otherFeat=[]-->, belongsTo=parr 326 Triton X-100) containing lipid activator (100 g/ml phosphati-, otherFeat=[]-->, belongsTo=parr 327 dylserine, 200 pM phorbol myristate acetate, 0.03% Triton, otherFeat=[]-->, belongsTo=parr 328 X-100, 100 M dithiothreitol), 100 M CaCl2, 200 M ATP, 1, otherFeat=[]-->, belongsTo=parr 329 mM magnesium acetate, and 10 ng of PKC (Upstate) in a 30 ?C, otherFeat=[]-->, belongsTo=parr 330 water bath for 30 min. After SDS sample buffer was added,, otherFeat=[]-->, belongsTo=parr 331 samples were heated to 65 ?C for 15 min, subjected to SDS-, otherFeat=[]-->, belongsTo=parr 332 PAGE, and analyzed by immunoblotting., otherFeat=[]-->, belongsTo=parr 333 Site-directed Mutagenesis--Mutations were introduced by, otherFeat=[]-->, belongsTo=parr 334 site-directed mutagenesis using a QuikChangeTM mutagenesis, otherFeat=[]-->, belongsTo=parr 335 kit (Stratagene) according to the instructions of the supplier., otherFeat=[]-->, belongsTo=parr 336 The complete sequences of the topo I open reading frames were, otherFeat=[]-->, belongsTo=parr 337 verified by sequencing after every mutagenesis. S-Topo I was, otherFeat=[]-->, belongsTo=parr 338 mutated to restore the enzyme active site (Tyr723) and to create, otherFeat=[]-->, belongsTo=parr 339 phosphorylation site mutants, including the quadruple phos-, otherFeat=[]-->, belongsTo=parr 340 phomutant "4A" (S10A/S21A/S112A/S394A), the triple, otherFeat=[]-->, belongsTo=parr 341 mutant "3A" (S10A/S112A/S394A), and the S21A mutant., otherFeat=[]-->, belongsTo=parr 342 Immunofluorescence--K562 cells were transiently trans-, otherFeat=[]-->, belongsTo=parr 343 fected with plasmids encoding wild type or phosphomutant, otherFeat=[]-->, belongsTo=parr 344 active (Tyr723) S-Topo I. 24 h later, cells were washed in PBS, otherFeat=[]-->, belongsTo=parr 345 and spun onto glass slides at 60 g for 5 min using a Shandon, otherFeat=[]-->, belongsTo=parr 346 cytocentrifuge. Cells were fixed in ice-cold methanol for 10, otherFeat=[]-->, belongsTo=parr 347 min, rehydrated in PBS, and blocked in TSM (150 mM NaCl, 10, otherFeat=[]-->, belongsTo=parr 348 mM Tris-HCl (pH 7.4) containing 10% (w/v) nonfat milk pow-, otherFeat=[]-->, belongsTo=parr 349 der, 100 units/ml penicillin G, 100 g/ml streptomycin, 1 mM, otherFeat=[]-->, belongsTo=parr 350 sodium azide) at 20 ?22 ?C for 1 h. After anti-S peptide antibody, otherFeat=[]-->, belongsTo=parr 351 was added (10 g/ml in TSM), samples were incubated at 4 ?C, otherFeat=[]-->, belongsTo=parr 352 overnight. Slides were washed six times with PBS and treated, otherFeat=[]-->, belongsTo=parr 353 with fluorescein isothiocyanate-conjugated anti-mouse IgG, otherFeat=[]-->, belongsTo=parr 354 secondary antibody (20 g/ml in TSM; Kirkegaard & Perry, otherFeat=[]-->, belongsTo=parr 355 Laboratories, Inc. (KPL), Gaithersburg, MD) for 45 min at, otherFeat=[]-->, belongsTo=parr 356 37 ?C. Slides were then washed six times with PBS and treated, otherFeat=[]-->, belongsTo=parr 357 with 1 g/ml Hoechst 33258 to locate nuclei and chromo-, otherFeat=[]-->, belongsTo=parr 358 somes. Cells were visualized using a Zeiss LSM 510 confocal, otherFeat=[]-->, belongsTo=parr 359 microscope., otherFeat=[]-->, belongsTo=parr 360 Binding Partner Analysis--Plasmids encoding wild type or, otherFeat=[]-->, belongsTo=parr 361 4A S-Topo I were transfected into K562 cells. Beginning 6 h, otherFeat=[]-->, belongsTo=parr 362 after transfection, 100 nM paclitaxel was added to cells for 18 h, otherFeat=[]-->, belongsTo=parr 363 to induce mitotic arrest. After incubation, topo I was recovered, otherFeat=[]-->, belongsTo=parr 364 with S protein beads, subjected to SDS-PAGE, and analyzed by, otherFeat=[]-->, belongsTo=parr 365 Mitotic Phosphorylation of Human Topoisomerase I, otherFeat=[]-->, belongsTo=nota_cab_pie 366 JUNE 13, 2008 ? VOLUME 283 ? NUMBER 24, otherFeat=['U']-->, belongsTo=nota_cab_pie 367 JOURNAL OF BIOLOGICAL CHEMISTRY 16713, otherFeat=[]-->, belongsTo=nota_cab_pie 368 at, otherFeat=[u'a']-->, belongsTo=nota_cab_pie 369 Centro, otherFeat=[]-->, belongsTo=nota_cab_pie 370 Nacional, otherFeat=[]-->, belongsTo=nota_cab_pie 371 de, otherFeat=[]-->, belongsTo=nota_cab_pie 372 Investigaciones, otherFeat=[]-->, belongsTo=nota_cab_pie 373 Oncol?gicas,, otherFeat=[]-->, belongsTo=nota_cab_pie 374 on, otherFeat=[]-->, belongsTo=nota_cab_pie 375 May, otherFeat=[]-->, belongsTo=nota_cab_pie 376 28,, otherFeat=[]-->, belongsTo=nota_cab_pie 377 2010, otherFeat=[]-->, belongsTo=nota_cab_pie 378 www.jbc.org, otherFeat=[]-->, belongsTo=nota_cab_pie 379 Downloaded, otherFeat=[]-->, belongsTo=nota_cab_pie 380 from, otherFeat=[]-->, belongsTo=nota_cab_pie 381 http://www.jbc.org/content/suppl/2008/04/16/M802246200.DC1.html, otherFeat=[]-->, belongsTo=nota_cab_pie 382 Supplemental Material can be found at:, otherFeat=[]-->, belongsTo=nota_cab_pie 383 immunoblotting with antibodies against various topo I binding, otherFeat=[]-->, belongsTo=parr 384 partners., otherFeat=[]-->, belongsTo=parr 385 Topo I Activity Assays --Plasmids encoding wild type or phos-, otherFeat=[]-->, belongsTo=parr 386 phorylation site mutant S-Topo I were transfected into K562, otherFeat=[]-->, belongsTo=parr 387 cells. Beginning 6 h after transfection, 100 nM paclitaxel was, otherFeat=[]-->, belongsTo=parr 388 added to cells to induce a mitotic arrest. After an 18-h incuba-, otherFeat=[]-->, belongsTo=parr 389 tion in paclitaxel, topo I was recovered with S protein-agarose., otherFeat=[]-->, belongsTo=parr 390 Beads were washed four times in 0.1% Nonidet P-40 in PBS, otherFeat=[]-->, belongsTo=parr 391 because RIPA buffer was found to abolish topo I activity. A, otherFeat=[]-->, belongsTo=parr 392 fraction of the beads was removed for immunoblotting to quan-, otherFeat=[]-->, belongsTo=parr 393 tify S-Topo I expression. Where indicated, 10 units of purified, otherFeat=[]-->, belongsTo=parr 394 calf intestine alkaline phosphatase (Invitrogen) was added, and, otherFeat=[]-->, belongsTo=parr 395 beads were incubated at 37 ?C for 30 min. Plasmid relaxation, otherFeat=[]-->, belongsTo=parr 396 activity of the immobilized topo I was then analyzed as, otherFeat=[]-->, belongsTo=parr 397 described by Hann et al. (31). In brief, the beads were diluted in, otherFeat=[]-->, belongsTo=parr 398 2-fold increments and resuspended in topo I activity buffer (50, otherFeat=[]-->, belongsTo=parr 399 mM Tris-HCl (pH 7.5), 2.5 mM MgCl2, 110 mM NaCl, 500 M, otherFeat=[]-->, belongsTo=parrnote 400 EDTA, 6 g/ml bovine serum albumin, 0.01% (w/v), otherFeat=[]-->, belongsTo=parr 401 dithiothreitol)., otherFeat=[]-->, belongsTo=parr 402 Alternatively, after treatment of K562 cells with 0.1% DMSO, otherFeat=[]-->, belongsTo=parr 403 or 100 nM paclitaxel for 16 h, cells were sedimented, washed, otherFeat=[]-->, belongsTo=parr 404 once in PBS, and resuspended in nuclear isolation buffer con-, otherFeat=[]-->, belongsTo=parr 405 sisting of 10 mM NaCl, 10 mM Tris-HCl (pH 7.4), 3 mM MgSO4, otherFeat=[]-->, belongsTo=parrnote 406 containing 1 mM dithiothreitol, 100 units/ml Trasylol, 1 mM, otherFeat=[]-->, belongsTo=parr 407 PMSF, 0.5 mM EDTA, 10 mM NaF, 2 mM Na2P2O7,1mM, otherFeat=[]-->, belongsTo=parrnote 408 Na2VO4,20nM microcystin. After a 20-min incubation on ice,, otherFeat=[]-->, belongsTo=parr 409 samples were subjected to 30 strokes in a tight fitting Dounce, otherFeat=[]-->, belongsTo=parr 410 homogenizer, examined microscopically to confirm cell dis-, otherFeat=[]-->, belongsTo=parr 411 ruption, and sedimented at 16,000 g for 15 min to sediment, otherFeat=[]-->, belongsTo=parr 412 nuclei and mitotic chromosomes, respectively. After a wash, otherFeat=[]-->, belongsTo=parr 413 with nuclear isolation buffer, pellets were resuspended in 75 l, otherFeat=[]-->, belongsTo=parr 414 of topoisomerase extraction buffer consisting of 100 mM, otherFeat=[]-->, belongsTo=parr 415 sodium phosphate (pH 7.4), 1 mM EDTA, 0.1 mM dithiothreitol,, otherFeat=[]-->, belongsTo=parrnote 416 1mM PMSF, 10 mM NaF, 2 mM Na2P2O7,1mM Na2VO4,20nM, otherFeat=[]-->, belongsTo=parrnote 417 microcystin; treated with an equal volume of topoisomerase, otherFeat=[]-->, belongsTo=parr 418 extraction buffer containing 1.2 M KCl; and incubated on ice for, otherFeat=[]-->, belongsTo=parr 419 15 min. After DNA was precipitated from the extracts using, otherFeat=[]-->, belongsTo=parr 420 polyethylene glycol (32), supernatants were adjusted to equal, otherFeat=[]-->, belongsTo=parr 421 protein concentrations. Aliquots recovered by precipitation in, otherFeat=[]-->, belongsTo=parr 422 20% (w/v) ice-cold trichloroacetic acid were subjected to SDS-, otherFeat=[]-->, belongsTo=parr 423 PAGE and immunoblotting for topo I content. For end point, otherFeat=[]-->, belongsTo=parr 424 assays, aliquots containing serial 2-fold dilutions of the extracts, otherFeat=[]-->, belongsTo=parr 425 in topo I activity buffer supplemented with the phosphatase, otherFeat=[]-->, belongsTo=parr 426 inhibitors 10 mM NaF, 1 mM Na2P2O7, and 20 nM microcystin, otherFeat=[]-->, belongsTo=parrnote 427 were assayed for ability to relax supercoiled plasmid. Reactions, otherFeat=[]-->, belongsTo=parr 428 were initiated by adding 500 ng of O6#7 plasmid DNA and incu-, otherFeat=[]-->, belongsTo=parr 429 bated at 37 ?C for 30 min. After dilution with 1/10 volume con-, otherFeat=[]-->, belongsTo=parr 430 taining 1 g of proteinase K in 10% (w/v) SDS, samples were, otherFeat=[]-->, belongsTo=parr 431 incubated at 37 ?C for another 15?30 min. For time course, otherFeat=[]-->, belongsTo=parr 432 experiments, reactions containing equal amounts of topo I as, otherFeat=[]-->, belongsTo=parr 433 assessed by immunoblotting were incubated with 5 g of plas-, otherFeat=[]-->, belongsTo=parr 434 mid O6#7 in 180 l of topo I activity buffer with phosphatase, otherFeat=[]-->, belongsTo=parr 435 inhibitors for 0 ?30 min. At the indicated times, 20- l aliquots, otherFeat=[]-->, belongsTo=parr 436 were treated with 1 g of proteinase K and 1% SDS (final con-, otherFeat=[]-->, belongsTo=parr 437 centration) at 50 ?C to stop the reaction. Plasmids were sepa-, otherFeat=[]-->, belongsTo=parr 438 rated on 1% agarose gels in TPE buffer (36 mM Tris, 30 mM, otherFeat=[]-->, belongsTo=parr 439 NaH2PO4,1mM EDTA, pH 7.8) containing 10 g/ml chloro-, otherFeat=[]-->, belongsTo=parr 440 quine, stained with 500 ng/ml ethidium bromide, and visual-, otherFeat=[]-->, belongsTo=parr 441 ized under 260 nm illumination., otherFeat=[]-->, belongsTo=parr 442 Topo I Cleavage Half-reaction--Based on the results of Pour-, otherFeat=[]-->, belongsTo=parr 443 quier et al. (33), substrate was generated by annealing a 16-mer, otherFeat=[]-->, belongsTo=parr 444 upper strand (5 -GATCTAAAAGACTTGG-3 ) to a 36-mer, otherFeat=[]-->, belongsTo=parr 445 bottom strand (5 -GATCTTTTTTAAAAATTTTTCCAAG-, otherFeat=[]-->, belongsTo=parr 446 TCTTTTACATC-3 ). The annealed substrate was labeled with, otherFeat=[]-->, belongsTo=parr 447 800 Ci/mol [ -32P]dATP (PerkinElmer Life Sciences) and 5, otherFeat=[]-->, belongsTo=parr 448 units of Klenow fragment (New England Biolabs) to add five, otherFeat=[]-->, belongsTo=parr 449 radiolabeled adenosines to the upper strand. The radiolabeled, otherFeat=[]-->, belongsTo=parr 450 duplex was separated from unincorporated nucleotide on a, otherFeat=[]-->, belongsTo=parr 451 Sephadex TE-10 spin column., otherFeat=[]-->, belongsTo=parr 452 After K562 cells transiently transfected with plasmid encod-, otherFeat=[]-->, belongsTo=parr 453 ing wild type or S21A topo I were incubated with 100 nM pacli-, otherFeat=[]-->, belongsTo=parrnote 454 taxel for 16 h, topo I was pulled down using S protein-agarose as, otherFeat=[]-->, belongsTo=parr 455 described above. An aliquot containing 40% of the beads was, otherFeat=[]-->, belongsTo=parr 456 incubated with 300 fmol of radiolabeled substrate in topo I, otherFeat=[]-->, belongsTo=parr 457 activity buffer at 37 ?C. At the indicated times, aliquots contain-, otherFeat=[]-->, belongsTo=parr 458 ing 50 fmol of substrate were removed, diluted with formamide, otherFeat=[]-->, belongsTo=parr 459 to a final concentration of 60%, heated to 100 ?C for 10 min,, otherFeat=[]-->, belongsTo=parr 460 cooled on ice, and separated on a denaturing 10% polyacrylam-, otherFeat=[]-->, belongsTo=parr 461 ide gel (19:1 acrylamide:bisacrylamide, 7.5 M urea). Substrate, otherFeat=[]-->, belongsTo=parr 462 incubated for 45 min at 37 ?C in buffer lacking topo I and bona, otherFeat=[]-->, belongsTo=parr 463 fide 7-mer (6-mer annealed to the bottom strand and extended, otherFeat=[]-->, belongsTo=parr 464 with Klenow as described above) were included on each gel as, otherFeat=[]-->, belongsTo=parr 465 markers. Gels were examined on a PhosphorImager as, otherFeat=[]-->, belongsTo=parr 466 described above. The remainder of each pulldown was utilized, otherFeat=[]-->, belongsTo=parr 467 for immunoblotting., otherFeat=[]-->, belongsTo=parr 468 Band Depletion Assays--K562 cells transiently transfected, otherFeat=[]-->, belongsTo=parr 469 with plasmids encoding wild type or S21A S-Topo I were, otherFeat=[]-->, belongsTo=parr 470 treated with 0.1% DMSO or 100 nM paclitaxel for 16 h as, otherFeat=[]-->, belongsTo=parr 471 described above. Alternatively untransfected K562 cells were, otherFeat=[]-->, belongsTo=parr 472 treated with 0.1% DMSO or 100 nM paclitaxel for 16 h to exam-, otherFeat=[]-->, belongsTo=parr 473 ine endogenous topo I. Cells were sedimented at 100 g for 10, otherFeat=[]-->, belongsTo=parr 474 min, resuspended in serum-free RPMI 1640 medium contain-, otherFeat=[]-->, belongsTo=parr 475 ing 10 mM HEPES (pH 7.4 at 21 ?C), diluent or 100 nM pacli-, otherFeat=[]-->, belongsTo=parrnote 476 taxel, and diluent versus 1.5, 5, 15, or 50 M CPT. After a 45-min, otherFeat=[]-->, belongsTo=parr 477 incubation at 37 ?C, cells were briefly sedimented at 9600 g,, otherFeat=[]-->, belongsTo=parr 478 resuspended in lysis buffer (6 M guanidine hydrochloride, 250, otherFeat=[]-->, belongsTo=parr 479 mM Tris-HCl (pH 8.5 at 21 ?C), 10 mM EDTA, 1% (v/v) freshly, otherFeat=[]-->, belongsTo=parr 480 added -mercaptoethanol, 1 mM PMSF), and sonicated. Sam-, otherFeat=[]-->, belongsTo=parr 481 ples were alkylated with iodoacetamide, dialyzed, and lyophi-, otherFeat=[]-->, belongsTo=parr 482 lized as described previously (34). After SDS-PAGE and immu-, otherFeat=[]-->, belongsTo=parr 483 noblotting, bands were quantified with ImageJ software., otherFeat=[]-->, belongsTo=parr 484 RESULTS, otherFeat=[]-->, belongsTo=title 485 Topo I Is Phosphorylated in Cells--When topo I was immu-, otherFeat=[]-->, belongsTo=parr 486 noprecipitated from [32P]orthophosphate-labeled log phase, otherFeat=[]-->, belongsTo=parr 487 K562 human leukemia cells using a human anti-topo I anti-, otherFeat=[]-->, belongsTo=parr 488 serum (35), subsequent SDS-PAGE and autoradiography indi-, otherFeat=[]-->, belongsTo=parr 489 cated that the topo I polypeptide contained covalently bound, otherFeat=[]-->, belongsTo=parr 490 32P (Fig. 1A) in agreement with previous results suggesting that, otherFeat=[]-->, belongsTo=parr 491 topo I is phosphorylated in intact cells (13, 16, 17, 20 ?22). To, otherFeat=[]-->, belongsTo=parr 492 permit isolation of the large amounts of purified topo I polypep-, otherFeat=[]-->, belongsTo=parr 493 tide required for mapping the phosphorylation sites, the S pep-, otherFeat=[]-->, belongsTo=parr 494 tide epitope tag and a linker were inserted on either the C ter-, otherFeat=[]-->, belongsTo=parr 495 minus (Topo I-S) or N terminus (S-Topo I) of the topo I open, otherFeat=[]-->, belongsTo=parr 496 reading frame (25). Because of the potential toxicity associated, otherFeat=[]-->, belongsTo=parr 497 Mitotic Phosphorylation of Human Topoisomerase I, otherFeat=[]-->, belongsTo=nota_cab_pie 498 16714 JOURNAL OF BIOLOGICAL CHEMISTRY, otherFeat=['U']-->, belongsTo=nota_cab_pie 499 VOLUME 283 ? NUMBER 24 ? JUNE 13, 2008, otherFeat=['U']-->, belongsTo=nota_cab_pie 500 at, otherFeat=[u'a']-->, belongsTo=nota_cab_pie 501 Centro, otherFeat=[]-->, belongsTo=nota_cab_pie 502 Nacional, otherFeat=[]-->, belongsTo=nota_cab_pie 503 de, otherFeat=[]-->, belongsTo=nota_cab_pie 504 Investigaciones, otherFeat=[]-->, belongsTo=nota_cab_pie 505 Oncol?gicas,, otherFeat=[]-->, belongsTo=nota_cab_pie 506 on, otherFeat=[]-->, belongsTo=nota_cab_pie 507 May, otherFeat=[]-->, belongsTo=nota_cab_pie 508 28,, otherFeat=[]-->, belongsTo=nota_cab_pie 509 2010, otherFeat=[]-->, belongsTo=nota_cab_pie 510 www.jbc.org, otherFeat=[]-->, belongsTo=nota_cab_pie 511 Downloaded, otherFeat=[]-->, belongsTo=nota_cab_pie 512 from, otherFeat=[]-->, belongsTo=nota_cab_pie 513 http://www.jbc.org/content/suppl/2008/04/16/M802246200.DC1.html, otherFeat=[]-->, belongsTo=nota_cab_pie 514 Supplemental Material can be found at:, otherFeat=[]-->, belongsTo=nota_cab_pie 515 with overexpressing active topo I in intact cells (4, 31), a plas-, otherFeat=[]-->, belongsTo=parr 516 mid encoding Topo I-S with an inactivating Y723F mutation, otherFeat=[]-->, belongsTo=parr 517 was stably transfected into K562 cells. When the stable line was, otherFeat=[]-->, belongsTo=parr 518 radiolabeled with [32P]orthophosphate and the tagged topo I, otherFeat=[]-->, belongsTo=parr 519 was pulled down using S protein-agarose beads, autoradiogra-, otherFeat=[]-->, belongsTo=parr 520 phy showed that the tagged construct also incorporated 32P, otherFeat=[]-->, belongsTo=parr 521 (Fig. 1B). Consistent with an earlier report that murine topo I is, otherFeat=[]-->, belongsTo=parr 522 differentially phosphorylated during mitosis (22), phosphoryl-, otherFeat=[]-->, belongsTo=parr 523 ation of Topo I-S (Y723F) increased in mitotic cells (Fig. 1C)., otherFeat=[]-->, belongsTo=parr 524 Two-dimensional tryptic mapping suggested the possibility of, otherFeat=[]-->, belongsTo=parr 525 four phosphorylated peptides in these mitotic cells (Fig. 1D)., otherFeat=[]-->, belongsTo=parr 526 Identification of Four Novel Phosphorylation Sites on Human, otherFeat=[]-->, belongsTo=parr 527 Topo I--To identify topo I phosphorylation sites, unlabeled, otherFeat=[]-->, belongsTo=parr 528 Topo I-S (Y723F) was isolated from stably transfected K562, otherFeat=[]-->, belongsTo=parr 529 cells and purified by SDS-PAGE. This procedure yielded a band, otherFeat=[]-->, belongsTo=parr 530 of purified polypeptide that was easily visible after Coomassie, otherFeat=[]-->, belongsTo=parr 531 Blue staining (Fig. 1E). Several samples prepared in this manner,, otherFeat=[]-->, belongsTo=parr 532 including samples from untreated cells, cells arrested in mitosis by, otherFeat=[]-->, belongsTo=parr 533 an 18-h treatment with 100 nM paclitaxel, and cells treated for 15, otherFeat=[]-->, belongsTo=parr 534 min with 50 nM phorbol myristate acetate, were subjected to tryp-, otherFeat=[]-->, belongsTo=parr 535 sin digestion followed by quadrupole time-of-flight tandem mass, otherFeat=[]-->, belongsTo=parr 536 spectrometry analysis. Results obtained with each of the three, otherFeat=[]-->, belongsTo=parr 537 samples suggested that Ser10, Ser112, and Ser394 of topo I are phos-, otherFeat=[]-->, belongsTo=parr 538 phorylated in cells (supplemental Fig. S1, A?C)., otherFeat=[]-->, belongsTo=parr 539 Further analysis of the mass spectrometry data indicated that, otherFeat=[]-->, belongsTo=parr 540 peptides corresponding to 20 ?30% of the polypeptide, espe-, otherFeat=[]-->, belongsTo=parr 541 cially the lysine-rich N-terminal domain, had not been, otherFeat=[]-->, belongsTo=parr 542 detected. To maximize coverage of the polypeptide, additional, otherFeat=[]-->, belongsTo=parr 543 samples were digested with the protease Arg-C instead of tryp-, otherFeat=[]-->, belongsTo=parr 544 sin and subjected to mass spectrometry. Results of this analysis, otherFeat=[]-->, belongsTo=parr 545 suggested that topo I is also phosphorylated at Ser21 (supple-, otherFeat=[]-->, belongsTo=parr 546 mental Fig. S1D)., otherFeat=[]-->, belongsTo=parr 547 The region around the phosphorylation sites was analyzed, otherFeat=[]-->, belongsTo=parr 548 for sequence conservation. All four putative phosphorylation, otherFeat=[]-->, belongsTo=parr 549 sites are conserved in higher eukaryotes, including mouse,, otherFeat=[]-->, belongsTo=parr 550 chicken, and human, but not in yeast (Fig. 2, A?D). These find-, otherFeat=[]-->, belongsTo=parr 551 ings suggest that these phosphorylations may be important for, otherFeat=[]-->, belongsTo=parr 552 some function of the higher eukaryotic forms of topo I., otherFeat=[]-->, belongsTo=parr 553 Ser10, Ser21, Ser112, and Ser394 Are Phosphorylated during, otherFeat=[]-->, belongsTo=parr 554 Mitosis--Phosphoepitope-specific anti-topo I antisera were, otherFeat=[]-->, belongsTo=parr 555 generated to assess whether these four sites are phosphoryl-, otherFeat=[]-->, belongsTo=parr 556 ated on the endogenous topo I polypeptide in cells and to, otherFeat=[]-->, belongsTo=parr 557 enable further analysis of the phosphorylation events. After, otherFeat=[]-->, belongsTo=parr 558 affinity purification, each antiserum selectively detected, otherFeat=[]-->, belongsTo=parr 559 phosphorylated polypeptide (Fig. 3A). Each phospho-, otherFeat=[]-->, belongsTo=parr 560 epitope-specific antiserum detected topo I in