Threonine 1342 in Human Topoisomerase IIα Is Phosphorylated Throughout the Cell Cycle

Abstract

To investigate the relationship between the modulation of topoisomerase II activity and its phosphorylation state during the cell cycle, a monoclonal antibody against C-terminal peptide (residues 1335-1350) of topoisomerase II␣ containing a consensus sequence of casein kinase II, TDDE and its phosphorylated threonine were prepared. In an enzyme-linked immunosorbent assay, the antibody, named PT1342, recognized the immunogenic phosphopeptide but not the non-phosphorylated form of the peptide. The PT1342 antibody reacted only with a 170-kDa protein from HeLa cells and recognized anti-topoisomerase II␣ immunoprecipitants. Furthermore, the antibody did not react with the human topoisomerase II␣ mutated at codon 1342 from threonine to alanine, showing that PT1342 was directed against the phosphorylated threonine 1342. To examine the level of phosphorylation of threonine 1342 of topoisomerase II␣ through the cell cycle, HeLa cells were stained simultaneously for phosphorylated topoisomerase II␣ and DNA and analyzed by flow cytometry. Cells in the G 2 -M phase contained about double the PT1341-reacted topoisomerase II␣ than did cells in G 1 or S phases. The antibody stained the nuclei in interphase and mitotic chromosomes and its periphery, as seen with anti-topoisomerase II␣ antibody. Thus, threonine 1342 in topoisomerase II␣ is phosphorylated throughout the cell cycle.DNA topoisomerases are enzymes that play an important role in DNA replication and transcription by relieving torsional or interlocking constraints of DNA accumulating during processes of macromolecular syntheses (1-3). Topoisomerases I and II relax supercoiled DNA through transient single-and double-strand breaks, respectively, and topoisomerase II unknots or decatenates the knotted or catenated DNA. The latter activity of topoisomerase II is presumably related to chromosome dynamics in mitosis, where topoisomerase II is absolutely required (4 -7). In addition to catalytic activity, topoisomerase II may function to anchor chromosomal DNA loops to the nuclear scaffold (8, 9); topoisomerase II is a major non-histone protein present in nuclear scaffold fractions (10, 11), and DNA sequences that bind preferentially to the nuclear matrix/chromosomal scaffold (MAR/SAR) contain topoisomerase II cleavage consensus (12). In contrast, the role of topoisomerase II in the maintenance of chromosomes was not supported in studies done using Xenopus egg extracts or Xenopus embryos (13, 14). Topoisomerase II has been considered as one of the targets for anticancer drugs (15). Topoisomerase II inhibitors, such as etoposide or 4Ј-(9-acridinylamino) methane sulfon-m-anisidide, stabilize a catalytic reaction intermediate termed " cleavable complex" in which the enzyme binds covalently to 5Ј-phosphoryl termini of broken DNA (15). There are two known isoforms of topoisomerase II in mammalian cells, topoisomerase II␣ and II␤. They have molecular masses of 170 kDa and 180 kDa, respectively. These isoforms are mapped to different genes on chromosomes 17 and 3 (16), respectively,...

Interactions between Protein Kinase CK2 and Pin1

“…In these yeast, topoisomerase II is hypo-phosphorylated and inactive, suggesting that it is a bona fide physiological target for CK2. Additionally, it was recently demonstrated in mammalian cells that CK2 phosphorylates residues on topoisomerase II␣, including Thr-1342, which are residues that are maximally phosphorylated in mitotic cells (32)(33)(34). In this study, we demonstrated that Pin1 inhibits the in vitro phosphorylation of Thr-1342 on topoisomerase II␣ by CK2 and that Pin1 interacts with topoisomerase II␣.…”

“…Incorporation of 32 P into Topoisomerase II-The wild-type Y2C fragment of topoisomerase II␣ and the mutant form with the Thr-1342 to alanine substitution T1342A were produced in bacteria and then purified and blotted to polyvinylidene difluoride membrane as described above (33). The membranes were blocked in 5% BSA in TBST.…”

“…Alternatively, these kinase assays were performed using casein or GST fusion proteins encoding known protein substrates of CK2 in the presence of the indicated amounts of Pin1. For the latter assays, kinase assays were performed in buffer containing 50 mM Tris, pH 7.5, 10 mM MgCl 2 , 100 mM NaCl, 0.2 mM DTT, 100 M [␥- 32 P]ATP (specific activity 400 -1000 cpm/pmol) using each of the indicated proteins as substrate. Following incubation for 10 min at 30°C, kinase reactions were terminated by the additional SDS-PAGE sample buffer and boiling.…”

“…A and B, 32 P-labeled GST-CK2␣-C126 was examined for interactions with Histagged Pin1 that had been immobilized on Ni-NTA. 32 P-Labeled GST fusion protein that was retained on His-Pin1 was visualized with a PhosphorImager following electrophoretic separation. The input lanes represent the phosphorylated 32 P-labeled GST-CK2␣-C126 fusion proteins that were utilized for these interactions.…”

Mitotic Phosphorylation of DNA Topoisomerase II α by Protein Kinase CK2 Creates the MPM-2 Phosphoepitope on Ser-1469

“…Residue numbers are taken from Medline files. A third phosphorylation site has been described for Thr-1343 (44). Recent studies report that the MPM-2 epitope on topoisomerase II␣ is present on Thr-666 whereas the 3F3/2 epitope corresponds to Thr-1343 (15,45).…”