K562 lysates by, otherFeat=[]-->, belongsTo=parr 561 immunoblotting (Fig. 3B), indicating the endogenous phos-, otherFeat=[]-->, belongsTo=parr 562 phorylation at these sites in cells. Competition experiments, otherFeat=[]-->, belongsTo=parr 563 (Fig. 3C) demonstrated that each affinity-purified reagent, otherFeat=[]-->, belongsTo=parr 564 was specific for only its phosphoepitope., otherFeat=[]-->, belongsTo=parr 565 To determine the phosphorylation state at these sites during, otherFeat=[]-->, belongsTo=parr 566 various phases of the cell cycle, K562 cells were arrested at, otherFeat=[]-->, belongsTo=parr 567 various stages of the cell cycle as verified by flow cytometry, otherFeat=[]-->, belongsTo=parr 568 (supplemental Fig. S2) using aphidicolin (G1/S), hydroxyurea, otherFeat=[]-->, belongsTo=parr 569 (G1/S), etoposide (G2), and paclitaxel or nocodazole (M)., otherFeat=[]-->, belongsTo=parr 570 Immunoblotting demonstrated that phosphorylation of Ser10,, otherFeat=[]-->, belongsTo=parr 571 FIGURE 1. Endogenous and S peptide-tagged topo I is phosphorylated in, otherFeat=[]-->, belongsTo=fig_caption 572 cells. A, after 2.5 107 K562 human leukemia cells were radiolabeled for 4 h, otherFeat=[]-->, belongsTo=fig_caption 573 with 0.25 mCi/ml [32P]orthophosphate, topo I was isolated by immunopre-, otherFeat=[]-->, belongsTo=fig_caption 574 cipitation using human anti-topo I antiserum, subjected to SDS-PAGE, and, otherFeat=[]-->, belongsTo=fig_caption 575 analyzed by autoradiography. B, after 2.5 10 7 K562 cells stably expressing, otherFeat=[]-->, belongsTo=fig_caption 576 topo I-S (Y723F) were labeled for 4 h with 0.25 mCi/ml [32P]orthophosphate,, otherFeat=[]-->, belongsTo=fig_caption 577 the tagged topo I was isolated on S protein-agarose beads. Samples were, otherFeat=[]-->, belongsTo=fig_caption 578 subjected to SDS-PAGE and analyzed by autoradiography. C, 3.0 10 7 K562, otherFeat=[]-->, belongsTo=fig_caption 579 cells transfected with a plasmid encoding S-topo I (Tyr723) were treated with, otherFeat=[]-->, belongsTo=fig_caption 580 100 nM paclitaxel or diluent for 16 h followed by 0.25 mCi/ml [32P]orthophos-, otherFeat=[]-->, belongsTo=fig_caption 581 phate for 4 h. Topo I was isolated on S protein-agarose beads, subjected to, otherFeat=[]-->, belongsTo=fig_caption 582 SDS-PAGE, and analyzed by autoradiography as well as blotting with anti-S, otherFeat=[]-->, belongsTo=fig_caption 583 peptide antibody (25). The panel contains lanes from a single film exposure., otherFeat=[]-->, belongsTo=fig_caption 584 Dashes indicate removal of unrelated lanes. D, after metabolic labeling with, otherFeat=[]-->, belongsTo=fig_caption 585 [32P]orthophosphate, topo I was isolated by pulldown from 5.0 107 pacli-, otherFeat=[]-->, belongsTo=fig_caption 586 taxel-treated cells stably expressing topo I-S (Y723F) as described in B, sub-, otherFeat=[]-->, belongsTo=fig_caption 587 jected to SDS-PAGE, and stained with Coomassie Brilliant Blue. The excised, otherFeat=[]-->, belongsTo=fig_caption 588 topo I band was subjected to two-dimensional tryptic mapping and Phos-, otherFeat=[]-->, belongsTo=fig_caption 589 phorImager analysis as described under "Experimental Procedures." The sam-, otherFeat=[]-->, belongsTo=fig_caption 590 ple origin is indicated by the circle. E, the stable line described in B was also, otherFeat=[]-->, belongsTo=fig_caption 591 used to obtain unlabeled, purified topo I for mass spectrometry as shown by, otherFeat=[]-->, belongsTo=fig_caption 592 a representative Coomassie Blue-stained gel. Results in B, C, D and E are rep-, otherFeat=[]-->, belongsTo=fig_caption 593 resentative of five, five, nine, and five experiments, respectively., otherFeat=[]-->, belongsTo=fig_caption 594 FIGURE 2. Conservation of the four mapped topo I phosphorylation sites., otherFeat=[]-->, belongsTo=fig_caption 595 A?D, sequence homology of the four phosphorylation sites. SC, Saccharomy-, otherFeat=[]-->, belongsTo=fig_caption 596 ces cerevisiae; SP, Schizosaccharomyces pombe; AT, Arabidopsis thaliana; X,, otherFeat=[]-->, belongsTo=fig_caption 597 Xenopus; M, mouse; CH, chicken; H, human; D, Drosophila melanogaster., otherFeat=[]-->, belongsTo=fig_caption 598 Mitotic Phosphorylation of Human Topoisomerase I, otherFeat=[]-->, belongsTo=nota_cab_pie 599 JUNE 13, 2008 ? VOLUME 283 ? NUMBER 24, otherFeat=['U']-->, belongsTo=nota_cab_pie 600 JOURNAL OF BIOLOGICAL CHEMISTRY 16715, otherFeat=[]-->, belongsTo=nota_cab_pie 601 at, otherFeat=[u'a']-->, belongsTo=nota_cab_pie 602 Centro, otherFeat=[]-->, belongsTo=nota_cab_pie 603 Nacional, otherFeat=[]-->, belongsTo=nota_cab_pie 604 de, otherFeat=[]-->, belongsTo=nota_cab_pie 605 Investigaciones, otherFeat=[]-->, belongsTo=nota_cab_pie 606 Oncol?gicas,, otherFeat=[]-->, belongsTo=nota_cab_pie 607 on, otherFeat=[]-->, belongsTo=nota_cab_pie 608 May, otherFeat=[]-->, belongsTo=nota_cab_pie 609 28,, otherFeat=[]-->, belongsTo=nota_cab_pie 610 2010, otherFeat=[]-->, belongsTo=nota_cab_pie 611 www.jbc.org, otherFeat=[]-->, belongsTo=nota_cab_pie 612 Downloaded, otherFeat=[]-->, belongsTo=nota_cab_pie 613 from, otherFeat=[]-->, belongsTo=nota_cab_pie 614 http://www.jbc.org/content/suppl/2008/04/16/M802246200.DC1.html, otherFeat=[]-->, belongsTo=nota_cab_pie 615 Supplemental Material can be found at:, otherFeat=[]-->, belongsTo=nota_cab_pie 616 Ser21, Ser112, and Ser394 was markedly increased only in cells, otherFeat=[]-->, belongsTo=parr 617 treated with paclitaxel or nocodazole, suggesting that these, otherFeat=[]-->, belongsTo=parr 618 phosphorylations occur predominantly or exclusively during, otherFeat=[]-->, belongsTo=parr 619 mitosis (Fig. 4A and data not shown). To confirm this finding, otherFeat=[]-->, belongsTo=parr 620 and verify that the mitotic phosphorylation of these sites was, otherFeat=[]-->, belongsTo=parr 621 not caused by the drug treatment, mitotic A549 human lung, otherFeat=[]-->, belongsTo=parr 622 cancer cells were separated from nonmitotic cells by mitotic, otherFeat=[]-->, belongsTo=parr 623 shake-off. Analysis of these cells demonstrated phosphoryla-, otherFeat=[]-->, belongsTo=parr 624 tion of topo I at Ser10, Ser21, Ser112, and Ser394 in mitotic cells, otherFeat=[]-->, belongsTo=parr 625 but not interphase cells (Fig. 4B)., otherFeat=[]-->, belongsTo=parr 626 In Vitro Phosphorylation Analysis--Analysis of the sequence, otherFeat=[]-->, belongsTo=parr 627 around the four phosphorylation sites (Fig. 2) indicated that, otherFeat=[]-->, belongsTo=parr 628 Ser10 and Ser21 are part of possible CKII and PKC consensus, otherFeat=[]-->, belongsTo=parr 629 sequences, respectively. In addition, Ser112 and Ser394 are, otherFeat=[]-->, belongsTo=parr 630 serine-proline sites, which are potential Cdk phosphoryla-, otherFeat=[]-->, belongsTo=parr 631 tion sites. Accordingly CKII, PKC , and Cdk1 were tested, otherFeat=[]-->, belongsTo=parr 632 for their ability to phosphorylate the sites in vitro. Topo I-S, otherFeat=[]-->, belongsTo=parr 633 was pulled down from stably transfected K562 cells, washed,, otherFeat=[]-->, belongsTo=parr 634 incubated with purified kinases in vitro, subjected to SDS-, otherFeat=[]-->, belongsTo=parr 635 PAGE, and analyzed by immunoblotting with the phospho-, otherFeat=[]-->, belongsTo=parr 636 epitope-specific antibodies. Alternatively purified topo I was, otherFeat=[]-->, belongsTo=parr 637 incubated with purified kinases in vitro and subjected to the, otherFeat=[]-->, belongsTo=parr 638 same protocol described above. Results demonstrated that, otherFeat=[]-->, belongsTo=parr 639 Ser10 can be phosphorylated by CKII, Ser21 can be phospho-, otherFeat=[]-->, belongsTo=parr 640 rylated by PKC , and Ser112 and Ser394 can be phosphoryla-, otherFeat=[]-->, belongsTo=parr 641 ted by Cdk1 in vitro (Fig. 5). Further analysis verified that, otherFeat=[]-->, belongsTo=parr 642 each of these sites was phosphorylated specifically by its, otherFeat=[]-->, belongsTo=parr 643 FIGURE 3. Development of phosphotopo I antibodies. A and B, polyclonal, otherFeat=[]-->, belongsTo=fig_caption 644 phosphoepitope-specific antibodies for Ser10, Ser21, Ser112, and Ser394 were, otherFeat=[]-->, belongsTo=fig_caption 645 generated in rabbits. Bleeds were affinity-purified as described under "Exper-, otherFeat=[]-->, belongsTo=fig_caption 646 imental Procedures" and screened by dot blots using phosphorylated (P) and, otherFeat=[]-->, belongsTo=fig_caption 647 nonphosphorylated (NP) peptides coupled to bovine serum albumin (A) and, otherFeat=[]-->, belongsTo=fig_caption 648 immunoblotting using K562 lysates (B). C, specificity of the anti-phospho-, otherFeat=[]-->, belongsTo=fig_caption 649 epitope antibodies. Affinity-purified antibodies were incubated with a 1, otherFeat=[]-->, belongsTo=fig_caption 650 g/ml concentration of the designated phosphopeptide during incubation, otherFeat=[]-->, belongsTo=fig_caption 651 with nitrocellulose-immobilized mitotic K562 lysates. Each panel contains, otherFeat=[]-->, belongsTo=fig_caption 652 lanes from a single film exposure. Dashes indicate removal of intervening, otherFeat=[]-->, belongsTo=fig_caption 653 lanes., otherFeat=[]-->, belongsTo=fig_caption 654 FIGURE 4. Ser10, Ser21, Ser112, and Ser394 are phosphorylated during mito-, otherFeat=[]-->, belongsTo=fig_caption 655 sis. A, K562 cells were treated for 16 h with a variety of drugs, including 10 M, otherFeat=[]-->, belongsTo=fig_caption 656 aphidicolin, 2 mM hydroxyurea, 100 nM paclitaxel, or 150 nM nocodazole, to, otherFeat=[]-->, belongsTo=fig_caption 657 cause arrest in various phases of the cell cycle. Changes in cell cycle distribu-, otherFeat=[]-->, belongsTo=fig_caption 658 tion were confirmed by flow cytometry (supplemental Fig. S2). Samples were, otherFeat=[]-->, belongsTo=fig_caption 659 then subjected to SDS-PAGE and immunoblotting using anti-phospho-topo I, otherFeat=[]-->, belongsTo=fig_caption 660 antibodies. Each panel contains lanes from a single film exposure. Dashes, otherFeat=[]-->, belongsTo=fig_caption 661 indicate removal of lanes from cells subjected to additional treatments that, otherFeat=[]-->, belongsTo=fig_caption 662 did not affect Ser10 phosphorylation. B, mitotic A549 cells were isolated by, otherFeat=[]-->, belongsTo=fig_caption 663 shake-off without drug treatment as described under "Experimental Proce-, otherFeat=[]-->, belongsTo=fig_caption 664 dures." Interphase cells were isolated at the completion of the shake-off, and, otherFeat=[]-->, belongsTo=fig_caption 665 the mitotic index of both interphase and mitotic cells was determined by, otherFeat=[]-->, belongsTo=fig_caption 666 microscopic examination after Hoechst 33258 staining. Samples were sub-, otherFeat=[]-->, belongsTo=fig_caption 667 jected to SDS-PAGE and immunoblotting. Representative topo I loading con-, otherFeat=[]-->, belongsTo=fig_caption 668 trol blots are shown for both panels. Results in both panels are representative, otherFeat=[]-->, belongsTo=fig_caption 669 of five separate experiments. P, phosphorylated., otherFeat=[]-->, belongsTo=fig_caption 670 Mitotic Phosphorylation of Human Topoisomerase I, otherFeat=[]-->, belongsTo=nota_cab_pie 671 16716 JOURNAL OF BIOLOGICAL CHEMISTRY, otherFeat=['U']-->, belongsTo=nota_cab_pie 672 VOLUME 283 ? NUMBER 24 ? JUNE 13, 2008, otherFeat=['U']-->, belongsTo=nota_cab_pie 673 at, otherFeat=[u'a']-->, belongsTo=nota_cab_pie 674 Centro, otherFeat=[]-->, belongsTo=nota_cab_pie 675 Nacional, otherFeat=[]-->, belongsTo=nota_cab_pie 676 de, otherFeat=[]-->, belongsTo=nota_cab_pie 677 Investigaciones, otherFeat=[]-->, belongsTo=nota_cab_pie 678 Oncol?gicas,, otherFeat=[]-->, belongsTo=nota_cab_pie 679 on, otherFeat=[]-->, belongsTo=nota_cab_pie 680 May, otherFeat=[]-->, belongsTo=nota_cab_pie 681 28,, otherFeat=[]-->, belongsTo=nota_cab_pie 682 2010, otherFeat=[]-->, belongsTo=nota_cab_pie 683 www.jbc.org, otherFeat=[]-->, belongsTo=nota_cab_pie 684 Downloaded, otherFeat=[]-->, belongsTo=nota_cab_pie 685 from, otherFeat=[]-->, belongsTo=nota_cab_pie 686 http://www.jbc.org/content/suppl/2008/04/16/M802246200.DC1.html, otherFeat=[]-->, belongsTo=nota_cab_pie 687 Supplemental Material can be found at:, otherFeat=[]-->, belongsTo=nota_cab_pie 688 indicated kinase and not by the other two kinases (supple-, otherFeat=[]-->, belongsTo=parr 689 mental Fig. S3)., otherFeat=[]-->, belongsTo=parr 690 Phosphorylation Does Not Affect Topo I Localization or Inter-, otherFeat=[]-->, belongsTo=parr 691 actions with Tested Binding Partners--To determine how, otherFeat=[]-->, belongsTo=parr 692 phosphorylation of these sites affects topo I, site-directed, otherFeat=[]-->, belongsTo=parr 693 mutagenesis was performed to create alanine mutants of, otherFeat=[]-->, belongsTo=parr 694 S-Topo I. Because all four sites are phosphorylated during, otherFeat=[]-->, belongsTo=parr 695 mitosis, a quadruple mutant (S10A/S21A/S112A/S394A, topo I, otherFeat=[]-->, belongsTo=parr 696 4A) construct was generated to remove all of these phosphoryl-, otherFeat=[]-->, belongsTo=parr 697 ation sites. Both catalytically active (Tyr723) and inactive, otherFeat=[]-->, belongsTo=parr 698 (Y723F) constructs were created., otherFeat=[]-->, belongsTo=parr 699 Immunofluorescence was performed to monitor subcellular, otherFeat=[]-->, belongsTo=parr 700 localization. After plasmids encoding wild type or 4A S-Topo I, otherFeat=[]-->, belongsTo=parr 701 (Tyr723) were transfected into K562 cells, localization of the, otherFeat=[]-->, belongsTo=parr 702 polypeptide was analyzed using anti-S peptide antibody (Fig., otherFeat=[]-->, belongsTo=parr 703 6A). 4A S-Topo I localized to the nucleus, including punctate, otherFeat=[]-->, belongsTo=parr 704 regions representing nucleoli in interphase cells (supplemental, otherFeat=[]-->, belongsTo=parr 705 Fig. S4A) and around the condensed chromosomes in mitotic, otherFeat=[]-->, belongsTo=parr 706 cells (Fig. 6A, lower panels). These patterns are identical to the, otherFeat=[]-->, belongsTo=parr 707 previously reported localization of endogenous topo I (36 ?39), otherFeat=[]-->, belongsTo=parr 708 and to the pattern seen with wild type S-Topo I in this study, otherFeat=[]-->, belongsTo=parr 709 (Fig. 6A, upper panels). Similar results were also seen in pacli-, otherFeat=[]-->, belongsTo=parr 710 taxel-treated K562 cells (supplemental Fig. S4B). These results, otherFeat=[]-->, belongsTo=parr 711 indicate that phosphorylation of these sites does not detectably, otherFeat=[]-->, belongsTo=parr 712 affect topo I localization during mitosis., otherFeat=[]-->, belongsTo=parr 713 To assess the effect of phosphorylation on the ability of topo, otherFeat=[]-->, belongsTo=parr 714 I to interact with reported binding partners, a pulldown assay, otherFeat=[]-->, belongsTo=parr 715 was performed. Following transfection with plasmids encoding, otherFeat=[]-->, belongsTo=parr 716 either wild type or the 4A S-Topo I (Tyr723), K562 cells were, otherFeat=[]-->, belongsTo=parr 717 arrested in mitosis with paclitaxel. After isolation of topo I, otherFeat=[]-->, belongsTo=parr 718 using S protein-agarose, samples were subjected to immuno-, otherFeat=[]-->, belongsTo=parr 719 blotting using antibodies against reported topo I binding, otherFeat=[]-->, belongsTo=parr 720 partners, including nucleolin, poly(ADP-ribose) polymerase,, otherFeat=[]-->, belongsTo=parr 721 TATA-binding protein, and topors (Fig. 6B). Wild type and 4A, otherFeat=[]-->, belongsTo=parr 722 S-Topo I bound similar amounts of these polypeptides, indicat-, otherFeat=[]-->, belongsTo=parr 723 ing that phosphorylation of these four sites does not affect these, otherFeat=[]-->, belongsTo=parr 724 protein-protein interactions., otherFeat=[]-->, belongsTo=parr 725 Phosphorylation of Ser21 Stimulates Topo I Activity in Vitro, otherFeat=[]-->, belongsTo=parr 726 and Enhances CPT-induced Cleavage Complex Stabilization in, otherFeat=[]-->, belongsTo=parr 727 Cells--To assess the effect of phosphorylation on topo I activ-, otherFeat=[]-->, belongsTo=parr 728 ity, endogenous topo I extracted from untreated or paclitaxel-, otherFeat=[]-->, belongsTo=parr 729 arrested K562 cells was assayed for enzymatic activity in vitro., otherFeat=[]-->, belongsTo=parr 730 In end point assays, serial 2-fold dilutions of the extracts were, otherFeat=[]-->, belongsTo=parr 731 tested for the ability to relax a supercoiled plasmid during a, otherFeat=[]-->, belongsTo=parr 732 30-min incubation. Topo I isolated from mitotic cells exhibited, otherFeat=[]-->, belongsTo=parr 733 2?3-fold more relaxation activity than topo I isolated from, otherFeat=[]-->, belongsTo=parr 734 untreated, interphase cells (Fig. 7A) as indicated by the dilution, otherFeat=[]-->, belongsTo=parr 735 that resulted in reappearance of substrate (Fig. 7A, graph). Like-, otherFeat=[]-->, belongsTo=parr 736 wise time course experiments demonstrated that nuclear, otherFeat=[]-->, belongsTo=parr 737 extracts from mitotic cells relaxed supercoiled substrate 2? 4-, otherFeat=[]-->, belongsTo=parr 738 fold more rapidly (Fig. 7B)., otherFeat=[]-->, belongsTo=parr 739 To verify that this change in activity is due to phosphoryla-, otherFeat=[]-->, belongsTo=parr 740 tion of topo I during mitosis, S-Topo I (Tyr723) isolated from, otherFeat=[]-->, belongsTo=parr 741 paclitaxel-arrested K562 cells was dephosphorylated with calf, otherFeat=[]-->, belongsTo=parr 742 intestine alkaline phosphatase and assayed for topo I enzymatic, otherFeat=[]-->, belongsTo=parr 743 activity using serial 2-fold dilutions of the pulldowns. Fractions, otherFeat=[]-->, belongsTo=parr 744 of pulldowns treated with buffer or calf intestine alkaline phos-, otherFeat=[]-->, belongsTo=parr 745 phatase were set aside and analyzed by immunoblotting to ver-, otherFeat=[]-->, belongsTo=parr 746 ify equal expression (Fig. 8A, inset). Treatment with calf intes-, otherFeat=[]-->, belongsTo=parr 747 tine alkaline phosphatase caused a 2-fold decrease in activity, otherFeat=[]-->, belongsTo=parr 748 (Fig. 8A)., otherFeat=[]-->, belongsTo=parr 749 To determine whether this 2-fold decrease was related to, otherFeat=[]-->, belongsTo=parr 750 dephosphorylation of one or more of the four phosphorylation, otherFeat=[]-->, belongsTo=parr 751 sites mapped in this study, the activities of wild type and 4A, otherFeat=[]-->, belongsTo=parr 752 S-Topo I (Tyr723) isolated from paclitaxel-treated K562 cells were, otherFeat=[]-->, belongsTo=parr 753 compared. The 4A mutant exhibited 2-fold lower levels of activity, otherFeat=[]-->, belongsTo=parr 754 with equal expression of the two constructs (Fig. 8B). Conversely, otherFeat=[]-->, belongsTo=parr 755 4A and wild type S-Topo I exhibited similar levels of activity when, otherFeat=[]-->, belongsTo=parr 756 4A had higher levels of expression (Fig. 8C). Collectively these, otherFeat=[]-->, belongsTo=parr 757 results indicate that one or more of the sites mapped in this study, otherFeat=[]-->, belongsTo=parr 758 enhances topo I activity when phosphorylated., otherFeat=[]-->, belongsTo=parr 759 To identify the phosphorylation(s) responsible for the altered, otherFeat=[]-->, belongsTo=parr 760 activity, additional topo I mutants were created and analyzed., otherFeat=[]-->, belongsTo=parr 761 When isolated from mitotic cells, the 3A mutant (S10A/S112A/, otherFeat=[]-->, belongsTo=parr 762 S394A) and wild type S-Topo I exhibited similar levels of activ-, otherFeat=[]-->, belongsTo=parr 763 ity (Fig. 8D), whereas the S21A mutant exhibited a 2-fold, otherFeat=[]-->, belongsTo=parr 764 decrease in activity relative to wild type topo I (Fig. 8E). Impor-, otherFeat=[]-->, belongsTo=parr 765 tantly this difference in activity was not observed when wild, otherFeat=[]-->, belongsTo=parr 766 type and S21A were pulled down from interphase cells (Fig. 8F),, otherFeat=[]-->, belongsTo=parr 767 which lack Ser21 phosphorylation (Fig. 4B). Therefore, the, otherFeat=[]-->, belongsTo=parr 768 decrease in relaxation activity seen in the 4A mutant versus wild, otherFeat=[]-->, belongsTo=parr 769 type (Fig. 8B) and the wild type S-Topo I treated with calf intes-, otherFeat=[]-->, belongsTo=parr 770 tine alkaline phosphatase (Fig. 8A) after isolation from mitotic, otherFeat=[]-->, belongsTo=parr 771 cells is likely due to the absence or dephosphorylation, respec-, otherFeat=[]-->, belongsTo=parr 772 tively, of Ser21. These results indicate that phosphorylation of, otherFeat=[]-->, belongsTo=parr 773 Ser21 during mitosis stimulates the DNA relaxation activity of, otherFeat=[]-->, belongsTo=parr 774 topo I 2-fold in vitro., otherFeat=[]-->, belongsTo=parr 775 To assess the possibility that the increased plasmid relax-, otherFeat=[]-->, belongsTo=parr 776 ation observed after Ser21 phosphorylation reflects increased, otherFeat=[]-->, belongsTo=parr 777 DNA cleavage activity of the enzyme, the cleavage half-reaction, otherFeat=[]-->, belongsTo=parr 778 was assayed using the strategy of Svejstrup et al. (40). In brief,, otherFeat=[]-->, belongsTo=parr 779 FIGURE 5. In vitro kinase analysis. Examination of the sequence around, otherFeat=[]-->, belongsTo=fig_caption 780 the phosphorylation sites (Fig. 2, A?D) tentatively identified kinases that, otherFeat=[]-->, belongsTo=fig_caption 781 could phosphorylate these sites. Topo I-S was pulled down from stably trans-, otherFeat=[]-->, belongsTo=fig_caption 782 fected K562 cells (Fig. 1E) and incubated with 200 M ATP and 10 units of, otherFeat=[]-->, belongsTo=fig_caption 783 purified CKII, 10 units of Cdk1, or 10 ng of purified PKC under conditions, otherFeat=[]-->, belongsTo=fig_caption 784 described under "Experimental Procedures." Alternatively purified topo I, otherFeat=[]-->, belongsTo=fig_caption 785 (Y723F) was incubated with 200 M ATP and 10 units of Cdk1. At the comple-, otherFeat=[]-->, belongsTo=fig_caption 786 tion of the incubation, samples were solubilized in SDS sample buffer and, otherFeat=[]-->, belongsTo=fig_caption 787 analyzed by immunoblotting using the anti-phospho-topo I antibodies. CKII, otherFeat=[]-->, belongsTo=fig_caption 788 was found to phosphorylate Ser10 (A), PKC was found to phosphorylate Ser21, otherFeat=[]-->, belongsTo=fig_caption 789 (B), and Cdk1 was found to phosphorylate Ser112 (C) and Ser394 (D) in vitro., otherFeat=[]-->, belongsTo=fig_caption 790 Results are representative of five separate experiments. P, phosphorylated., otherFeat=[]-->, belongsTo=fig_caption 791 Mitotic Phosphorylation of Human Topoisomerase I, otherFeat=[]-->, belongsTo=nota_cab_pie 792 JUNE 13, 2008 ? VOLUME 283 ? NUMBER 24, otherFeat=['U']-->, belongsTo=nota_cab_pie 793 JOURNAL OF BIOLOGICAL CHEMISTRY 16717, otherFeat=[]-->, belongsTo=nota_cab_pie 794 at, otherFeat=[u'a']-->, belongsTo=nota_cab_pie 795 Centro, otherFeat=[]-->, belongsTo=nota_cab_pie 796 Nacional, otherFeat=[]-->, belongsTo=nota_cab_pie 797 de, otherFeat=[]-->, belongsTo=nota_cab_pie 798 Investigaciones, otherFeat=[]-->, belongsTo=nota_cab_pie 799 Oncol?gicas,, otherFeat=[]-->, belongsTo=nota_cab_pie 800 on, otherFeat=[]-->, belongsTo=nota_cab_pie 801 May, otherFeat=[]-->, belongsTo=nota_cab_pie 802 28,, otherFeat=[]-->, belongsTo=nota_cab_pie 803 2010, otherFeat=[]-->, belongsTo=nota_cab_pie 804 www.jbc.org, otherFeat=[]-->, belongsTo=nota_cab_pie 805 Downloaded, otherFeat=[]-->, belongsTo=nota_cab_pie 806 from, otherFeat=[]-->, belongsTo=nota_cab_pie 807 http://www.jbc.org/content/suppl/2008/04/16/M802246200.DC1.html, otherFeat=[]-->, belongsTo=nota_cab_pie 808 Supplemental Material can be found at:, otherFeat=[]-->, belongsTo=nota_cab_pie 809 S-Topo I pulled down from mitotic cells was incubated with, otherFeat=[]-->, belongsTo=parr 810 radiolabeled suicide cleavage substrate as indicated in Fig. 8G., otherFeat=[]-->, belongsTo=parr 811 With the amounts of topo I recovered after transient transfec-, otherFeat=[]-->, belongsTo=parr 812 tion, progressive cleavage of the substrate was observed over, otherFeat=[]-->, belongsTo=parr 813 5? 45 min. Importantly 2-fold larger amounts of S21A topo I, otherFeat=[]-->, belongsTo=parr 814 were required to yield the same cleavage rate as wild type topo, otherFeat=[]-->, belongsTo=parr 815 I, suggesting that Ser21 phosphorylation is enhancing the rate of, otherFeat=[]-->, belongsTo=parr 816 DNA cleavage., otherFeat=[]-->, belongsTo=parr 817 To determine whether the in vitro changes in topo I activity, otherFeat=[]-->, belongsTo=parr 818 correspond to an alteration in the behavior of topo I in intact, otherFeat=[]-->, belongsTo=parr 819 cells, band depletion assays were performed to assess CPT-in-, otherFeat=[]-->, belongsTo=parr 820 duced stabilization of covalent topo I-DNA complexes (34)., otherFeat=[]-->, belongsTo=parr 821 K562 cells transiently transfected with plasmids encoding wild, otherFeat=[]-->, belongsTo=parr 822 type or S21A S-Topo I were treated with paclitaxel for 16 h to, otherFeat=[]-->, belongsTo=parr 823 allow mitotic phosphorylation and, otherFeat=[]-->, belongsTo=parr 824 then exposed briefly to varying CPT, otherFeat=[]-->, belongsTo=parr 825 concentrations in the continued, otherFeat=[]-->, belongsTo=parr 826 presence of paclitaxel. The S21A, otherFeat=[]-->, belongsTo=parr 827 mutant required higher CPT con-, otherFeat=[]-->, belongsTo=parr 828 centrations than wild type S-Topo I, otherFeat=[]-->, belongsTo=parr 829 to trap the same amount of topo I in, otherFeat=[]-->, belongsTo=parr 830 covalent topo I-DNA complexes, otherFeat=[]-->, belongsTo=parr 831 (Fig. 9A). In contrast, CPT stabilized, otherFeat=[]-->, belongsTo=parr 832 similar levels of cleavage complexes, otherFeat=[]-->, belongsTo=parr 833 involving the two constructs when, otherFeat=[]-->, belongsTo=parr 834 K562 cells were not arrested in, otherFeat=[]-->, belongsTo=parr 835 mitosis (Fig. 9B). Consistent with, otherFeat=[]-->, belongsTo=parr 836 these results, comparison of dilu-, otherFeat=[]-->, belongsTo=parr 837 ent- versus paclitaxel-treated cells, otherFeat=[]-->, belongsTo=parr 838 revealed that stabilization of topo, otherFeat=[]-->, belongsTo=parr 839 I-DNA complexes required lower, otherFeat=[]-->, belongsTo=parr 840 CPT concentrations in mitotic than, otherFeat=[]-->, belongsTo=parr 841 in log phase cells (Fig. 9C). These, otherFeat=[]-->, belongsTo=parr 842 results indicate that Ser21 phospho-, otherFeat=[]-->, belongsTo=parr 843 rylation concomitantly enhances, otherFeat=[]-->, belongsTo=parr 844 topo I activity (Fig. 8) and CPT sen-, otherFeat=[]-->, belongsTo=parr 845 sitivity (Fig. 9) specifically during, otherFeat=[]-->, belongsTo=parr 846 mitosis., otherFeat=[]-->, belongsTo=parr 847 DISCUSSION, otherFeat=[]-->, belongsTo=title 848 Results of the present study iden-, otherFeat=[]-->, belongsTo=parr 849 tified four phosphorylation sites on, otherFeat=[]-->, belongsTo=parr 850 human topo I: Ser10, Ser21, Ser112,, otherFeat=[]-->, belongsTo=parr 851 and Ser394. Further experiments, otherFeat=[]-->, belongsTo=parr 852 demonstrated that all four sites are, otherFeat=[]-->, belongsTo=parr 853 phosphorylated exclusively during, otherFeat=[]-->, belongsTo=parr 854 mitosis. CKII and PKC phospho-, otherFeat=[]-->, belongsTo=parr 855 rylated Ser10 and Ser21, respectively,, otherFeat=[]-->, belongsTo=parr 856 and Cdk1 phosphorylated Ser112, otherFeat=[]-->, belongsTo=parr 857 and Ser394 in vitro. Mutation of all, otherFeat=[]-->, belongsTo=parr 858 four sites to alanine did not alter the, otherFeat=[]-->, belongsTo=parr 859 mitotic localization or the assayed, otherFeat=[]-->, belongsTo=parr 860 protein-protein interactions of topo, otherFeat=[]-->, belongsTo=parr 861 I. Comprehensive mass spectromet-, otherFeat=[]-->, belongsTo=parr 862 ric analyses also demonstrated that, otherFeat=[]-->, belongsTo=parr 863 wild type and the 4A S-topo I, otherFeat=[]-->, belongsTo=parr 864 mutant have similar binding part-, otherFeat=[]-->, belongsTo=parr 865 ners.3 However, both wild type topo, otherFeat=[]-->, belongsTo=parr 866 I dephosphorylated by calf intestine alkaline phosphatase and, otherFeat=[]-->, belongsTo=parr 867 the 4A mutant exhibited a 2-fold reduction in DNA relaxation, otherFeat=[]-->, belongsTo=parr 868 activity in vitro relative to wild type topo I isolated from mitotic, otherFeat=[]-->, belongsTo=parr 869 cells. Additional analysis determined that Ser21 phosphoryla-, otherFeat=[]-->, belongsTo=parr 870 tion is responsible for this change in enzymatic activity. Collec-, otherFeat=[]-->, belongsTo=parr 871 tively these results suggest that phosphorylation plays a role in, otherFeat=[]-->, belongsTo=parr 872 regulating topo I enzymatic activity and interaction with DNA, otherFeat=[]-->, belongsTo=parr 873 during mitosis., otherFeat=[]-->, belongsTo=parr 874 Mass spectrometry identified Ser10, Ser21, Ser112, and Ser394, otherFeat=[]-->, belongsTo=parr 875 as four sites of topo I phosphorylation in intact cells (Fig. 2 and, otherFeat=[]-->, belongsTo=parr 876 supplemental Fig. S1). Although phosphorylation of Ser10 had, otherFeat=[]-->, belongsTo=parr 877 been suggested previously based on the ability of CKII to phos-, otherFeat=[]-->, belongsTo=parr 878 3 J. S. Hackbarth and S. H. Kaufmann, unpublished observations., otherFeat=[]-->, belongsTo=parrnote 879 FIGURE 6. Mutation of the four sites does not detectably affect localization or the assayed protein-, otherFeat=[]-->, belongsTo=fig_caption 880 protein interactions of topo I. A, 24 h after transfection with plasmids encoding 4A or wild type (wt) S-topo I,, otherFeat=[]-->, belongsTo=fig_caption 881 log phase K562 cells were fixed with methanol and stained with anti-S peptide antibody followed by fluores-, otherFeat=[]-->, belongsTo=fig_caption 882 cein-conjugated anti-mouse IgG and Hoechst 33258. Representative images of mitotic cells are shown. Note, otherFeat=[]-->, belongsTo=fig_caption 883 that the mitotic index is low because no spindle poisons were added. B, 6 h after transfection with plasmids, otherFeat=[]-->, belongsTo=fig_caption 884 encoding wild type (wt) or 4A S-topo I, K562 cells were treated for 16 h with 100 nM paclitaxel to induce mitotic, otherFeat=[]-->, belongsTo=fig_caption 885 arrest. Tagged S-topo I was isolated using S protein-agarose, subjected to SDS-PAGE, and analyzed by immu-, otherFeat=[]-->, belongsTo=fig_caption 886 noblotting using antibodies that recognize known binding partners of topo I. Untransfected (untransf.) K562, otherFeat=[]-->, belongsTo=fig_caption 887 cells were included as a negative control. Results are representative of three (A) or four (B) separate experi-, otherFeat=[]-->, belongsTo=fig_caption 888 ments. PARP, poly(ADP-ribose) polymerase; TBP, TATA-binding protein., otherFeat=[]-->, belongsTo=fig_caption 889 Mitotic Phosphorylation of Human Topoisomerase I, otherFeat=[]-->, belongsTo=nota_cab_pie 890 16718 JOURNAL OF BIOLOGICAL CHEMISTRY, otherFeat=['U']-->, belongsTo=nota_cab_pie 891 VOLUME 283 ? NUMBER 24 ? JUNE 13, 2008, otherFeat=['U']-->, belongsTo=nota_cab_pie 892 at, otherFeat=[u'a']-->, belongsTo=nota_cab_pie 893 Centro, otherFeat=[]-->, belongsTo=nota_cab_pie 894 Nacional, otherFeat=[]-->, belongsTo=nota_cab_pie 895 de, otherFeat=[]-->, belongsTo=nota_cab_pie 896 Investigaciones, otherFeat=[]-->, belongsTo=nota_cab_pie 897 Oncol?gicas,, otherFeat=[]-->, belongsTo=nota_cab_pie 898 on, otherFeat=[]-->, belongsTo=nota_cab_pie 899 May, otherFeat=[]-->, belongsTo=nota_cab_pie 900 28,, otherFeat=[]-->, belongsTo=nota_cab_pie 901 2010, otherFeat=[]-->, belongsTo=nota_cab_pie 902 www.jbc.org, otherFeat=[]-->, belongsTo=nota_cab_pie 903 Downloaded, otherFeat=[]-->, belongsTo=nota_cab_pie 904 from, otherFeat=[]-->, belongsTo=nota_cab_pie 905 http://www.jbc.org/content/suppl/2008/04/16/M802246200.DC1.html, otherFeat=[]-->, belongsTo=nota_cab_pie 906 Supplemental Material can be found at:, otherFeat=[]-->, belongsTo=nota_cab_pie 907 phorylate a small peptide corresponding to the N terminus of, otherFeat=[]-->, belongsTo=parr 908 topo I (23), the other three sites had not been identified previ-, otherFeat=[]-->, belongsTo=parr 909 ously. All sites identified in the present study were located on, otherFeat=[]-->, belongsTo=parr 910 serines in agreement with previous phosphoamino acid analysis, otherFeat=[]-->, belongsTo=parr 911 (13, 18, 21). At least two previous studies, however, suggested, otherFeat=[]-->, belongsTo=parr 912 that topo I can also be phosphorylated on tyrosine residues., otherFeat=[]-->, belongsTo=parr 913 Tse-Dinh et al. (41) found that tyrosine phosphorylation of, otherFeat=[]-->, belongsTo=parr 914 topo I in vitro can alter its enzymatic activity. More recently, Yu, otherFeat=[]-->, belongsTo=parr 915 et al. (24) reported that c-Abl can phosphorylate topo I on, otherFeat=[]-->, belongsTo=parr 916 Tyr268 in vitro. Our mass spectrometry analysis did not detect, otherFeat=[]-->, belongsTo=parr 917 phosphorylation of Tyr268, although it is conceivable that the, otherFeat=[]-->, belongsTo=parr 918 phosphorylated peptide was present in such low abundance, otherFeat=[]-->, belongsTo=parr 919 that it could not be detected., otherFeat=[]-->, belongsTo=parr 920 In further experiments we examined the kinases that can, otherFeat=[]-->, belongsTo=parr 921 phosphorylate topo I on the sites we identified. Data base, otherFeat=[]-->, belongsTo=parr 922 searches indicated that Ser10 and Ser21 conform to consensus, otherFeat=[]-->, belongsTo=parr 923 phosphorylation sites for CKII and PKC, respectively. In vitro, otherFeat=[]-->, belongsTo=parr 924 kinase assays (Fig. 5) verified this prediction, confirming and, otherFeat=[]-->, belongsTo=parr 925 extending previous reports that CKII and PKC can phospho-, otherFeat=[]-->, belongsTo=parr 926 rylate topo I (16, 17). In addition, sequence analysis indicated, otherFeat=[]-->, belongsTo=parr 927 that Ser112 and Ser394 are part of Ser-Pro sequences, which can, otherFeat=[]-->, belongsTo=parr 928 be phosphorylated by Cdks or the mitogen-activated protein, otherFeat=[]-->, belongsTo=parr 929 kinase family. Both sites were phosphorylated by Cdk1 in vitro,, otherFeat=[]-->, belongsTo=parr 930 providing the first indication of a possible interaction between, otherFeat=[]-->, belongsTo=parr 931 topo I and Cdk1. Cdk1 is active only during mitosis, which, otherFeat=[]-->, belongsTo=parr 932 correlates with the finding that Ser112 and Ser394 are predomi-, otherFeat=[]-->, belongsTo=parr 933 nantly or exclusively phosphorylated during that phase of the, otherFeat=[]-->, belongsTo=parr 934 cell cycle. Confirmation that each of, otherFeat=[]-->, belongsTo=parr 935 these kinases is responsible for, otherFeat=[]-->, belongsTo=parr 936 phosphorylating the respective sites, otherFeat=[]-->, belongsTo=parr 937 in cells is not possible with the pres-, otherFeat=[]-->, belongsTo=parr 938 ent technology. CKII /, otherFeat=[]-->, belongsTo=parr 939 or, otherFeat=[]-->, belongsTo=parr 940 Cdk1 / cell lines do not exist due, otherFeat=[]-->, belongsTo=parr 941 to the importance of these kinases, otherFeat=[]-->, belongsTo=parr 942 for cell cycle progression. Moreover, otherFeat=[]-->, belongsTo=parr 943 cells treated with inhibitors or small, otherFeat=[]-->, belongsTo=parr 944 interfering RNA targeting these, otherFeat=[]-->, belongsTo=parr 945 kinases are unable to enter mitosis., otherFeat=[]-->, belongsTo=parr 946 Thus, the identification of the kinases, otherFeat=[]-->, belongsTo=parr 947 responsible for phosphorylating topo, otherFeat=[]-->, belongsTo=parr 948 I must be considered tentative until, otherFeat=[]-->, belongsTo=parr 949 new tools become available., otherFeat=[]-->, belongsTo=parr 950 Experiments using phosphospe-, otherFeat=[]-->, belongsTo=parr 951 cific topo I antibodies demonstrated, otherFeat=[]-->, belongsTo=parr 952 that phosphorylation of all four sites, otherFeat=[]-->, belongsTo=parr 953 was markedly increased during mito-, otherFeat=[]-->, belongsTo=parr 954 sis. These findings were observed, otherFeat=[]-->, belongsTo=parr 955 both in cells treated with spindle poi-, otherFeat=[]-->, belongsTo=parr 956 sons such as nocodazole and pacli-, otherFeat=[]-->, belongsTo=parr 957 taxel (Fig. 4A) and in untreated, otherFeat=[]-->, belongsTo=parr 958 mitotic cells isolated by shake-off (Fig., otherFeat=[]-->, belongsTo=parr 959 4B). These observations demon-, otherFeat=[]-->, belongsTo=parr 960 strated that human topo I is phospho-, otherFeat=[]-->, belongsTo=parr 961 rylated in a cell-cycle dependent man-, otherFeat=[]-->, belongsTo=parr 962 ner consistent with previous findings, otherFeat=[]-->, belongsTo=parr 963 indicating that the murine topo I has a, otherFeat=[]-->, belongsTo=parr 964 mitosis-specific phosphorylated form, otherFeat=[]-->, belongsTo=parr 965 (22). Additional experiments that, otherFeat=[]-->, belongsTo=parr 966 examined whether any of these sites are phosphorylated after var-, otherFeat=[]-->, belongsTo=parr 967 ious types of DNA damage (not shown) failed to demonstrate, otherFeat=[]-->, belongsTo=parr 968 phosphorylation of these four sites after treatment with CPT or the, otherFeat=[]-->, belongsTo=parr 969 topo II poison etoposide. In contrast, Ser10 was phosphorylated, otherFeat=[]-->, belongsTo=parr 970 after treatment with N-methyl-N -nitro-N-nitrosoguanidine or, otherFeat=[]-->, belongsTo=parr 971 ionizing radiation consistent with the ability of DNA damage to, otherFeat=[]-->, belongsTo=parr 972 activate CKII (42, 43)., otherFeat=[]-->, belongsTo=parr 973 Phosphorylation was reported previously to alter topo I enzy-, otherFeat=[]-->, belongsTo=parr 974 matic activity in vitro. Specifically dephosphorylation reportedly, otherFeat=[]-->, belongsTo=parr 975 decreased or abolished topo I activity (13?15), and subsequent, otherFeat=[]-->, belongsTo=parr 976 treatment with CKII or PKC stimulated activity (14, 15). On the, otherFeat=[]-->, belongsTo=parr 977 other hand, recombinant topo I, which has no detectable phos-, otherFeat=[]-->, belongsTo=parr 978 phorylation, was observed to be enzymatically active (44 ? 46),, otherFeat=[]-->, belongsTo=parr 979 casting doubt on the prior claim that phosphorylation is required, otherFeat=[]-->, belongsTo=parr 980 for activity. Results of the present study were consistent with both, otherFeat=[]-->, belongsTo=parr 981 sets of findings. After isolation from mitotic cells, dephosphoryla-, otherFeat=[]-->, belongsTo=parr 982 tion of topo I by calf intestine alkaline phosphatase caused a 2-fold, otherFeat=[]-->, belongsTo=parr 983 decrease in plasmid relaxation activity (Fig. 8A). Because of the, otherFeat=[]-->, belongsTo=parr 984 nonlinearity of the topo I relaxation assays, this result could poten-, otherFeat=[]-->, belongsTo=parr 985 tially be interpreted as a more dramatic decrease if serial dilutions, otherFeat=[]-->, belongsTo=parr 986 are not examined, possibly explaining earlier claims of more exten-, otherFeat=[]-->, belongsTo=parr 987 sive activity loss upon dephosphorylation., otherFeat=[]-->, belongsTo=parr 988 In further experiments, the 4A mutant exhibited the same, otherFeat=[]-->, belongsTo=parr 989 2-fold decrease relative to wild type enzyme after isolation from, otherFeat=[]-->, belongsTo=parr 990 mitotic cells (Fig. 8B and C), indicating that one or more of the, otherFeat=[]-->, belongsTo=parr 991 four mapped sites contributes to activity when phosphorylated, otherFeat=[]-->, belongsTo=parr 992 during mitosis. Further analysis determined that Ser21 is the, otherFeat=[]-->, belongsTo=parr 993 FIGURE 7. DNA relaxation activity of wild type topo I from interphase and mitotic cells. A, endogenous topo I, otherFeat=[]-->, belongsTo=fig_caption 994 was extracted from K562 cells treated with 0.1% DMSO or 100 nM paclitaxel to induce mitotic arrest as described, otherFeat=[]-->, belongsTo=fig_caption 995 under "Experimental Procedures." Serial 2-fold dilutions of the extracts were subjected to immunoblotting for topo, otherFeat=[]-->, belongsTo=fig_caption 996 I or used in a DNA relaxation assay to examine conversion from supercoiled plasmid (SC) to relaxed forms (R)., otherFeat=[]-->, belongsTo=fig_caption 997 Aliquots of the most concentrated extracts (first lanes after dashed lines) contained equal amounts of topo I (inset)., otherFeat=[]-->, belongsTo=fig_caption 998 The "0" lane contains plasmid that was incubated without extract. Dashed lines indicate removal of intervening, otherFeat=[]-->, belongsTo=fig_caption 999 lanes that contained unequal amounts of topo I. The graph shows substrate remaining at the end of the 30-min, otherFeat=[]-->, belongsTo=fig_caption 1000 incubation. Error bars, mean S.E. of three independent experiments. B, extracts adjusted to contain equal amounts, otherFeat=[]-->, belongsTo=fig_caption 1001 of topo I polypeptide (inset) from interphase or paclitaxel-arrested K562 cells were assayed for plasmid relaxation, otherFeat=[]-->, belongsTo=fig_caption 1002 activity over time (lanes 2?10). Lane 1, untreated substrate. The dashed line indicates juxtaposition of two separate, otherFeat=[]-->, belongsTo=fig_caption 1003 agarose gels from the same assay. The graph shows substrate remaining on the gel at each time point and the first, otherFeat=[]-->, belongsTo=fig_caption 1004 order regression line. Results are representative of assays using three independently derived extracts. N, location of, otherFeat=[]-->, belongsTo=fig_caption 1005 nicked and, in some assays, relaxed plasmid., otherFeat=[]-->, belongsTo=fig_caption 1006 Mitotic Phosphorylation of Human Topoisomerase I, otherFeat=[]-->, belongsTo=nota_cab_pie 1007 JUNE 13, 2008 ? VOLUME 283 ? NUMBER 24, otherFeat=['U']-->, belongsTo=nota_cab_pie 1008 JOURNAL OF BIOLOGICAL CHEMISTRY 16719, otherFeat=[]-->, belongsTo=nota_cab_pie 1009 at, otherFeat=[u'a']-->, belongsTo=nota_cab_pie 1010 Centro, otherFeat=[]-->, belongsTo=nota_cab_pie 1011 Nacional, otherFeat=[]-->, belongsTo=nota_cab_pie 1012 de, otherFeat=[]-->, belongsTo=nota_cab_pie 1013 Investigaciones, otherFeat=[]-->, belongsTo=nota_cab_pie 1014 Oncol?gicas,, otherFeat=[]-->, belongsTo=nota_cab_pie 1015 on, otherFeat=[]-->, belongsTo=nota_cab_pie 1016 May, otherFeat=[]-->, belongsTo=nota_cab_pie 1017 28,, otherFeat=[]-->, belongsTo=nota_cab_pie 1018 2010, otherFeat=[]-->, belongsTo=nota_cab_pie 1019 www.jbc.org, otherFeat=[]-->, belongsTo=nota_cab_pie 1020 Downloaded, otherFeat=[]-->, belongsTo=nota_cab_pie 1021 from, otherFeat=[]-->, belongsTo=nota_cab_pie 1022 http://www.jbc.org/content/suppl/2008/04/16/M802246200.DC1.html, otherFeat=[]-->, belongsTo=nota_cab_pie 1023 Supplemental Material can be found at:, otherFeat=[]-->, belongsTo=nota_cab_pie 1024 FIGURE 8. Effect of Ser21 phosphorylation on topo I activity. A, K562 cells were transiently transfected with wild type (wt) S-topo I (Tyr723) and arrested, otherFeat=[]-->, belongsTo=fig_caption 1025 in mitosis with 100 nM paclitaxel. S protein-agarose precipitates were treated with buffer or 10 units of calf intestine alkaline phosphatase (CIAP)as, otherFeat=[]-->, belongsTo=fig_caption 1026 described under "Experimental Procedures." The beads were subjected to 2-fold dilutions in topo I assay buffer and used in a DNA relaxation assay., otherFeat=[]-->, belongsTo=fig_caption 1027 Aliquots of the immobilized topo I were also subjected to immunoblotting with anti-S peptide antibody to verify equal loading (see insets). The inset, otherFeat=[]-->, belongsTo=fig_caption 1028 contains lanes from a single film exposure. B?E, wild type and 4A S-Topo I (B and C), wild type and 3A S-Topo I (D), or wild type and S21A S-Topo I (E) were, otherFeat=[]-->, belongsTo=fig_caption 1029 isolated from paclitaxel-treated K562 cells and assayed as described in A. F, wild type and S21A S-Topo I were isolated from interphase K562 cells and, otherFeat=[]-->, belongsTo=fig_caption 1030 assayed as described in A. Because of variability in transfection efficiency, experiments shown in A?F were conducted separately and cannot be directly, otherFeat=[]-->, belongsTo=fig_caption 1031 compared. G, wild type and S21A S-Topo I isolated from paclitaxel-treated K562 cells were incubated with radiolabeled suicide substrate (inset) for the, otherFeat=[]-->, belongsTo=fig_caption 1032 indicated length of time. *, radiolabeled nucleotide in substrate. At the completion of the reaction, the 21-mer substrate and 7-mer product were, otherFeat=[]-->, belongsTo=fig_caption 1033 separated and visualized by phosphorimaging. Two separate gels from a single assay were imaged simultaneously. Inset, S peptide blot showing, otherFeat=[]-->, belongsTo=fig_caption 1034 corresponding topo I contents of the pulldowns. Dashes indicate removal of extraneous lanes. Results are representative of three (A), 14 (B and C), four, otherFeat=[]-->, belongsTo=fig_caption 1035 (D), three (E), five (F), and three (G) assays. SC, supercoiled; R, relaxed; N, location of nicked and, in some assays, relaxed plasmid., otherFeat=[]-->, belongsTo=fig_caption 1036 Mitotic Phosphorylation of Human Topoisomerase I, otherFeat=[]-->, belongsTo=nota_cab_pie 1037 16720 JOURNAL OF BIOLOGICAL CHEMISTRY, otherFeat=['U']-->, belongsTo=nota_cab_pie 1038 VOLUME 283 ? NUMBER 24 ? JUNE 13, 2008, otherFeat=['U']-->, belongsTo=nota_cab_pie 1039 at, otherFeat=[u'a']-->, belongsTo=nota_cab_pie 1040 Centro, otherFeat=[]-->, belongsTo=nota_cab_pie 1041 Nacional, otherFeat=[]-->, belongsTo=nota_cab_pie 1042 de, otherFeat=[]-->, belongsTo=nota_cab_pie 1043 Investigaciones, otherFeat=[]-->, belongsTo=nota_cab_pie 1044 Oncol?gicas,, otherFeat=[]-->, belongsTo=nota_cab_pie 1045 on, otherFeat=[]-->, belongsTo=nota_cab_pie 1046 May, otherFeat=[]-->, belongsTo=nota_cab_pie 1047 28,, otherFeat=[]-->, belongsTo=nota_cab_pie 1048 2010, otherFeat=[]-->, belongsTo=nota_cab_pie 1049 www.jbc.org, otherFeat=[]-->, belongsTo=nota_cab_pie 1050 Downloaded, otherFeat=[]-->, belongsTo=nota_cab_pie 1051 from, otherFeat=[]-->, belongsTo=nota_cab_pie 1052 http://www.jbc.org/content/suppl/2008/04/16/M802246200.DC1.html, otherFeat=[]-->, belongsTo=nota_cab_pie 1053 Supplemental Material can be found at:, otherFeat=[]-->, belongsTo=nota_cab_pie 1054 site that enhances activity when phosphorylated. A Ser21Ala, otherFeat=[]-->, belongsTo=parr 1055 mutant exhibited decreased ability to relax supercoiled plasmid, otherFeat=[]-->, belongsTo=parr 1056 (Fig. 8E) or cleave suicide substrate (Fig. 8G) in vitro as well as, otherFeat=[]-->, belongsTo=parr 1057 decreased CPT-induced cleavage complexes in mitotic cells, otherFeat=[]-->, belongsTo=parr 1058 (Fig. 9A) compared with wild type topo I. Importantly, these, otherFeat=[]-->, belongsTo=parr 1059 differences were not observed in interphase cells (Figs. 8F and, otherFeat=[]-->, belongsTo=parr 1060 9B), where Ser21 is not phosphorylated (Fig. 4B)., otherFeat=[]-->, belongsTo=parr 1061 Additional observations suggest that the effect of Ser21 phos-, otherFeat=[]-->, belongsTo=parr 1062 phorylation is not limited to cells transfected with epitope-, otherFeat=[]-->, belongsTo=parr 1063 tagged topo I. Extracts from untransfected cells exhibited an, otherFeat=[]-->, belongsTo=parr 1064 increase in topo I activity during mitosis (Fig. 7, A and B) and, otherFeat=[]-->, belongsTo=parr 1065 enhanced sensitivity to CPT-induced stabilization of cleavage, otherFeat=[]-->, belongsTo=parr 1066 complexes (Fig. 9C) compared with interphase cells. Although, otherFeat=[]-->, belongsTo=parr 1067 the ability of phosphorylation in the N-terminal domain to affect, otherFeat=[]-->, belongsTo=parr 1068 events at the topo I active site might seem counterintuitive, previ-, otherFeat=[]-->, belongsTo=parr 1069 ous studies have demonstrated that removal of the N-terminal, otherFeat=[]-->, belongsTo=parr 1070 domain modestly decreases topo I enzymatic activity and CPT, otherFeat=[]-->, belongsTo=parr 1071 sensitivity (47, 48). Crystallization of topo I with an intact N termi-, otherFeat=[]-->, belongsTo=parr 1072 nus, a feat that has not been reported to date, appears to be, otherFeat=[]-->, belongsTo=parr 1073 required to determine whether phos-, otherFeat=[]-->, belongsTo=parr 1074 phorylation of Ser21 enhances activity, otherFeat=[]-->, belongsTo=parr 1075 and CPT sensitivity through an inter-, otherFeat=[]-->, belongsTo=parr 1076 action with portions of the N-termi-, otherFeat=[]-->, belongsTo=parr 1077 nal domain previously implicated in, otherFeat=[]-->, belongsTo=parr 1078 topo I activation or through interac-, otherFeat=[]-->, belongsTo=parr 1079 tion with different regions of the, otherFeat=[]-->, belongsTo=parr 1080 polypeptide., otherFeat=[]-->, belongsTo=parr 1081 The results presented in this study, otherFeat=[]-->, belongsTo=parr 1082 reveal interesting similarities between, otherFeat=[]-->, belongsTo=parr 1083 the phosphorylation of topo I and, otherFeat=[]-->, belongsTo=parr 1084 topo II (49, 50), a structurally and, otherFeat=[]-->, belongsTo=parr 1085 mechanistically unrelated member of, otherFeat=[]-->, belongsTo=parr 1086 the topoisomerase family. Topo II is, otherFeat=[]-->, belongsTo=parr 1087 phosphorylated at numerous serine, otherFeat=[]-->, belongsTo=parr 1088 and threonine sites in cells and is, otherFeat=[]-->, belongsTo=parr 1089 phosphorylated by CKII, PKC, and, otherFeat=[]-->, belongsTo=parr 1090 Cdk1 in vitro. In addition, topo II has, otherFeat=[]-->, belongsTo=parr 1091 sites that are phosphorylated specifi-, otherFeat=[]-->, belongsTo=parr 1092 cally during mitosis (51), and topo II, otherFeat=[]-->, belongsTo=parr 1093 activity peaks during mitosis (52, 53),, otherFeat=[]-->, belongsTo=parr 1094 presumably to facilitate chromosome, otherFeat=[]-->, belongsTo=parr 1095 separation. Although numerous stud-, otherFeat=[]-->, belongsTo=parr 1096 ies have examined whether there is a, otherFeat=[]-->, belongsTo=parr 1097 link between topo II phosphorylation, otherFeat=[]-->, belongsTo=parr 1098 and its enzymatic activity, the results, otherFeat=[]-->, belongsTo=parr 1099 are conflicting especially when com-, otherFeat=[]-->, belongsTo=parr 1100 pared between species (54 ?58). The, otherFeat=[]-->, belongsTo=parr 1101 present study demonstrates that topo, otherFeat=[]-->, belongsTo=parr 1102 I activity likewise increases during, otherFeat=[]-->, belongsTo=parr 1103 mitosis, indicating that topo I might, otherFeat=[]-->, belongsTo=parr 1104 also participate in some way during, otherFeat=[]-->, belongsTo=parr 1105 that phase of the cell cycle., otherFeat=[]-->, belongsTo=parr 1106 In summary, the present study, otherFeat=[]-->, belongsTo=parr 1107 located and examined the four sites of, otherFeat=[]-->, belongsTo=parr 1108 endogenous phosphorylation on, otherFeat=[]-->, belongsTo=parr 1109 human topo I. All of these sites were, otherFeat=[]-->, belongsTo=parr 1110 phosphorylated predominantly dur-, otherFeat=[]-->, belongsTo=parr 1111 ing mitosis, demonstrating that topo I, otherFeat=[]-->, belongsTo=parr 1112 is phosphorylated in a cell cycle-dependent manner. Two of these, otherFeat=[]-->, belongsTo=parr 1113 sites, Ser112 and Ser394, were phosphorylated in vitro by Cdk1, pro-, otherFeat=[]-->, belongsTo=parr 1114 viding the first indication that a Cdk can modify topo I. Although, otherFeat=[]-->, belongsTo=parr 1115 the phosphorylation of these four sites did not detectably alter, otherFeat=[]-->, belongsTo=parr 1116 topo I localization or protein-protein interactions during mito-, otherFeat=[]-->, belongsTo=parr 1117 sis, Ser21 phosphorylation enhanced topo I relaxation activity in, otherFeat=[]-->, belongsTo=parr 1118 vitro and CPT-induced stabilization of cleavage complexes in, otherFeat=[]-->, belongsTo=parr 1119 cells. These observations provide new understanding of the cir-, otherFeat=[]-->, belongsTo=parr 1120 cumstances and effect of topo I phosphorylation in cells., otherFeat=[]-->, belongsTo=parr 1121 Acknowledgments--We gratefully acknowledge gifts of the topo I plas-, otherFeat=[]-->, belongsTo=parrnote 1122 mid from M.-A. Bjornsti; antibodies from Guy Poirier, Naomi Roth-, otherFeat=[]-->, belongsTo=parrnote 1123 field, and Yung-Chi Cheng; assistance and analysis from Ross, otherFeat=[]-->, belongsTo=parrnote 1124 Tomaino and the Taplin Biological Mass Spectrometry Facility as, otherFeat=[]-->, belongsTo=parrnote 1125 well as the Flow Cytometry and Optical Morphology Shared Resource, otherFeat=[]-->, belongsTo=parrnote 1126 and the Peptide Synthesis Core at the Mayo Clinic; technical advice, otherFeat=[]-->, belongsTo=parrnote 1127 from L. James Maher and Robert McDonald; and editorial assistance, otherFeat=[]-->, belongsTo=parrnote 1128 from Deb Strauss., otherFeat=[]-->, belongsTo=parrnote 1129 FIGURE 9. The S21A topo I mutant is less sensitive to CPT-induced trapping on DNA in intact mitotic cells., otherFeat=[]-->, belongsTo=fig_caption 1130 K562 cells transfected with plasmids encoding wild type (wt) or S21A S-topo I (Tyr723) were treated with 100 nM, otherFeat=[]-->, belongsTo=fig_caption 1131 paclitaxel for 16 h (A) or left untreated (B) before subjecting cells to a band depletion assay to assess CPT-, otherFeat=[]-->, belongsTo=fig_caption 1132 induced stabilization of topo I-DNA complexes as described under "Experimental Procedures." The blots were, otherFeat=[]-->, belongsTo=fig_caption 1133 probed with anti-S peptide antibody to specifically detect transfected constructs. C, K562 cells were treated, otherFeat=[]-->, belongsTo=fig_caption 1134 with 100 nM paclitaxel or diluent for 16 h and then subjected to band depletion assay. Samples were subjected, otherFeat=[]-->, belongsTo=fig_caption 1135 to SDS-PAGE, immunoblotting with anti-topo I to detect endogenous polypeptide, and densitometry using, otherFeat=[]-->, belongsTo=fig_caption 1136 ImageJ. Dashes indicate removal of extraneous lanes. Error bars, mean S.E. of three (A), three (B), and five (C), otherFeat=[]-->, belongsTo=fig_caption 1137 experiments, respectively. PARP, poly(ADP-ribose) polymerase., otherFeat=[]-->, belongsTo=fig_caption 1138 Mitotic Phosphorylation of Human Topoisomerase I, otherFeat=[]-->, belongsTo=nota_cab_pie 1139 JUNE 13, 2008 ? VOLUME 283 ? NUMBER 24, otherFeat=['U']-->, belongsTo=nota_cab_pie 1140 JOURNAL OF BIOLOGICAL CHEMISTRY 16721, otherFeat=[]-->, belongsTo=nota_cab_pie 1141 at, otherFeat=[u'a']-->, belongsTo=nota_cab_pie 1142 Centro, otherFeat=[]-->, belongsTo=nota_cab_pie 1143 Nacional, otherFeat=[]-->, belongsTo=nota_cab_pie 1144 de, otherFeat=[]-->, belongsTo=nota_cab_pie 1145 Investigaciones, otherFeat=[]-->, belongsTo=nota_cab_pie 1146 Oncol?gicas,, otherFeat=[]-->, belongsTo=nota_cab_pie 1147 on, otherFeat=[]-->, belongsTo=nota_cab_pie 1148 May, otherFeat=[]-->, belongsTo=nota_cab_pie 1149 28,, otherFeat=[]-->, belongsTo=nota_cab_pie 1150 2010, otherFeat=[]-->, belongsTo=nota_cab_pie 1151 www.jbc.org, otherFeat=[]-->, belongsTo=nota_cab_pie 1152 Downloaded, otherFeat=[]-->, belongsTo=nota_cab_pie 1153 from, otherFeat=[]-->, belongsTo=nota_cab_pie 1154 http://www.jbc.org/content/suppl/2008/04/16/M802246200.DC1.html, otherFeat=[]-->, belongsTo=nota_cab_pie 1155 Supplemental Material can be found at:, otherFeat=[]-->, belongsTo=nota_cab_pie 1156 REFERENCES, otherFeat=[]-->, belongsTo=parrnote 1157 1. 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JUNE 13, 2008, otherFeat=['U']-->, belongsTo=nota_cab_pie 1264 at, otherFeat=[u'a']-->, belongsTo=nota_cab_pie 1265 Centro, otherFeat=[]-->, belongsTo=nota_cab_pie 1266 Nacional, otherFeat=[]-->, belongsTo=nota_cab_pie 1267 de, otherFeat=[]-->, belongsTo=nota_cab_pie 1268 Investigaciones, otherFeat=[]-->, belongsTo=nota_cab_pie 1269 Oncol?gicas,, otherFeat=[]-->, belongsTo=nota_cab_pie 1270 on, otherFeat=[]-->, belongsTo=nota_cab_pie 1271 May, otherFeat=[]-->, belongsTo=nota_cab_pie 1272 28,, otherFeat=[]-->, belongsTo=nota_cab_pie 1273 2010, otherFeat=[]-->, belongsTo=nota_cab_pie 1274 www.jbc.org, otherFeat=[]-->, belongsTo=nota_cab_pie 1275 Downloaded, otherFeat=[]-->, belongsTo=nota_cab_pie 1276 from, otherFeat=[]-->, belongsTo=nota_cab_pie 1277 http://www.jbc.org/content/suppl/2008/04/16/M802246200.DC1.html, otherFeat=[]-->, belongsTo=nota_cab_pie 1278 Supplemental Material can be found at:, otherFeat=[]-->, belongsTo=nota_cab_pie ============================== 0 Mitotic Phosphorylation Stimulates DNA Relaxation Activity-->id=0, page=0, size=27, fam=Times, col=#231f20, type=title, textLines=1--->[]--->title Mitotic Phosphorylat>>> Relaxation Activity 1 of Human Topoisomerase I*S-->id=2, page=0, size=11, fam=Times, col=#231f20, type=parrnote, textLines=67--->[]--->note of Human Topoisomera>>>an Topoisomerase I*S 2 Received for publication, September 14, 2007, and in revised form, March 20, 2008 Published, JBC Papers in Press, April 11, 2008, DOI 10.1074/jbc.M802246200-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[]--->note Received for publica>>>.1074/jbc.M802246200 3 Jennifer S. Hackbarth, Marina Galvez-Peralta§, Nga T. Dai§, David A. Loegering§, Kevin L. Peterson§,-->id=6, page=0, size=7, fam=Times, col=#231f20, type=parrnote, textLines=45--->[]--->note Jennifer S. Hackbart>>> Kevin L. Peterson§, 4 Xue W. Meng§, Larry M. Karnitz§, and Scott H. Kaufmann§1 From the Department of Biochemistry and Molecular Biology and §Division of Oncology Research, Mayo Clinic, Mayo Graduate School, Rochester, Minnesota 55905-->id=5, page=0, size=13, fam=Times, col=#231f20, type=title, textLines=9--->[]--->note Xue W. Meng§, Larry >>>ter, Minnesota 55905 5 Human DNA topoisomerase I (topo I) is an essential mammalian enzyme that regulates DNA supercoiling during transcription and replication. In addition, topo I is specifically targeted by the anticancer compound camptothecin and its derivatives. Previous studies have indicated that topo I is a phosphoprotein and that phosphorylation stimulates its DNA relaxation activity. The locations of most topo I phosphorylation sites have not been identified, preventing a more detailed examination of this modification. To address this issue, mass spectrometry was used to identify four topo I residues that are phosphorylated in intact cells: Ser10, Ser21, Ser112, and Ser394. Immunoblotting using anti-phosphoepitope antibodies demonstrated that these sites are phosphorylated during mitosis. In vitro kinase assays demonstrated that Ser10 can be phosphorylated by casein kinase II, Ser21 can be phosphorylated by protein kinase C , and Ser112 and Ser394 can be phosphorylated by Cdk1. When wild type topo I was pulled down from mitotic cells and dephosphorylated with alkaline phosphatase, topo I activity decreased 2-fold. Likewise, topo I polypeptide with all four phosphorylation sites mutated to alanine exhibited 2-fold lower DNA relaxation activity than wild type topo I after isolation from mitotic cells. Further mutational analysis demonstrated that Ser21 phosphorylation was responsible for this change. Consistent with these results, wild type topo I (but not S21A topo I) exhibited increased sensitivity to camptothecin-induced trapping on DNA during mitosis. Collectively these results indicate that topo I is phosphorylated during mitosis at multiple sites, one of which enhances DNA relaxation activity in vitro and interaction with DNA in cells.-->id=2, page=0, size=11, fam=Times, col=#231f20, type=parrnote, textLines=67--->[]--->parr Human DNA topoisomer>>>n with DNA in cells. 6 Human topo I2 is a type IB topoisomerase that relieves positive and negative DNA supercoiling caused by transcription, replication, and chromosome condensation (1). The 91-kDa, 765-amino acid polypeptide contains four domains: a poorly conserved lysine-rich N-terminal domain that contains nuclear and nucleolar localization signals, a linker region, and the core and C-terminal domains that contain the residues important for DNA interaction and relaxation of supercoils (2). A transesterification reaction at the active site of topo I ligates Tyr723 of the enzyme to the 3 phosphate of the DNA, thereby creating a nick in the DNA backbone (3). This nick allows controlled rotation of the DNA to relieve supercoils. Mutation of Tyr723 prevents the transesterification reaction and abolishes all relaxation activity (4). The anticancer drug CPT and its derivatives slow topo I-mediated DNA relaxation (5, 6) and inhibit the religation reaction step of the enzyme (7, 8), trapping topo I on DNA (9, 10) and causing cell death (11, 12).-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=904--->[]--->parr Human topo I2 is a t>>>cell death (11, 12). 7 A number of observations have raised the possibility that phosphorylation can modulate the activity and CPT sensitivity of topo I. Treatment with calf intestine alkaline phosphatase decreases topo I enzymatic activity in vitro (13­15). Conversely subsequent treatment with PKC or CKII, two kinases that copurify with topo I and phosphorylate it in vitro (16 ­18), stimulates topo I activity 2­3-fold (14, 15, 19) and enhances the ability of CPT to trap covalent topo I-DNA cleavage complexes (20), suggesting that phosphorylation by these kinases might make topo I more sensitive to CPT.-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=904--->[]--->parr A number of observat>>>re sensitive to CPT. 8 Despite its potential importance, many aspects of topo I phosphorylation remain poorly understood. The number of phosphorylation sites, for example, remains unclear because the number of phosphopeptides detected after metabolic labeling and immunoprecipitation has varied from one (16) to as many as five (21) or six (17). The effect of cell cycle progression on phosphorylation has likewise been unclear. Initial studies suggested that topo I is phosphorylated in interphase cells as evidenced by its labeling in unsynchronized cell populations and a rapid increase in phosphorylation after certain treatments (13, 16, 17, 20, 21). In contrast, a more recent study found that topo I in rodent cells quantitatively shifts to a slower migrating, phosphorylated state exclusively during mitosis (22). Finally the location of topo I phosphorylation sites has not been resolved. Cardellini et al. (23) reported that a 17-amino acid peptide from the N terminus of topo I could be phosphorylated at Ser10 by CKII in vitro, but phosphorylation of this site was not verified in the full-length polypeptide in vitro or in vivo. More recently, Yu et al. (24) reported that topo I is phosphorylated on Tyr268 by c-Abl in vitro, but again the phosphorylation of this site in intact cells was not confirmed. The location of other possible phosphorylation sites on topo I, the cellular conditions that cause phosphorylation, and the effects of individual phosphorylation events on topo I remain largely unknown.-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=904--->[]--->parr Despite its potentia>>>ain largely unknown. 9 * This work was supported, in whole or in part, by National Institutes of Health Grant R01 CA73709. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[]--->parr * This work was supp>>> indicate this fact. 10 S The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1­S4.-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[]--->parr S The on-line versio>>>mental Figs. S1­S4. 11 1 To whom correspondence should be addressed: Division of Oncology Research, Guggenheim 1342C, Mayo Clinic, 200 First St. S.W., Rochester, MN 55905. Tel.: 507-284-8950; Fax: 507-284-3906; E-mail: Kaufmann. scott@mayo.edu.-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[]--->parr 1 To whom correspond>>>ann. scott@mayo.edu. 12 2 The abbreviations used are: topo, topoisomerase; Cdk, cyclin-dependent kinase; CKII, casein kinase II; CPT, camptothecin; PBS, calcium- and magnesium-free Dulbecco's phosphate-buffered saline; PKC, protein kinase C; PMSF, -phenylmethylsulfonyl fluoride.-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[]--->parr 2 The abbreviations >>>ylsulfonyl fluoride. 13 THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 283, NO. 24, pp. 16711­16722, June 13, 2008-->id=6, page=0, size=7, fam=Times, col=#231f20, type=parrnote, textLines=45--->[]--->note THE JOURNAL OF BIOLO>>>16722, June 13, 2008 14 © 2008 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.-->id=6, page=0, size=7, fam=Times, col=#231f20, type=parrnote, textLines=45--->[]--->note © 2008 by The Americ>>>rinted in the U.S.A. 15 JUNE 13, 2008 · VOLUME 283 · NUMBER 24-->id=2, page=0, size=11, fam=Times, col=#231f20, type=parrnote, textLines=67--->['U']--->note JUNE 13, 2008 · VOLU>>>LUME 283 · NUMBER 24 16 JOURNAL OF BIOLOGICAL CHEMISTRY 16711-->id=2, page=0, size=11, fam=Times, col=#231f20, type=parrnote, textLines=67--->[]--->note JOURNAL OF BIOLOGICA>>>ICAL CHEMISTRY 16711 17 at-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[u'a']--->note at>>>at 18 Centro-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[]--->note Centro>>>Centro 19 Nacional-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[]--->note Nacional>>>Nacional 20 de-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[]--->note de>>>de 21 Investigaciones-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[]--->note Investigaciones>>>Investigaciones 22 Oncológicas,-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[]--->note Oncológicas,>>>Oncológicas, 23 on-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[]--->note on>>>on 24 May-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[]--->note May>>>May 25 28,-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[]--->note 28,>>>28, 26 2010-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[]--->note 2010>>>2010 27 www.jbc.org-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[]--->note www.jbc.org>>>www.jbc.org 28 Downloaded-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[]--->note Downloaded>>>Downloaded 29 from-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[]--->note from>>>from 30 http://www.jbc.org/content/suppl/2008/04/16/M802246200.DC1.html-->id=6, page=0, size=7, fam=Times, col=#231f20, type=parrnote, textLines=45--->[]--->note http://www.jbc.org/c>>>/M802246200.DC1.html 31 Supplemental Material can be found at:-->id=6, page=0, size=7, fam=Times, col=#231f20, type=parrnote, textLines=45--->[]--->note Supplemental Materia>>>ial can be found at: 32 In the present study, we utilized mass spectrometry to map four phosphorylation sites on topo I, generated phosphoepitope-specific antibodies, and used these antibodies to study the conditions leading to topo I phosphorylation in vitro and in intact cells. In addition, we performed site-directed mutagenesis of these sites to assess the impact on localization and activity. Results of this analysis suggest that topo I is phosphorylated during mitosis in untreated cells and that one of these mitotic phosphorylations modestly enhances topo I activity in vitro as well as sensitivity to CPT-induced trapping on DNA in intact cells.-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=904--->[]--->parr In the present study>>>DNA in intact cells. 33 EXPERIMENTAL PROCEDURES-->id=5, page=0, size=13, fam=Times, col=#231f20, type=title, textLines=9--->['U']--->title EXPERIMENTAL PROCEDU>>>ERIMENTAL PROCEDURES 34 Materials--Reagents were obtained from the following suppliers: CPT, paclitaxel, aphidicolin, hydroxyurea, Hoechst 33258, PMSF, phorbol myristate acetate, and chloroquine from Sigma; nocodazole from Aldrich; and DMSO from Fisher. Antibodies to the following antigens were obtained from the indicated suppliers: nucleolin from Santa Cruz Biotechnology, Inc., TATA-binding protein from BD Transduction Laboratories, and topors from Novus. Anti-S peptide antibody was raised and characterized as described previously (25). Antibodies to other antigens were kind gifts from the following investigators: murine monoclonal anti-poly(ADP-ribose) polymerase from Guy Poirier (Laval University, Ste. Foy, Quebec, Canada), human anti-topo I autoantiserum from Naomi Rothfield (University of Connecticut, Farmington, CT), and C21 murine monoclonal antitopo I from Y.-C. Cheng (Yale University Medical School, New Haven, CT).-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=904--->[]--->parr Materials--Reagents >>>ool, New Haven, CT). 35 Cell Culture--A549 human lung cancer cells and K562 human leukemia cells were grown in RPMI 1640 medium containing 10% heat-inactivated fetal bovine serum, 100 units/ml-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=904--->[]--->parr Cell Culture--A549 h>>> serum, 100 units/ml 36 penicillin G, 100 g/ml streptomycin, 2 mML-glutamine-->id=3, page=0, size=8, fam=Times, col=#231f20, type=parrnote, textLines=20--->[]--->parr penicillin G, 100 g/>>>cin, 2 mML-glutamine 37 (medium A). Cells were incubated at 37 °C in a humidified incubator with 5% CO2.-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=904--->[]--->parr (medium A). Cells we>>>cubator with 5% CO2. 38 In the indicated experiments, cells were treated with 100 nM paclitaxel for 16 h, resulting in arrest of 80% of cells in G2/M as assessed by flow cytometry and 75­ 80% of cells in metaphase as assessed by fluorescence microscopy after Hoechst 33258 staining. Longer exposures to paclitaxel were avoided because of adaptation of the mitotic checkpoint and exit of these cells into a multinucleated, tetraploid G1 state (26).-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=904--->[]--->parr In the indicated exp>>>ploid G1 state (26). 39 Plasmid Construction--The yeast shuttle vector hGal1 containing cDNA encoding catalytically inactive (Y723F) human topo I was a kind gift from M.-A. Bjornsti (St. Jude Children's Hospital, Memphis, TN). The cDNA was subjected to PCR to add the S peptide and linker sequence (in brackets) [GAGAGAGAGGAP]MKETAAAKFERQHMDS or MKETAAAKFERQHMDS[GAGAGAGAGGAP] to either the C-terminal (Topo I-S) or N-terminal (S-Topo I) end of the topo I open reading frame, respectively. After ligation of Topo I-S or S-Topo I into pcDNA 3.1 (Invitrogen), the entire insert was sequenced. Topo I-S was used for generating a stably transfected K562 cell line, which was then used for mass spectrometry and in vitro kinase assays as described below. S-Topo I was used for all other experiments.-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=904--->[]--->parr Plasmid Construction>>>l other experiments. 40 Transient and Stable Transfection--10 ­15 106 K562 cells were washed in sterile PBS and resuspended in cytomix buffer (120 mM KCl, 150 M CaCl2,2mM EGTA, 5 mM MgCl2,25mM HEPES, 10 mM K2HPO4/KH2PO4 (pH 7.6)) containing 40 gof plasmid. Cells were electroporated at 320 V for 10 ms in a BTX ECM 830 square wave electroporator, incubated for 15 min at room temperature, and diluted in medium A without antibiotics. After 24 h, cells were utilized for the assays described below. Alternatively 48 h after electroporation, cells transfected with Topo I-S (Y723F) were treated with 800 g/ml Geneticin to select stable transfectants. Once Geneticin-resistant cells grew, clones were isolated by limiting dilution and assayed for Topo I-S expression by immunoblotting using anti-S peptide antibody. A resulting stable line with high expression was maintained in medium A containing 400 g/ml Geneticin.-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=904--->[]--->parr Transient and Stable>>> 400 g/ml Geneticin. 41 Immunoprecipitation and Pulldown--Endogenous topo I was isolated from K562 cells using human anti-topo I autoantibody and protein A-Sepharose beads. All steps were performed at 4 °C. Cells were washed twice in ice-cold PBS, incubated in lysis buffer with protease and phosphatase inhibitors (1% (w/v) Triton X-100, 400 mM NaCl, 50 mM HEPES (pH 7.5),-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=904--->[]--->parr Immunoprecipitation >>>0 mM HEPES (pH 7.5), 42 10% (w/v) glycerol, 5 mM MgSO4,1mM EDTA, 1 mM sodium-->id=3, page=0, size=8, fam=Times, col=#231f20, type=parrnote, textLines=20--->[]--->parr 10% (w/v) glycerol, >>>mM EDTA, 1 mM sodium 43 orthovanadate, 10 mM sodium pyrophosphate, 100 mM NaF, 1 mM PMSF, 10 g/ml leupeptin, 100 units/ml Trasylol, 1% (w/v) thiodiglycol, 20 nM microcystin) for 15 min, and sedimented at 12,000 g for 15 min. Topo I antibody was added to the resulting supernatant, which was rotated end over end overnight. Samples were supplemented with protein A beads and rotated for an additional 4 h. Beads were spun down at 12,000 g for 1 min and washed four times with RIPA buffer containing phosphatase inhibitors (1% (w/v) Triton X-100, 1% (w/v) sodium-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=904--->[]--->parr orthovanadate, 10 mM>>>100, 1% (w/v) sodium 44 deoxycholate, 0.1% (w/v) SDS, 150 mM NaCl, 10 mM sodium-->id=3, page=0, size=8, fam=Times, col=#231f20, type=parrnote, textLines=20--->[]--->parr deoxycholate, 0.1% (>>>M NaCl, 10 mM sodium 45 phosphate (pH 7.2), 2 mM EDTA, 1 mM sodium orthovanadate, 100 units/ml Trasylol, 50 mM NaF). To elute protein for SDSPAGE, beads were resuspended in sample buffer (4 M urea, 2% (w/v) SDS, 62.5 mM Tris-HCl (pH 6.8), 1 mM EDTA, 5% (v/v) 2-mercaptoethanol, 0.1% (w/v) bromphenol blue) and heated to 65 °C for 20 min.-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=904--->[]--->parr phosphate (pH 7.2), >>>to 65 °C for 20 min. 46 S-Topo I or Topo I-S was isolated using a similar procedure. In brief, lysates were prepared as described above and sedimented at 12,000 g for 15 min. S protein beads (Novagen) were added to the resulting supernatant, which was rotated end over end overnight. Beads were then sedimented and washed four times with RIPA buffer containing phosphatase inhibitors prior to SDS-PAGE or incubation with kinases. Alternatively immobilized topo I was washed with 0.1% Nonidet P-40 in PBS rather than RIPA buffer and assayed for topo I activity.-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=904--->[]--->parr S-Topo I or Topo I-S>>>for topo I activity. 47 Metabolic Labeling--2.5 107 K562 cells were washed once and resuspended in 10 ml of phosphate- and serum-free RPMI 1640 medium (BIOSOURCE). After cells were incubated for 30 min at 37 °C, 2.5 mCi of [32P]orthophosphate (Amersham Biosciences) and dialyzed fetal calf serum (10% (v/v) final concentration) were added for 4 h. Topo I was recovered from radiolabeled cells by immunoprecipitation or pulldown with S protein-agarose as described above, subjected to SDS-PAGE, and visualized by autoradiography.-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=904--->[]--->parr Metabolic Labeling-->>> by autoradiography. 48 Two-dimensional Tryptic Mapping--5.0 107 K562 cells stably transfected with Topo I-S (Y723F) were treated for 16 h with 100 nM paclitaxel and then radiolabeled with [32P]orthophosphate as described above. Topo I pulled down with S protein-agarose was identified by SDS-PAGE followed by autoradiography. The radiolabeled topo I band was excised and subjected to reduction, alkylation, and digestion with 2 g of trypsin as described by Meisenhelder et al. (27). The resulting sample was spotted onto a 20 20-cm, 100- m cellulose thin layer chromatography plate (EM Science), subjected to electrophoresis at 1000 V for 30 min in pH 1.9 buffer, and exposed to ascending chromatography overnight in buffer consisting of 37.5% (v/v) n-butyl alcohol, 25% (v/v) pyridine, 7.5% (v/v) acetic acid. Radiolabeled spots were detected using a Storm 840 PhosphorImager (GE Healthcare).-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=904--->[]--->parr Two-dimensional Tryp>>>ger (GE Healthcare). 49 Mitotic Phosphorylation of Human Topoisomerase I-->id=1, page=0, size=16, fam=Times, col=#231f20, type=title, textLines=11--->[]--->note Mitotic Phosphorylat>>>uman Topoisomerase I 50 16712 JOURNAL OF BIOLOGICAL CHEMISTRY-->id=2, page=0, size=11, fam=Times, col=#231f20, type=parrnote, textLines=67--->['U']--->note 16712 JOURNAL OF BIO>>>BIOLOGICAL CHEMISTRY 51 VOLUME 283 · NUMBER 24 · JUNE 13, 2008-->id=2, page=0, size=11, fam=Times, col=#231f20, type=parrnote, textLines=67--->['U']--->note VOLUME 283 · NUMBER >>>R 24 · JUNE 13, 2008 52 at-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[u'a']--->note at>>>at 53 Centro-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[]--->note Centro>>>Centro 54 Nacional-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[]--->note Nacional>>>Nacional 55 de-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[]--->note de>>>de 56 Investigaciones-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[]--->note Investigaciones>>>Investigaciones 57 Oncológicas,-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[]--->note Oncológicas,>>>Oncológicas, 58 on-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[]--->note on>>>on 59 May-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[]--->note May>>>May 60 28,-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[]--->note 28,>>>28, 61 2010-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[]--->note 2010>>>2010 62 www.jbc.org-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[]--->note www.jbc.org>>>www.jbc.org 63 Downloaded-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[]--->note Downloaded>>>Downloaded 64 from-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[]--->note from>>>from 65 http://www.jbc.org/content/suppl/2008/04/16/M802246200.DC1.html-->id=6, page=0, size=7, fam=Times, col=#231f20, type=parrnote, textLines=45--->[]--->note http://www.jbc.org/c>>>/M802246200.DC1.html 66 Supplemental Material can be found at:-->id=6, page=0, size=7, fam=Times, col=#231f20, type=parrnote, textLines=45--->[]--->note Supplemental Materia>>>ial can be found at: 67 Mass Spectrometry--Topo I-S was isolated from 1 107 stably transfected K562 cells, subjected to SDS-PAGE, and stained with Coomassie Blue. The topo I band was excised, digested using either trypsin or Arg-C, and analyzed by quadrupole time-of-flight tandem mass spectrometry at the Taplin Biological Mass Spectrometry Facility (Harvard Medical School, Boston, MA).-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=904--->[]--->parr Mass Spectrometry--T>>>School, Boston, MA). 68 Generation of Phosphospecific Antibodies--Phosphorylated and nonphosphorylated peptides surrounding Ser10-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=904--->[]--->parr Generation of Phosph>>>es surrounding Ser10 69 (CDHLHNDSQIEADFR), Ser21 (CADFRLNDSHKHKDKH),-->id=6, page=0, size=7, fam=Times, col=#231f20, type=parrnote, textLines=45--->[]--->parr (CDHLHNDSQIEADFR), S>>> (CADFRLNDSHKHKDKH), 70 Ser112 (CEKENGFSSPPQIKDE), and Ser394 (CSKDAKVPSPPPGHKW) were synthesized. Phosphorylated peptides were conjugated to keyhole limpet hemocyanin through their N-terminal cysteines and utilized to immunize rabbits. Bleeds were screened by immunoblotting using whole cell lysates of K562 cells and dot blots of both the phosphorylated and nonphosphorylated peptides coupled to bovine serum albumin at concentrations from 1 g to 100 pg. Bleeds showing evidence of specificity for phosphorylated peptides were affinity-purified by sequential passage over SulfoLink columns (Pierce) derivatized with nonphosphorylated and phosphorylated peptides. Antibodies were eluted in 100 mM glycine (pH 2.5), neutralized with 1 M Tris-HCl (pH 9.5), assayed by dot blot and whole cell lysate immunoblotting, diluted 1:1 in glycerol, and stored at 20 °C. The specificity of the antibodies was tested by a peptide competition assay in which blots of mitotic K562 lysates were probed with each phosphospecific antibody in the absence or presence of 1 g/ml synthetic phosphopeptide as indicated.-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=904--->[]--->parr Ser112 (CEKENGFSSPPQ>>>eptide as indicated. 71 Immunoblotting--Western blotting of whole cell lysates, immunoprecipitates, or pulldown samples was performed on nitrocellulose membranes as described previously (28). Either ECL (Amersham Biosciences) or SuperSignal (Pierce) enhanced chemiluminescence reagents were used depending on the strength of the antibody.-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=904--->[]--->parr Immunoblotting--West>>>gth of the antibody. 72 Cell Cycle Analysis of Topo I Phosphorylation--K562 cells-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=904--->[]--->parr Cell Cycle Analysis >>>rylation--K562 cells 73 were treated for 16 h with 10 M aphidicolin, 2 mM hydroxyu--->id=3, page=0, size=8, fam=Times, col=#231f20, type=parrnote, textLines=20--->[]--->parr were treated for 16 >>>olin, 2 mM hydroxyu- 74 rea, 100 nM paclitaxel, 150 nM nocodazole, or diluent (0.1% DMSO). At the end of the incubation, cells were harvested, subjected to SDS-PAGE, and analyzed by immunoblotting using phosphoepitope-specific anti-topo I antibodies. Duplicate aliquots were fixed in 50% ethanol, treated with RNase A, stained with propidium iodide, and subjected to flow cytometry as described previously (29).-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=904--->[]--->parr rea, 100 nM paclitax>>>bed previously (29). 75 To isolate mitotic cells without the use of pharmacological agents, mitotic shake-off (30) was performed. T175 flasks of log phase A549 cells were vigorously agitated for l min. After the loosened cells and medium were removed, fresh medium was added to the flasks. The process was repeated every 30 min for 9 h. The initial aliquot of dislodged cells was discarded. Thereafter cells were sedimented at 100 g, resuspended in ice-cold RPMI 1640 medium, 10 mM HEPES (pH 7.4), pooled, and stored on ice. The isolated mitotic cells and remaining control adherent cells were stained with Hoechst 33258 to determine mitotic index and were subjected to immunoblotting.-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=904--->[]--->parr To isolate mitotic c>>>d to immunoblotting. 76 In Vitro Kinase Assay--Topo I-S pulled down from stably transfected K562 cells using S protein beads or commercially available purified topo I (Topogen, Columbus, OH) was mixed with 200 M ATP and 10 units of purified CKII or Cdk1 (New England Biolabs) in the CKII or Cdk1 reaction buffers provided by the supplier. Alternatively the beads were incubated with PKC reaction buffer (20 mM HEPES-NaOH (pH 7.4), 0.03% Triton X-100) containing lipid activator (100 g/ml phosphatidylserine, 200 pM phorbol myristate acetate, 0.03% Triton X-100, 100 M dithiothreitol), 100 M CaCl2, 200 M ATP, 1 mM magnesium acetate, and 10 ng of PKC (Upstate) in a 30 °C water bath for 30 min. After SDS sample buffer was added, samples were heated to 65 °C for 15 min, subjected to SDSPAGE, and analyzed by immunoblotting.-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=904--->[]--->parr In Vitro Kinase Assa>>>d by immunoblotting. 77 Site-directed Mutagenesis--Mutations were introduced by site-directed mutagenesis using a QuikChangeTM mutagenesis kit (Stratagene) according to the instructions of the supplier. The complete sequences of the topo I open reading frames were verified by sequencing after every mutagenesis. S-Topo I was mutated to restore the enzyme active site (Tyr723) and to create phosphorylation site mutants, including the quadruple phosphomutant "4A" (S10A/S21A/S112A/S394A), the triple mutant "3A" (S10A/S112A/S394A), and the S21A mutant.-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=904--->[]--->parr Site-directed Mutage>>>and the S21A mutant. 78 Immunofluorescence--K562 cells were transiently transfected with plasmids encoding wild type or phosphomutant active (Tyr723) S-Topo I. 24 h later, cells were washed in PBS and spun onto glass slides at 60 g for 5 min using a Shandon cytocentrifuge. Cells were fixed in ice-cold methanol for 10 min, rehydrated in PBS, and blocked in TSM (150 mM NaCl, 10 mM Tris-HCl (pH 7.4) containing 10% (w/v) nonfat milk powder, 100 units/ml penicillin G, 100 g/ml streptomycin, 1 mM sodium azide) at 20 ­22 °C for 1 h. After anti-S peptide antibody was added (10 g/ml in TSM), samples were incubated at 4 °C overnight. Slides were washed six times with PBS and treated with fluorescein isothiocyanate-conjugated anti-mouse IgG secondary antibody (20 g/ml in TSM; Kirkegaard & Perry Laboratories, Inc. (KPL), Gaithersburg, MD) for 45 min at 37 °C. Slides were then washed six times with PBS and treated with 1 g/ml Hoechst 33258 to locate nuclei and chromosomes. Cells were visualized using a Zeiss LSM 510 confocal microscope.-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=904--->[]--->parr Immunofluorescence-->>>confocal microscope. 79 Binding Partner Analysis--Plasmids encoding wild type or 4A S-Topo I were transfected into K562 cells. Beginning 6 h after transfection, 100 nM paclitaxel was added to cells for 18 h to induce mitotic arrest. After incubation, topo I was recovered with S protein beads, subjected to SDS-PAGE, and analyzed by immunoblotting with antibodies against various topo I binding partners.-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=904--->[]--->parr Binding Partner Anal>>> I binding partners. 80 Mitotic Phosphorylation of Human Topoisomerase I-->id=1, page=0, size=16, fam=Times, col=#231f20, type=title, textLines=11--->[]--->note Mitotic Phosphorylat>>>uman Topoisomerase I 81 JUNE 13, 2008 · VOLUME 283 · NUMBER 24-->id=2, page=0, size=11, fam=Times, col=#231f20, type=parrnote, textLines=67--->['U']--->note JUNE 13, 2008 · VOLU>>>LUME 283 · NUMBER 24 82 JOURNAL OF BIOLOGICAL CHEMISTRY 16713-->id=2, page=0, size=11, fam=Times, col=#231f20, type=parrnote, textLines=67--->[]--->note JOURNAL OF BIOLOGICA>>>ICAL CHEMISTRY 16713 83 at-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[u'a']--->note at>>>at 84 Centro-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[]--->note Centro>>>Centro 85 Nacional-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[]--->note Nacional>>>Nacional 86 de-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[]--->note de>>>de 87 Investigaciones-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[]--->note Investigaciones>>>Investigaciones 88 Oncológicas,-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[]--->note Oncológicas,>>>Oncológicas, 89 on-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[]--->note on>>>on 90 May-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[]--->note May>>>May 91 28,-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[]--->note 28,>>>28, 92 2010-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[]--->note 2010>>>2010 93 www.jbc.org-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[]--->note www.jbc.org>>>www.jbc.org 94 Downloaded-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[]--->note Downloaded>>>Downloaded 95 from-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[]--->note from>>>from 96 http://www.jbc.org/content/suppl/2008/04/16/M802246200.DC1.html-->id=6, page=0, size=7, fam=Times, col=#231f20, type=parrnote, textLines=45--->[]--->note http://www.jbc.org/c>>>/M802246200.DC1.html 97 Supplemental Material can be found at:-->id=6, page=0, size=7, fam=Times, col=#231f20, type=parrnote, textLines=45--->[]--->note Supplemental Materia>>>ial can be found at: 98 Topo I Activity Assays --Plasmids encoding wild type or phosphorylation site mutant S-Topo I were transfected into K562 cells. Beginning 6 h after transfection, 100 nM paclitaxel was added to cells to induce a mitotic arrest. After an 18-h incubation in paclitaxel, topo I was recovered with S protein-agarose. Beads were washed four times in 0.1% Nonidet P-40 in PBS because RIPA buffer was found to abolish topo I activity. A fraction of the beads was removed for immunoblotting to quantify S-Topo I expression. Where indicated, 10 units of purified calf intestine alkaline phosphatase (Invitrogen) was added, and beads were incubated at 37 °C for 30 min. Plasmid relaxation activity of the immobilized topo I was then analyzed as described by Hann et al. (31). In brief, the beads were diluted in 2-fold increments and resuspended in topo I activity buffer (50-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=904--->[]--->parr Topo I Activity Assa>>> activity buffer (50 99 mM Tris-HCl (pH 7.5), 2.5 mM MgCl2, 110 mM NaCl, 500 M-->id=3, page=0, size=8, fam=Times, col=#231f20, type=parrnote, textLines=20--->[]--->parr mM Tris-HCl (pH 7.5)>>>, 110 mM NaCl, 500 M 100 EDTA, 6 g/ml bovine serum albumin, 0.01% (w/v) dithiothreitol).-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=904--->[]--->parr EDTA, 6 g/ml bovine >>>/v) dithiothreitol). 101 Alternatively, after treatment of K562 cells with 0.1% DMSO or 100 nM paclitaxel for 16 h, cells were sedimented, washed once in PBS, and resuspended in nuclear isolation buffer con--->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=904--->[]--->parr Alternatively, after>>>solation buffer con- 102 sisting of 10 mM NaCl, 10 mM Tris-HCl (pH 7.4), 3 mM MgSO4-->id=3, page=0, size=8, fam=Times, col=#231f20, type=parrnote, textLines=20--->[]--->parr sisting of 10 mM NaC>>>(pH 7.4), 3 mM MgSO4 103 containing 1 mM dithiothreitol, 100 units/ml Trasylol, 1 mM-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=904--->[]--->parr containing 1 mM dith>>>ts/ml Trasylol, 1 mM 104 PMSF, 0.5 mM EDTA, 10 mM NaF, 2 mM Na2P2O7,1mM-->id=3, page=0, size=8, fam=Times, col=#231f20, type=parrnote, textLines=20--->[]--->parr PMSF, 0.5 mM EDTA, 1>>>aF, 2 mM Na2P2O7,1mM 105 Na2VO4,20nM microcystin. After a 20-min incubation on ice, samples were subjected to 30 strokes in a tight fitting Dounce homogenizer, examined microscopically to confirm cell disruption, and sedimented at 16,000 g for 15 min to sediment nuclei and mitotic chromosomes, respectively. After a wash with nuclear isolation buffer, pellets were resuspended in 75 l of topoisomerase extraction buffer consisting of 100 mM-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=904--->[]--->parr Na2VO4,20nM microcys>>>consisting of 100 mM 106 sodium phosphate (pH 7.4), 1 mM EDTA, 0.1 mM dithiothreitol, 1mM PMSF, 10 mM NaF, 2 mM Na2P2O7,1mM Na2VO4,20nM-->id=3, page=0, size=8, fam=Times, col=#231f20, type=parrnote, textLines=20--->[]--->parr sodium phosphate (pH>>>P2O7,1mM Na2VO4,20nM 107 microcystin; treated with an equal volume of topoisomerase extraction buffer containing 1.2 M KCl; and incubated on ice for 15 min. After DNA was precipitated from the extracts using polyethylene glycol (32), supernatants were adjusted to equal protein concentrations. Aliquots recovered by precipitation in 20% (w/v) ice-cold trichloroacetic acid were subjected to SDSPAGE and immunoblotting for topo I content. For end point assays, aliquots containing serial 2-fold dilutions of the extracts in topo I activity buffer supplemented with the phosphatase-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=904--->[]--->parr microcystin; treated>>>with the phosphatase 108 inhibitors 10 mM NaF, 1 mM Na2P2O7, and 20 nM microcystin-->id=3, page=0, size=8, fam=Times, col=#231f20, type=parrnote, textLines=20--->[]--->parr inhibitors 10 mM NaF>>>nd 20 nM microcystin 109 were assayed for ability to relax supercoiled plasmid. Reactions were initiated by adding 500 ng of O6#7 plasmid DNA and incubated at 37 °C for 30 min. After dilution with 1/10 volume containing 1 g of proteinase K in 10% (w/v) SDS, samples were incubated at 37 °C for another 15­30 min. For time course experiments, reactions containing equal amounts of topo I as assessed by immunoblotting were incubated with 5 g of plasmid O6#7 in 180 l of topo I activity buffer with phosphatase inhibitors for 0 ­30 min. At the indicated times, 20- l aliquots were treated with 1 g of proteinase K and 1% SDS (final concentration) at 50 °C to stop the reaction. Plasmids were separated on 1% agarose gels in TPE buffer (36 mM Tris, 30 mM NaH2PO4,1mM EDTA, pH 7.8) containing 10 g/ml chloroquine, stained with 500 ng/ml ethidium bromide, and visualized under 260 nm illumination.-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=904--->[]--->parr were assayed for abi>>>260 nm illumination. 110 Topo I Cleavage Half-reaction--Based on the results of Pourquier et al. (33), substrate was generated by annealing a 16-mer upper strand (5 -GATCTAAAAGACTTGG-3 ) to a 36-mer bottom strand (5 -GATCTTTTTTAAAAATTTTTCCAAGTCTTTTACATC-3 ). The annealed substrate was labeled with 800 Ci/mol [ -32P]dATP (PerkinElmer Life Sciences) and 5 units of Klenow fragment (New England Biolabs) to add five radiolabeled adenosines to the upper strand. The radiolabeled duplex was separated from unincorporated nucleotide on a Sephadex TE-10 spin column.-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=904--->[]--->parr Topo I Cleavage Half>>>x TE-10 spin column. 111 After K562 cells transiently transfected with plasmid encod--->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=904--->[]--->parr After K562 cells tra>>> with plasmid encod- 112 ing wild type or S21A topo I were incubated with 100 nM pacli--->id=3, page=0, size=8, fam=Times, col=#231f20, type=parrnote, textLines=20--->[]--->parr ing wild type or S21>>>d with 100 nM pacli- 113 taxel for 16 h, topo I was pulled down using S protein-agarose as described above. An aliquot containing 40% of the beads was incubated with 300 fmol of radiolabeled substrate in topo I activity buffer at 37 °C. At the indicated times, aliquots containing 50 fmol of substrate were removed, diluted with formamide to a final concentration of 60%, heated to 100 °C for 10 min, cooled on ice, and separated on a denaturing 10% polyacrylamide gel (19:1 acrylamide:bisacrylamide, 7.5 M urea). Substrate incubated for 45 min at 37 °C in buffer lacking topo I and bona fide 7-mer (6-mer annealed to the bottom strand and extended with Klenow as described above) were included on each gel as markers. Gels were examined on a PhosphorImager as described above. The remainder of each pulldown was utilized for immunoblotting.-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=904--->[]--->parr taxel for 16 h, topo>>> for immunoblotting. 114 Band Depletion Assays--K562 cells transiently transfected with plasmids encoding wild type or S21A S-Topo I were treated with 0.1% DMSO or 100 nM paclitaxel for 16 h as described above. Alternatively untransfected K562 cells were treated with 0.1% DMSO or 100 nM paclitaxel for 16 h to examine endogenous topo I. Cells were sedimented at 100 g for 10 min, resuspended in serum-free RPMI 1640 medium containwith overexpressing active topo I in intact cells (4, 31), a plasmid encoding Topo I-S with an inactivating Y723F mutation was stably transfected into K562 cells. When the stable line was radiolabeled with [32P]orthophosphate and the tagged topo I was pulled down using S protein-agarose beads, autoradiography showed that the tagged construct also incorporated 32P (Fig. 1B). Consistent with an earlier report that murine topo I is differentially phosphorylated during mitosis (22), phosphorylation of Topo I-S (Y723F) increased in mitotic cells (Fig. 1C). Two-dimensional tryptic mapping suggested the possibility of four phosphorylated peptides in these mitotic cells (Fig. 1D).-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=904--->[]--->parr Band Depletion Assay>>>tic cells (Fig. 1D). 115 ing 10 mM HEPES (pH 7.4 at 21 °C), diluent or 100 nM pacli--->id=3, page=0, size=8, fam=Times, col=#231f20, type=parrnote, textLines=20--->[]--->parr ing 10 mM HEPES (pH >>>ent or 100 nM pacli- 116 taxel, and diluent versus 1.5, 5, 15, or 50 M CPT. After a 45-min incubation at 37 °C, cells were briefly sedimented at 9600 g, resuspended in lysis buffer (6 M guanidine hydrochloride, 250 mM Tris-HCl (pH 8.5 at 21 °C), 10 mM EDTA, 1% (v/v) freshly added -mercaptoethanol, 1 mM PMSF), and sonicated. Samples were alkylated with iodoacetamide, dialyzed, and lyophilized as described previously (34). After SDS-PAGE and immunoblotting, bands were quantified with ImageJ software.-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=904--->[]--->parr taxel, and diluent v>>>ith ImageJ software. 117 RESULTS-->id=5, page=0, size=13, fam=Times, col=#231f20, type=title, textLines=9--->[]--->title RESULTS>>>RESULTS 118 Topo I Is Phosphorylated in Cells--When topo I was immunoprecipitated from [32P]orthophosphate-labeled log phase K562 human leukemia cells using a human anti-topo I antiserum (35), subsequent SDS-PAGE and autoradiography indicated that the topo I polypeptide contained covalently bound 32P (Fig. 1A) in agreement with previous results suggesting that topo I is phosphorylated in intact cells (13, 16, 17, 20 ­22). To permit isolation of the large amounts of purified topo I polypeptide required for mapping the phosphorylation sites, the S peptide epitope tag and a linker were inserted on either the C terminus (Topo I-S) or N terminus (S-Topo I) of the topo I open reading frame (25). Because of the potential toxicity associated-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=904--->[]--->parr Topo I Is Phosphoryl>>> toxicity associated 119 Mitotic Phosphorylation of Human Topoisomerase I-->id=1, page=0, size=16, fam=Times, col=#231f20, type=title, textLines=11--->[]--->note Mitotic Phosphorylat>>>uman Topoisomerase I 120 16714 JOURNAL OF BIOLOGICAL CHEMISTRY-->id=2, page=0, size=11, fam=Times, col=#231f20, type=parrnote, textLines=67--->['U']--->note 16714 JOURNAL OF BIO>>>BIOLOGICAL CHEMISTRY 121 VOLUME 283 · NUMBER 24 · JUNE 13, 2008-->id=2, page=0, size=11, fam=Times, col=#231f20, type=parrnote, textLines=67--->['U']--->note VOLUME 283 · NUMBER >>>R 24 · JUNE 13, 2008 122 at-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[u'a']--->note at>>>at 123 Centro-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[]--->note Centro>>>Centro 124 Nacional-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[]--->note Nacional>>>Nacional 125 de-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[]--->note de>>>de 126 Investigaciones-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[]--->note Investigaciones>>>Investigaciones 127 Oncológicas,-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[]--->note Oncológicas,>>>Oncológicas, 128 on-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[]--->note on>>>on 129 May-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[]--->note May>>>May 130 28,-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[]--->note 28,>>>28, 131 2010-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[]--->note 2010>>>2010 132 www.jbc.org-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[]--->note www.jbc.org>>>www.jbc.org 133 Downloaded-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[]--->note Downloaded>>>Downloaded 134 from-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[]--->note from>>>from 135 http://www.jbc.org/content/suppl/2008/04/16/M802246200.DC1.html-->id=6, page=0, size=7, fam=Times, col=#231f20, type=parrnote, textLines=45--->[]--->note http://www.jbc.org/c>>>/M802246200.DC1.html 136 Supplemental Material can be found at:-->id=6, page=0, size=7, fam=Times, col=#231f20, type=parrnote, textLines=45--->[]--->note Supplemental Materia>>>ial can be found at: 137 Identification of Four Novel Phosphorylation Sites on Human Topo I--To identify topo I phosphorylation sites, unlabeled Topo I-S (Y723F) was isolated from stably transfected K562 cells and purified by SDS-PAGE. This procedure yielded a band of purified polypeptide that was easily visible after Coomassie Blue staining (Fig. 1E). Several samples prepared in this manner, including samples from untreated cells, cells arrested in mitosis by an 18-h treatment with 100 nM paclitaxel, and cells treated for 15 min with 50 nM phorbol myristate acetate, were subjected to trypsin digestion followed by quadrupole time-of-flight tandem mass spectrometry analysis. Results obtained with each of the three samples suggested that Ser10, Ser112, and Ser394 of topo I are phosphorylated in cells (supplemental Fig. S1, A­C).-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=904--->[]--->parr Identification of Fo>>>ntal Fig. S1, A­C). 138 Further analysis of the mass spectrometry data indicated that peptides corresponding to 20 ­30% of the polypeptide, especially the lysine-rich N-terminal domain, had not been detected. To maximize coverage of the polypeptide, additional samples were digested with the protease Arg-C instead of trypsin and subjected to mass spectrometry. Results of this analysis suggested that topo I is also phosphorylated at Ser21 (supplemental Fig. S1D).-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=904--->[]--->parr Further analysis of >>>lemental Fig. S1D). 139 The region around the phosphorylation sites was analyzed for sequence conservation. All four putative phosphorylation sites are conserved in higher eukaryotes, including mouse, chicken, and human, but not in yeast (Fig. 2, A­D). These findings suggest that these phosphorylations may be important for some function of the higher eukaryotic forms of topo I.-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=904--->[]--->parr The region around th>>>tic forms of topo I. 140 Ser10, Ser21, Ser112, and Ser394 Are Phosphorylated during Mitosis--Phosphoepitope-specific anti-topo I antisera were generated to assess whether these four sites are phosphorylated on the endogenous topo I polypeptide in cells and to enable further analysis of the phosphorylation events. After affinity purification, each antiserum selectively detected phosphorylated polypeptide (Fig. 3A). Each phosphoepitope-specific antiserum detected topo I in K562 lysates by immunoblotting (Fig. 3B), indicating the endogenous phosphorylation at these sites in cells. Competition experiments (Fig. 3C) demonstrated that each affinity-purified reagent was specific for only its phosphoepitope.-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=904--->[]--->parr Ser10, Ser21, Ser112>>> its phosphoepitope. 141 To determine the phosphorylation state at these sites during various phases of the cell cycle, K562 cells were arrested at various stages of the cell cycle as verified by flow cytometry (supplemental Fig. S2) using aphidicolin (G1/S), hydroxyurea (G1/S), etoposide (G2), and paclitaxel or nocodazole (M). Immunoblotting demonstrated that phosphorylation of Ser10, Ser21, Ser112, and Ser394 was markedly increased only in cells treated with paclitaxel or nocodazole, suggesting that these phosphorylations occur predominantly or exclusively during mitosis (Fig. 4A and data not shown). To confirm this finding and verify that the mitotic phosphorylation of these sites was not caused by the drug treatment, mitotic A549 human lung cancer cells were separated from nonmitotic cells by mitotic shake-off. Analysis of these cells demonstrated phosphorylation of topo I at Ser10, Ser21, Ser112, and Ser394 in mitotic cells but not interphase cells (Fig. 4B).-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=904--->[]--->parr To determine the pho>>>ase cells (Fig. 4B). 142 FIGURE 1. Endogenous and S peptide-tagged topo I is phosphorylated in cells. A, after 2.5 107 K562 human leukemia cells were radiolabeled for 4 h with 0.25 mCi/ml [32P]orthophosphate, topo I was isolated by immunoprecipitation using human anti-topo I antiserum, subjected to SDS-PAGE, and analyzed by autoradiography. B, after 2.5 10 7 K562 cells stably expressing topo I-S (Y723F) were labeled for 4 h with 0.25 mCi/ml [32P]orthophosphate, the tagged topo I was isolated on S protein-agarose beads. Samples were subjected to SDS-PAGE and analyzed by autoradiography. C, 3.0 10 7 K562 cells transfected with a plasmid encoding S-topo I (Tyr723) were treated with 100 nM paclitaxel or diluent for 16 h followed by 0.25 mCi/ml [32P]orthophosphate for 4 h. Topo I was isolated on S protein-agarose beads, subjected to SDS-PAGE, and analyzed by autoradiography as well as blotting with anti-S peptide antibody (25). The panel contains lanes from a single film exposure. Dashes indicate removal of unrelated lanes. D, after metabolic labeling with [32P]orthophosphate, topo I was isolated by pulldown from 5.0 107 paclitaxel-treated cells stably expressing topo I-S (Y723F) as described in B, subjected to SDS-PAGE, and stained with Coomassie Brilliant Blue. The excised topo I band was subjected to two-dimensional tryptic mapping and PhosphorImager analysis as described under "Experimental Procedures." The sample origin is indicated by the circle. E, the stable line described in B was also used to obtain unlabeled, purified topo I for mass spectrometry as shown by a representative Coomassie Blue-stained gel. Results in B, C, D and E are representative of five, five, nine, and five experiments, respectively.-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[]--->capfig FIGURE 1. Endogenous>>>ments, respectively. 143 FIGURE 2. Conservation of the four mapped topo I phosphorylation sites. A­D, sequence homology of the four phosphorylation sites. SC, Saccharomyces cerevisiae; SP, Schizosaccharomyces pombe; AT, Arabidopsis thaliana; X, Xenopus; M, mouse; CH, chicken; H, human; D, Drosophila melanogaster.-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[]--->capfig FIGURE 2. Conservati>>>ophila melanogaster. 144 Mitotic Phosphorylation of Human Topoisomerase I-->id=1, page=0, size=16, fam=Times, col=#231f20, type=title, textLines=11--->[]--->note Mitotic Phosphorylat>>>uman Topoisomerase I 145 JUNE 13, 2008 · VOLUME 283 · NUMBER 24-->id=2, page=0, size=11, fam=Times, col=#231f20, type=parrnote, textLines=67--->['U']--->note JUNE 13, 2008 · VOLU>>>LUME 283 · NUMBER 24 146 JOURNAL OF BIOLOGICAL CHEMISTRY 16715-->id=2, page=0, size=11, fam=Times, col=#231f20, type=parrnote, textLines=67--->[]--->note JOURNAL OF BIOLOGICA>>>ICAL CHEMISTRY 16715 147 at-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[u'a']--->note at>>>at 148 Centro-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[]--->note Centro>>>Centro 149 Nacional-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[]--->note Nacional>>>Nacional 150 de-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[]--->note de>>>de 151 Investigaciones-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[]--->note Investigaciones>>>Investigaciones 152 Oncológicas,-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[]--->note Oncológicas,>>>Oncológicas, 153 on-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[]--->note on>>>on 154 May-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[]--->note May>>>May 155 28,-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[]--->note 28,>>>28, 156 2010-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[]--->note 2010>>>2010 157 www.jbc.org-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[]--->note www.jbc.org>>>www.jbc.org 158 Downloaded-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[]--->note Downloaded>>>Downloaded 159 from-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[]--->note from>>>from 160 http://www.jbc.org/content/suppl/2008/04/16/M802246200.DC1.html-->id=6, page=0, size=7, fam=Times, col=#231f20, type=parrnote, textLines=45--->[]--->note http://www.jbc.org/c>>>/M802246200.DC1.html 161 Supplemental Material can be found at:-->id=6, page=0, size=7, fam=Times, col=#231f20, type=parrnote, textLines=45--->[]--->note Supplemental Materia>>>ial can be found at: 162 In Vitro Phosphorylation Analysis--Analysis of the sequence around the four phosphorylation sites (Fig. 2) indicated that Ser10 and Ser21 are part of possible CKII and PKC consensus sequences, respectively. In addition, Ser112 and Ser394 are serine-proline sites, which are potential Cdk phosphorylation sites. Accordingly CKII, PKC , and Cdk1 were tested for their ability to phosphorylate the sites in vitro. Topo I-S was pulled down from stably transfected K562 cells, washed, incubated with purified kinases in vitro, subjected to SDSPAGE, and analyzed by immunoblotting with the phosphoepitope-specific antibodies. Alternatively purified topo I was incubated with purified kinases in vitro and subjected to the same protocol described above. Results demonstrated that Ser10 can be phosphorylated by CKII, Ser21 can be phosphorylated by PKC , and Ser112 and Ser394 can be phosphorylated by Cdk1 in vitro (Fig. 5). Further analysis verified that each of these sites was phosphorylated specifically by its indicated kinase and not by the other two kinases (supplemental Fig. S3).-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=904--->[]--->parr In Vitro Phosphoryla>>>plemental Fig. S3). 163 FIGURE 3. Development of phosphotopo I antibodies. A and B, polyclonal phosphoepitope-specific antibodies for Ser10, Ser21, Ser112, and Ser394 were generated in rabbits. Bleeds were affinity-purified as described under "Experimental Procedures" and screened by dot blots using phosphorylated (P) and nonphosphorylated (NP) peptides coupled to bovine serum albumin (A) and immunoblotting using K562 lysates (B). C, specificity of the anti-phosphoepitope antibodies. Affinity-purified antibodies were incubated with a 1 g/ml concentration of the designated phosphopeptide during incubation with nitrocellulose-immobilized mitotic K562 lysates. Each panel contains lanes from a single film exposure. Dashes indicate removal of intervening lanes.-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[]--->capfig FIGURE 3. Developmen>>>f intervening lanes. 164 FIGURE 4. Ser10, Ser21, Ser112, and Ser394 are phosphorylated during mitosis. A, K562 cells were treated for 16 h with a variety of drugs, including 10 M aphidicolin, 2 mM hydroxyurea, 100 nM paclitaxel, or 150 nM nocodazole, to cause arrest in various phases of the cell cycle. Changes in cell cycle distribution were confirmed by flow cytometry (supplemental Fig. S2). Samples were then subjected to SDS-PAGE and immunoblotting using anti-phospho-topo I antibodies. Each panel contains lanes from a single film exposure. Dashes indicate removal of lanes from cells subjected to additional treatments that did not affect Ser10 phosphorylation. B, mitotic A549 cells were isolated by shake-off without drug treatment as described under "Experimental Procedures." Interphase cells were isolated at the completion of the shake-off, and the mitotic index of both interphase and mitotic cells was determined by microscopic examination after Hoechst 33258 staining. Samples were subjected to SDS-PAGE and immunoblotting. Representative topo I loading control blots are shown for both panels. Results in both panels are representative of five separate experiments. P, phosphorylated.-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[]--->capfig FIGURE 4. Ser10, Ser>>>. P, phosphorylated. 165 Mitotic Phosphorylation of Human Topoisomerase I-->id=1, page=0, size=16, fam=Times, col=#231f20, type=title, textLines=11--->[]--->note Mitotic Phosphorylat>>>uman Topoisomerase I 166 16716 JOURNAL OF BIOLOGICAL CHEMISTRY-->id=2, page=0, size=11, fam=Times, col=#231f20, type=parrnote, textLines=67--->['U']--->note 16716 JOURNAL OF BIO>>>BIOLOGICAL CHEMISTRY 167 VOLUME 283 · NUMBER 24 · JUNE 13, 2008-->id=2, page=0, size=11, fam=Times, col=#231f20, type=parrnote, textLines=67--->['U']--->note VOLUME 283 · NUMBER >>>R 24 · JUNE 13, 2008 168 at-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[u'a']--->note at>>>at 169 Centro-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[]--->note Centro>>>Centro 170 Nacional-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[]--->note Nacional>>>Nacional 171 de-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[]--->note de>>>de 172 Investigaciones-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[]--->note Investigaciones>>>Investigaciones 173 Oncológicas,-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[]--->note Oncológicas,>>>Oncológicas, 174 on-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[]--->note on>>>on 175 May-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[]--->note May>>>May 176 28,-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[]--->note 28,>>>28, 177 2010-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[]--->note 2010>>>2010 178 www.jbc.org-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[]--->note www.jbc.org>>>www.jbc.org 179 Downloaded-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[]--->note Downloaded>>>Downloaded 180 from-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[]--->note from>>>from 181 http://www.jbc.org/content/suppl/2008/04/16/M802246200.DC1.html-->id=6, page=0, size=7, fam=Times, col=#231f20, type=parrnote, textLines=45--->[]--->note http://www.jbc.org/c>>>/M802246200.DC1.html 182 Supplemental Material can be found at:-->id=6, page=0, size=7, fam=Times, col=#231f20, type=parrnote, textLines=45--->[]--->note Supplemental Materia>>>ial can be found at: 183 Phosphorylation Does Not Affect Topo I Localization or Interactions with Tested Binding Partners--To determine how phosphorylation of these sites affects topo I, site-directed mutagenesis was performed to create alanine mutants of S-Topo I. Because all four sites are phosphorylated during mitosis, a quadruple mutant (S10A/S21A/S112A/S394A, topo I 4A) construct was generated to remove all of these phosphorylation sites. Both catalytically active (Tyr723) and inactive (Y723F) constructs were created.-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=904--->[]--->parr Phosphorylation Does>>>tructs were created. 184 Immunofluorescence was performed to monitor subcellular localization. After plasmids encoding wild type or 4A S-Topo I (Tyr723) were transfected into K562 cells, localization of the polypeptide was analyzed using anti-S peptide antibody (Fig. 6A). 4A S-Topo I localized to the nucleus, including punctate regions representing nucleoli in interphase cells (supplemental Fig. S4A) and around the condensed chromosomes in mitotic cells (Fig. 6A, lower panels). These patterns are identical to the previously reported localization of endogenous topo I (36 ­39) and to the pattern seen with wild type S-Topo I in this study (Fig. 6A, upper panels). Similar results were also seen in paclitaxel-treated K562 cells (supplemental Fig. S4B). These results indicate that phosphorylation of these sites does not detectably affect topo I localization during mitosis.-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=904--->[]--->parr Immunofluorescence w>>>tion during mitosis. 185 To assess the effect of phosphorylation on the ability of topo I to interact with reported binding partners, a pulldown assay was performed. Following transfection with plasmids encoding either wild type or the 4A S-Topo I (Tyr723), K562 cells were arrested in mitosis with paclitaxel. After isolation of topo I using S protein-agarose, samples were subjected to immunoblotting using antibodies against reported topo I binding partners, including nucleolin, poly(ADP-ribose) polymerase, TATA-binding protein, and topors (Fig. 6B). Wild type and 4A S-Topo I bound similar amounts of these polypeptides, indicating that phosphorylation of these four sites does not affect these protein-protein interactions.-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=904--->[]--->parr To assess the effect>>>rotein interactions. 186 Phosphorylation of Ser21 Stimulates Topo I Activity in Vitro and Enhances CPT-induced Cleavage Complex Stabilization in Cells--To assess the effect of phosphorylation on topo I activity, endogenous topo I extracted from untreated or paclitaxelarrested K562 cells was assayed for enzymatic activity in vitro. In end point assays, serial 2-fold dilutions of the extracts were tested for the ability to relax a supercoiled plasmid during a 30-min incubation. Topo I isolated from mitotic cells exhibited 2­3-fold more relaxation activity than topo I isolated from untreated, interphase cells (Fig. 7A) as indicated by the dilution that resulted in reappearance of substrate (Fig. 7A, graph). Likewise time course experiments demonstrated that nuclear extracts from mitotic cells relaxed supercoiled substrate 2­ 4fold more rapidly (Fig. 7B).-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=904--->[]--->parr Phosphorylation of S>>>e rapidly (Fig. 7B). 187 To verify that this change in activity is due to phosphorylation of topo I during mitosis, S-Topo I (Tyr723) isolated from paclitaxel-arrested K562 cells was dephosphorylated with calf intestine alkaline phosphatase and assayed for topo I enzymatic activity using serial 2-fold dilutions of the pulldowns. Fractions of pulldowns treated with buffer or calf intestine alkaline phosphatase were set aside and analyzed by immunoblotting to verify equal expression (Fig. 8A, inset). Treatment with calf intestine alkaline phosphatase caused a 2-fold decrease in activity (Fig. 8A).-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=904--->[]--->parr To verify that this >>> activity (Fig. 8A). 188 To determine whether this 2-fold decrease was related to dephosphorylation of one or more of the four phosphorylation sites mapped in this study, the activities of wild type and 4A S-Topo I (Tyr723) isolated from paclitaxel-treated K562 cells were compared. The 4A mutant exhibited 2-fold lower levels of activity with equal expression of the two constructs (Fig. 8B). Conversely 4A and wild type S-Topo I exhibited similar levels of activity when 4A had higher levels of expression (Fig. 8C). Collectively these results indicate that one or more of the sites mapped in this study enhances topo I activity when phosphorylated.-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=904--->[]--->parr To determine whether>>>when phosphorylated. 189 To identify the phosphorylation(s) responsible for the altered activity, additional topo I mutants were created and analyzed. When isolated from mitotic cells, the 3A mutant (S10A/S112A/ S394A) and wild type S-Topo I exhibited similar levels of activity (Fig. 8D), whereas the S21A mutant exhibited a 2-fold decrease in activity relative to wild type topo I (Fig. 8E). Importantly this difference in activity was not observed when wild type and S21A were pulled down from interphase cells (Fig. 8F), which lack Ser21 phosphorylation (Fig. 4B). Therefore, the decrease in relaxation activity seen in the 4A mutant versus wild type (Fig. 8B) and the wild type S-Topo I treated with calf intestine alkaline phosphatase (Fig. 8A) after isolation from mitotic cells is likely due to the absence or dephosphorylation, respectively, of Ser21. These results indicate that phosphorylation of Ser21 during mitosis stimulates the DNA relaxation activity of topo I 2-fold in vitro.-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=904--->[]--->parr To identify the phos>>>o I 2-fold in vitro. 190 To assess the possibility that the increased plasmid relaxation observed after Ser21 phosphorylation reflects increased DNA cleavage activity of the enzyme, the cleavage half-reaction was assayed using the strategy of Svejstrup et al. (40). In brief, S-Topo I pulled down from mitotic cells was incubated with radiolabeled suicide cleavage substrate as indicated in Fig. 8G. With the amounts of topo I recovered after transient transfection, progressive cleavage of the substrate was observed over 5­ 45 min. Importantly 2-fold larger amounts of S21A topo I were required to yield the same cleavage rate as wild type topo I, suggesting that Ser21 phosphorylation is enhancing the rate of DNA cleavage.-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=904--->[]--->parr To assess the possib>>>ate of DNA cleavage. 191 FIGURE 5. In vitro kinase analysis. Examination of the sequence around the phosphorylation sites (Fig. 2, A­D) tentatively identified kinases that could phosphorylate these sites. Topo I-S was pulled down from stably transfected K562 cells (Fig. 1E) and incubated with 200 M ATP and 10 units of purified CKII, 10 units of Cdk1, or 10 ng of purified PKC under conditions described under "Experimental Procedures." Alternatively purified topo I (Y723F) was incubated with 200 M ATP and 10 units of Cdk1. At the completion of the incubation, samples were solubilized in SDS sample buffer and analyzed by immunoblotting using the anti-phospho-topo I antibodies. CKII was found to phosphorylate Ser10 (A), PKC was found to phosphorylate Ser21 (B), and Cdk1 was found to phosphorylate Ser112 (C) and Ser394 (D) in vitro. Results are representative of five separate experiments. P, phosphorylated.-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[]--->capfig FIGURE 5. In vitro k>>>. P, phosphorylated. 192 Mitotic Phosphorylation of Human Topoisomerase I-->id=1, page=0, size=16, fam=Times, col=#231f20, type=title, textLines=11--->[]--->note Mitotic Phosphorylat>>>uman Topoisomerase I 193 JUNE 13, 2008 · VOLUME 283 · NUMBER 24-->id=2, page=0, size=11, fam=Times, col=#231f20, type=parrnote, textLines=67--->['U']--->note JUNE 13, 2008 · VOLU>>>LUME 283 · NUMBER 24 194 JOURNAL OF BIOLOGICAL CHEMISTRY 16717-->id=2, page=0, size=11, fam=Times, col=#231f20, type=parrnote, textLines=67--->[]--->note JOURNAL OF BIOLOGICA>>>ICAL CHEMISTRY 16717 195 at-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[u'a']--->note at>>>at 196 Centro-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[]--->note Centro>>>Centro 197 Nacional-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[]--->note Nacional>>>Nacional 198 de-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[]--->note de>>>de 199 Investigaciones-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[]--->note Investigaciones>>>Investigaciones 200 Oncológicas,-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[]--->note Oncológicas,>>>Oncológicas, 201 on-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[]--->note on>>>on 202 May-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[]--->note May>>>May 203 28,-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[]--->note 28,>>>28, 204 2010-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[]--->note 2010>>>2010 205 www.jbc.org-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[]--->note www.jbc.org>>>www.jbc.org 206 Downloaded-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[]--->note Downloaded>>>Downloaded 207 from-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[]--->note from>>>from 208 http://www.jbc.org/content/suppl/2008/04/16/M802246200.DC1.html-->id=6, page=0, size=7, fam=Times, col=#231f20, type=parrnote, textLines=45--->[]--->note http://www.jbc.org/c>>>/M802246200.DC1.html 209 Supplemental Material can be found at:-->id=6, page=0, size=7, fam=Times, col=#231f20, type=parrnote, textLines=45--->[]--->note Supplemental Materia>>>ial can be found at: 210 To determine whether the in vitro changes in topo I activity correspond to an alteration in the behavior of topo I in intact cells, band depletion assays were performed to assess CPT-induced stabilization of covalent topo I-DNA complexes (34). K562 cells transiently transfected with plasmids encoding wild type or S21A S-Topo I were treated with paclitaxel for 16 h to allow mitotic phosphorylation and then exposed briefly to varying CPT concentrations in the continued presence of paclitaxel. The S21A mutant required higher CPT concentrations than wild type S-Topo I to trap the same amount of topo I in covalent topo I-DNA complexes (Fig. 9A). In contrast, CPT stabilized similar levels of cleavage complexes involving the two constructs when K562 cells were not arrested in mitosis (Fig. 9B). Consistent with these results, comparison of diluent- versus paclitaxel-treated cells revealed that stabilization of topo I-DNA complexes required lower CPT concentrations in mitotic than in log phase cells (Fig. 9C). These results indicate that Ser21 phosphorylation concomitantly enhances topo I activity (Fig. 8) and CPT sensitivity (Fig. 9) specifically during mitosis.-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=904--->[]--->parr To determine whether>>>ally during mitosis. 211 DISCUSSION-->id=5, page=0, size=13, fam=Times, col=#231f20, type=title, textLines=9--->[]--->title DISCUSSION>>>DISCUSSION 212 Results of the present study identified four phosphorylation sites on human topo I: Ser10, Ser21, Ser112, and Ser394. Further experiments demonstrated that all four sites are phosphorylated exclusively during mitosis. CKII and PKC phosphorylated Ser10 and Ser21, respectively, and Cdk1 phosphorylated Ser112 and Ser394 in vitro. Mutation of all four sites to alanine did not alter the mitotic localization or the assayed protein-protein interactions of topo I. Comprehensive mass spectrometric analyses also demonstrated that wild type and the 4A S-topo I mutant have similar binding partners.3 However, both wild type topo I dephosphorylated by calf intestine alkaline phosphatase and the 4A mutant exhibited a 2-fold reduction in DNA relaxation activity in vitro relative to wild type topo I isolated from mitotic cells. Additional analysis determined that Ser21 phosphorylation is responsible for this change in enzymatic activity. Collectively these results suggest that phosphorylation plays a role in regulating topo I enzymatic activity and interaction with DNA during mitosis.-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=904--->[]--->parr Results of the prese>>> DNA during mitosis. 213 Mass spectrometry identified Ser10, Ser21, Ser112, and Ser394 as four sites of topo I phosphorylation in intact cells (Fig. 2 and supplemental Fig. S1). Although phosphorylation of Ser10 had been suggested previously based on the ability of CKII to phosphorylate a small peptide corresponding to the N terminus of topo I (23), the other three sites had not been identified previously. All sites identified in the present study were located on serines in agreement with previous phosphoamino acid analysis (13, 18, 21). At least two previous studies, however, suggested that topo I can also be phosphorylated on tyrosine residues. Tse-Dinh et al. (41) found that tyrosine phosphorylation of topo I in vitro can alter its enzymatic activity. More recently, Yu et al. (24) reported that c-Abl can phosphorylate topo I on Tyr268 in vitro. Our mass spectrometry analysis did not detect phosphorylation of Tyr268, although it is conceivable that the phosphorylated peptide was present in such low abundance that it could not be detected.-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=904--->[]--->parr Mass spectrometry id>>>uld not be detected. 214 3 J. S. Hackbarth and S. H. Kaufmann, unpublished observations.-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[]--->parr 3 J. S. Hackbarth an>>>lished observations. 215 FIGURE 6. Mutation of the four sites does not detectably affect localization or the assayed proteinprotein interactions of topo I. A, 24 h after transfection with plasmids encoding 4A or wild type (wt) S-topo I, log phase K562 cells were fixed with methanol and stained with anti-S peptide antibody followed by fluorescein-conjugated anti-mouse IgG and Hoechst 33258. Representative images of mitotic cells are shown. Note that the mitotic index is low because no spindle poisons were added. B, 6 h after transfection with plasmids encoding wild type (wt) or 4A S-topo I, K562 cells were treated for 16 h with 100 nM paclitaxel to induce mitotic arrest. Tagged S-topo I was isolated using S protein-agarose, subjected to SDS-PAGE, and analyzed by immunoblotting using antibodies that recognize known binding partners of topo I. Untransfected (untransf.) K562 cells were included as a negative control. Results are representative of three (A) or four (B) separate experiments. PARP, poly(ADP-ribose) polymerase; TBP, TATA-binding protein.-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[]--->capfig FIGURE 6. Mutation o>>>ATA-binding protein. 216 Mitotic Phosphorylation of Human Topoisomerase I-->id=1, page=0, size=16, fam=Times, col=#231f20, type=title, textLines=11--->[]--->note Mitotic Phosphorylat>>>uman Topoisomerase I 217 16718 JOURNAL OF BIOLOGICAL CHEMISTRY-->id=2, page=0, size=11, fam=Times, col=#231f20, type=parrnote, textLines=67--->['U']--->note 16718 JOURNAL OF BIO>>>BIOLOGICAL CHEMISTRY 218 VOLUME 283 · NUMBER 24 · JUNE 13, 2008-->id=2, page=0, size=11, fam=Times, col=#231f20, type=parrnote, textLines=67--->['U']--->note VOLUME 283 · NUMBER >>>R 24 · JUNE 13, 2008 219 at-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[u'a']--->note at>>>at 220 Centro-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[]--->note Centro>>>Centro 221 Nacional-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[]--->note Nacional>>>Nacional 222 de-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[]--->note de>>>de 223 Investigaciones-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[]--->note Investigaciones>>>Investigaciones 224 Oncológicas,-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[]--->note Oncológicas,>>>Oncológicas, 225 on-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[]--->note on>>>on 226 May-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[]--->note May>>>May 227 28,-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[]--->note 28,>>>28, 228 2010-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[]--->note 2010>>>2010 229 www.jbc.org-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[]--->note www.jbc.org>>>www.jbc.org 230 Downloaded-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[]--->note Downloaded>>>Downloaded 231 from-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[]--->note from>>>from 232 http://www.jbc.org/content/suppl/2008/04/16/M802246200.DC1.html-->id=6, page=0, size=7, fam=Times, col=#231f20, type=parrnote, textLines=45--->[]--->note http://www.jbc.org/c>>>/M802246200.DC1.html 233 Supplemental Material can be found at:-->id=6, page=0, size=7, fam=Times, col=#231f20, type=parrnote, textLines=45--->[]--->note Supplemental Materia>>>ial can be found at: 234 In further experiments we examined the kinases that can phosphorylate topo I on the sites we identified. Data base searches indicated that Ser10 and Ser21 conform to consensus phosphorylation sites for CKII and PKC, respectively. In vitro kinase assays (Fig. 5) verified this prediction, confirming and extending previous reports that CKII and PKC can phosphorylate topo I (16, 17). In addition, sequence analysis indicated that Ser112 and Ser394 are part of Ser-Pro sequences, which can be phosphorylated by Cdks or the mitogen-activated protein kinase family. Both sites were phosphorylated by Cdk1 in vitro, providing the first indication of a possible interaction between topo I and Cdk1. Cdk1 is active only during mitosis, which correlates with the finding that Ser112 and Ser394 are predominantly or exclusively phosphorylated during that phase of the cell cycle. Confirmation that each of these kinases is responsible for phosphorylating the respective sites in cells is not possible with the present technology. CKII / or Cdk1 / cell lines do not exist due to the importance of these kinases for cell cycle progression. Moreover cells treated with inhibitors or small interfering RNA targeting these kinases are unable to enter mitosis. Thus, the identification of the kinases responsible for phosphorylating topo I must be considered tentative until new tools become available.-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=904--->[]--->parr In further experimen>>>ls become available. 235 Experiments using phosphospecific topo I antibodies demonstrated that phosphorylation of all four sites was markedly increased during mitosis. These findings were observed both in cells treated with spindle poisons such as nocodazole and paclitaxel (Fig. 4A) and in untreated mitotic cells isolated by shake-off (Fig. 4B). These observations demonstrated that human topo I is phosphorylated in a cell-cycle dependent manner consistent with previous findings indicating that the murine topo I has a mitosis-specific phosphorylated form (22). Additional experiments that examined whether any of these sites are phosphorylated after various types of DNA damage (not shown) failed to demonstrate phosphorylation of these four sites after treatment with CPT or the topo II poison etoposide. In contrast, Ser10 was phosphorylated after treatment with N-methyl-N -nitro-N-nitrosoguanidine or ionizing radiation consistent with the ability of DNA damage to activate CKII (42, 43).-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=904--->[]--->parr Experiments using ph>>>ivate CKII (42, 43). 236 Phosphorylation was reported previously to alter topo I enzymatic activity in vitro. Specifically dephosphorylation reportedly decreased or abolished topo I activity (13­15), and subsequent treatment with CKII or PKC stimulated activity (14, 15). On the other hand, recombinant topo I, which has no detectable phosphorylation, was observed to be enzymatically active (44 ­ 46), casting doubt on the prior claim that phosphorylation is required for activity. Results of the present study were consistent with both sets of findings. After isolation from mitotic cells, dephosphorylation of topo I by calf intestine alkaline phosphatase caused a 2-fold decrease in plasmid relaxation activity (Fig. 8A). Because of the nonlinearity of the topo I relaxation assays, this result could potentially be interpreted as a more dramatic decrease if serial dilutions are not examined, possibly explaining earlier claims of more extensive activity loss upon dephosphorylation.-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=904--->[]--->parr Phosphorylation was >>>n dephosphorylation. 237 In further experiments, the 4A mutant exhibited the same 2-fold decrease relative to wild type enzyme after isolation from mitotic cells (Fig. 8B and C), indicating that one or more of the four mapped sites contributes to activity when phosphorylated during mitosis. Further analysis determined that Ser21 is the-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=904--->[]--->parr In further experimen>>>ed that Ser21 is the 238 FIGURE 7. DNA relaxation activity of wild type topo I from interphase and mitotic cells. A, endogenous topo I was extracted from K562 cells treated with 0.1% DMSO or 100 nM paclitaxel to induce mitotic arrest as described under "Experimental Procedures." Serial 2-fold dilutions of the extracts were subjected to immunoblotting for topo I or used in a DNA relaxation assay to examine conversion from supercoiled plasmid (SC) to relaxed forms (R). Aliquots of the most concentrated extracts (first lanes after dashed lines) contained equal amounts of topo I (inset). The "0" lane contains plasmid that was incubated without extract. Dashed lines indicate removal of intervening lanes that contained unequal amounts of topo I. The graph shows substrate remaining at the end of the 30-min incubation. Error bars, mean S.E. of three independent experiments. B, extracts adjusted to contain equal amounts of topo I polypeptide (inset) from interphase or paclitaxel-arrested K562 cells were assayed for plasmid relaxation activity over time (lanes 2­10). Lane 1, untreated substrate. The dashed line indicates juxtaposition of two separate agarose gels from the same assay. The graph shows substrate remaining on the gel at each time point and the first order regression line. Results are representative of assays using three independently derived extracts. N, location of nicked and, in some assays, relaxed plasmid.-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[]--->capfig FIGURE 7. DNA relaxa>>>ys, relaxed plasmid. 239 Mitotic Phosphorylation of Human Topoisomerase I-->id=1, page=0, size=16, fam=Times, col=#231f20, type=title, textLines=11--->[]--->note Mitotic Phosphorylat>>>uman Topoisomerase I 240 JUNE 13, 2008 · VOLUME 283 · NUMBER 24-->id=2, page=0, size=11, fam=Times, col=#231f20, type=parrnote, textLines=67--->['U']--->note JUNE 13, 2008 · VOLU>>>LUME 283 · NUMBER 24 241 JOURNAL OF BIOLOGICAL CHEMISTRY 16719-->id=2, page=0, size=11, fam=Times, col=#231f20, type=parrnote, textLines=67--->[]--->note JOURNAL OF BIOLOGICA>>>ICAL CHEMISTRY 16719 242 at-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[u'a']--->note at>>>at 243 Centro-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[]--->note Centro>>>Centro 244 Nacional-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[]--->note Nacional>>>Nacional 245 de-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[]--->note de>>>de 246 Investigaciones-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[]--->note Investigaciones>>>Investigaciones 247 Oncológicas,-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[]--->note Oncológicas,>>>Oncológicas, 248 on-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[]--->note on>>>on 249 May-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[]--->note May>>>May 250 28,-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[]--->note 28,>>>28, 251 2010-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[]--->note 2010>>>2010 252 www.jbc.org-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[]--->note www.jbc.org>>>www.jbc.org 253 Downloaded-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[]--->note Downloaded>>>Downloaded 254 from-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[]--->note from>>>from 255 http://www.jbc.org/content/suppl/2008/04/16/M802246200.DC1.html-->id=6, page=0, size=7, fam=Times, col=#231f20, type=parrnote, textLines=45--->[]--->note http://www.jbc.org/c>>>/M802246200.DC1.html 256 Supplemental Material can be found at:-->id=6, page=0, size=7, fam=Times, col=#231f20, type=parrnote, textLines=45--->[]--->note Supplemental Materia>>>ial can be found at: 257 FIGURE 8. Effect of Ser21 phosphorylation on topo I activity. A, K562 cells were transiently transfected with wild type (wt) S-topo I (Tyr723) and arrested in mitosis with 100 nM paclitaxel. S protein-agarose precipitates were treated with buffer or 10 units of calf intestine alkaline phosphatase (CIAP)as described under "Experimental Procedures." The beads were subjected to 2-fold dilutions in topo I assay buffer and used in a DNA relaxation assay. Aliquots of the immobilized topo I were also subjected to immunoblotting with anti-S peptide antibody to verify equal loading (see insets). The inset contains lanes from a single film exposure. B­E, wild type and 4A S-Topo I (B and C), wild type and 3A S-Topo I (D), or wild type and S21A S-Topo I (E) were isolated from paclitaxel-treated K562 cells and assayed as described in A. F, wild type and S21A S-Topo I were isolated from interphase K562 cells and assayed as described in A. Because of variability in transfection efficiency, experiments shown in A­F were conducted separately and cannot be directly compared. G, wild type and S21A S-Topo I isolated from paclitaxel-treated K562 cells were incubated with radiolabeled suicide substrate (inset) for the indicated length of time. *, radiolabeled nucleotide in substrate. At the completion of the reaction, the 21-mer substrate and 7-mer product were separated and visualized by phosphorimaging. Two separate gels from a single assay were imaged simultaneously. Inset, S peptide blot showing corresponding topo I contents of the pulldowns. Dashes indicate removal of extraneous lanes. Results are representative of three (A), 14 (B and C), four (D), three (E), five (F), and three (G) assays. SC, supercoiled; R, relaxed; N, location of nicked and, in some assays, relaxed plasmid.-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[]--->capfig FIGURE 8. Effect of >>>ys, relaxed plasmid. 258 Mitotic Phosphorylation of Human Topoisomerase I-->id=1, page=0, size=16, fam=Times, col=#231f20, type=title, textLines=11--->[]--->note Mitotic Phosphorylat>>>uman Topoisomerase I 259 16720 JOURNAL OF BIOLOGICAL CHEMISTRY-->id=2, page=0, size=11, fam=Times, col=#231f20, type=parrnote, textLines=67--->['U']--->note 16720 JOURNAL OF BIO>>>BIOLOGICAL CHEMISTRY 260 VOLUME 283 · NUMBER 24 · JUNE 13, 2008-->id=2, page=0, size=11, fam=Times, col=#231f20, type=parrnote, textLines=67--->['U']--->note VOLUME 283 · NUMBER >>>R 24 · JUNE 13, 2008 261 at-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[u'a']--->note at>>>at 262 Centro-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[]--->note Centro>>>Centro 263 Nacional-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[]--->note Nacional>>>Nacional 264 de-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[]--->note de>>>de 265 Investigaciones-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[]--->note Investigaciones>>>Investigaciones 266 Oncológicas,-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[]--->note Oncológicas,>>>Oncológicas, 267 on-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[]--->note on>>>on 268 May-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[]--->note May>>>May 269 28,-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[]--->note 28,>>>28, 270 2010-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[]--->note 2010>>>2010 271 www.jbc.org-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[]--->note www.jbc.org>>>www.jbc.org 272 Downloaded-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[]--->note Downloaded>>>Downloaded 273 from-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[]--->note from>>>from 274 http://www.jbc.org/content/suppl/2008/04/16/M802246200.DC1.html-->id=6, page=0, size=7, fam=Times, col=#231f20, type=parrnote, textLines=45--->[]--->note http://www.jbc.org/c>>>/M802246200.DC1.html 275 Supplemental Material can be found at:-->id=6, page=0, size=7, fam=Times, col=#231f20, type=parrnote, textLines=45--->[]--->note Supplemental Materia>>>ial can be found at: 276 site that enhances activity when phosphorylated. A Ser21Ala mutant exhibited decreased ability to relax supercoiled plasmid (Fig. 8E) or cleave suicide substrate (Fig. 8G) in vitro as well as decreased CPT-induced cleavage complexes in mitotic cells (Fig. 9A) compared with wild type topo I. Importantly, these differences were not observed in interphase cells (Figs. 8F and 9B), where Ser21 is not phosphorylated (Fig. 4B).-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=904--->[]--->parr site that enhances a>>>horylated (Fig. 4B). 277 Additional observations suggest that the effect of Ser21 phosphorylation is not limited to cells transfected with epitopetagged topo I. Extracts from untransfected cells exhibited an increase in topo I activity during mitosis (Fig. 7, A and B) and enhanced sensitivity to CPT-induced stabilization of cleavage complexes (Fig. 9C) compared with interphase cells. Although the ability of phosphorylation in the N-terminal domain to affect events at the topo I active site might seem counterintuitive, previous studies have demonstrated that removal of the N-terminal domain modestly decreases topo I enzymatic activity and CPT sensitivity (47, 48). Crystallization of topo I with an intact N terminus, a feat that has not been reported to date, appears to be required to determine whether phosphorylation of Ser21 enhances activity and CPT sensitivity through an interaction with portions of the N-terminal domain previously implicated in topo I activation or through interaction with different regions of the polypeptide.-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=904--->[]--->parr Additional observati>>> of the polypeptide. 278 The results presented in this study reveal interesting similarities between the phosphorylation of topo I and topo II (49, 50), a structurally and mechanistically unrelated member of the topoisomerase family. Topo II is phosphorylated at numerous serine and threonine sites in cells and is phosphorylated by CKII, PKC, and Cdk1 in vitro. In addition, topo II has sites that are phosphorylated specifically during mitosis (51), and topo II activity peaks during mitosis (52, 53), presumably to facilitate chromosome separation. Although numerous studies have examined whether there is a link between topo II phosphorylation and its enzymatic activity, the results are conflicting especially when compared between species (54 ­58). The present study demonstrates that topo I activity likewise increases during mitosis, indicating that topo I might also participate in some way during that phase of the cell cycle.-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=904--->[]--->parr The results presente>>>e of the cell cycle. 279 In summary, the present study located and examined the four sites of endogenous phosphorylation on human topo I. All of these sites were phosphorylated predominantly during mitosis, demonstrating that topo I is phosphorylated in a cell cycle-dependent manner. Two of these sites, Ser112 and Ser394, were phosphorylated in vitro by Cdk1, providing the first indication that a Cdk can modify topo I. Although the phosphorylation of these four sites did not detectably alter topo I localization or protein-protein interactions during mitosis, Ser21 phosphorylation enhanced topo I relaxation activity in vitro and CPT-induced stabilization of cleavage complexes in cells. These observations provide new understanding of the circumstances and effect of topo I phosphorylation in cells.-->id=10, page=0, size=12, fam=Times, col=#231f20, type=parr, textLines=904--->[]--->parr In summary, the pres>>>horylation in cells. 280 Acknowledgments--We gratefully acknowledge gifts of the topo I plasmid from M.-A. Bjornsti; antibodies from Guy Poirier, Naomi Rothfield, and Yung-Chi Cheng; assistance and analysis from Ross Tomaino and the Taplin Biological Mass Spectrometry Facility as well as the Flow Cytometry and Optical Morphology Shared Resource and the Peptide Synthesis Core at the Mayo Clinic; technical advice from L. James Maher and Robert McDonald; and editorial assistance from Deb Strauss.-->id=2, page=0, size=11, fam=Times, col=#231f20, type=parrnote, textLines=67--->[]--->parr Acknowledgments--We >>>ce from Deb Strauss. 281 FIGURE 9. The S21A topo I mutant is less sensitive to CPT-induced trapping on DNA in intact mitotic cells. K562 cells transfected with plasmids encoding wild type (wt) or S21A S-topo I (Tyr723) were treated with 100 nM paclitaxel for 16 h (A) or left untreated (B) before subjecting cells to a band depletion assay to assess CPTinduced stabilization of topo I-DNA complexes as described under "Experimental Procedures." The blots were probed with anti-S peptide antibody to specifically detect transfected constructs. C, K562 cells were treated with 100 nM paclitaxel or diluent for 16 h and then subjected to band depletion assay. Samples were subjected to SDS-PAGE, immunoblotting with anti-topo I to detect endogenous polypeptide, and densitometry using ImageJ. Dashes indicate removal of extraneous lanes. Error bars, mean S.E. of three (A), three (B), and five (C) experiments, respectively. PARP, poly(ADP-ribose) polymerase.-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[]--->capfig FIGURE 9. The S21A t>>>-ribose) polymerase. 282 Mitotic Phosphorylation of Human Topoisomerase I-->id=1, page=0, size=16, fam=Times, col=#231f20, type=title, textLines=11--->[]--->note Mitotic Phosphorylat>>>uman Topoisomerase I 283 JUNE 13, 2008 · VOLUME 283 · NUMBER 24-->id=2, page=0, size=11, fam=Times, col=#231f20, type=parrnote, textLines=67--->['U']--->note JUNE 13, 2008 · VOLU>>>LUME 283 · NUMBER 24 284 JOURNAL OF BIOLOGICAL CHEMISTRY 16721-->id=2, page=0, size=11, fam=Times, col=#231f20, type=parrnote, textLines=67--->[]--->note JOURNAL OF BIOLOGICA>>>ICAL CHEMISTRY 16721 285 at-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[u'a']--->note at>>>at 286 Centro-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[]--->note Centro>>>Centro 287 Nacional-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[]--->note Nacional>>>Nacional 288 de-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[]--->note de>>>de 289 Investigaciones-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[]--->note Investigaciones>>>Investigaciones 290 Oncológicas,-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[]--->note Oncológicas,>>>Oncológicas, 291 on-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[]--->note on>>>on 292 May-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[]--->note May>>>May 293 28,-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[]--->note 28,>>>28, 294 2010-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[]--->note 2010>>>2010 295 www.jbc.org-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[]--->note www.jbc.org>>>www.jbc.org 296 Downloaded-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[]--->note Downloaded>>>Downloaded 297 from-->id=4, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=424--->[]--->note from>>>from 298 http://www.jbc.org/content/suppl/2008/04/16/M802246200.DC1.html-->id=6, page=0, size=7, fam=Times, col=#231f20, type=parrnote, textLines=45--->[]--->note http://www.jbc.org/c>>>/M802246200.DC1.html 299 Supplemental Material can be found at:-->id=6, page=0, size=7, fam=Times, col=#231f20, type=parrnote, textLines=45--->[]--->note Supplemental Materia>>>ial can be found at: 300 REFERENCES-->id=2, page=0, size=11, fam=Times, col=#231f20, type=parrnote, textLines=67--->[]--->parr REFERENCES>>>REFERENCES 301 1. 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