Cell Cycle-Specific and Transcription-Related Phosphorylation of Mammalian Topoisomerase I

Liu, Leroy F.

doi:10.1006/excr.1995.1071

Cited by 29 publications

(20 citation statements)

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“…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.…”

Section: Discussionsupporting

confidence: 89%

“…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).…”

Section: Topo I Is Phosphorylated In Cells-when Topo I Was Immunoprecsupporting

confidence: 93%

“…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.…”

Section: Human Topo Imentioning

confidence: 86%

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Mitotic Phosphorylation Stimulates DNA Relaxation Activity of Human Topoisomerase I

Hackbarth¹,

Gálvez-Peralta

Dai

et al. 2008

Journal of Biological Chemistry

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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. and Ser 394 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 Ser 21 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. 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 Tyr 723 of the enzyme to the 3Ј 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 Tyr 723 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).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 (13-15). 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 2-3-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.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 immunopreci...

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Section: Discussionsupporting

confidence: 89%

“…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).…”

Section: Topo I Is Phosphorylated In Cells-when Topo I Was Immunoprecsupporting

confidence: 93%

Section: Human Topo Imentioning

confidence: 86%

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Mitotic Phosphorylation Stimulates DNA Relaxation Activity of Human Topoisomerase I

Hackbarth¹,

Gálvez-Peralta

Dai

et al. 2008

Journal of Biological Chemistry

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“…For analysis of cleavable complexes by the band depletion method, CPTtreated cells were pelleted and lysed immediately with SDS-PAGE sample buffer. Immunoprecipitation of TOP1 was as described (17), except nuclei were prepared in the presence of 5 mM N-ethymaleimide for 15 min on ice, followed by the addition of 10 mM cysteine. In some experiments (see Fig.…”

Section: Methodsmentioning

confidence: 99%

“…This covalent TOP1-DNA cleavable product has slower mobility during SDS-PAGE compared with free TOP1 (20). In CHO cells exposed to 25 M CPT and immediately lysed with SDS, more than 90% of the TOP1 disappeared from its location at 100 kDa on Western blots where it is present as two differently phosphorylated forms (17), and it coincidentally reappeared as a higher molecular mass smear (Fig. 1).…”

Section: Multi-ub Conjugates Of Top1 In Cells Treated With Cpt-tomentioning

confidence: 99%

Ubiquitin-dependent Destruction of Topoisomerase I Is Stimulated by the Antitumor Drug Camptothecin

Desai¹,

Liu²,

Vázquez‐Abad³

et al. 1997

Journal of Biological Chemistry

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234

228

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Topoisomerase I (TOP1) relaxes superhelical DNA through a breakage/rejoining reaction in which the active site tyrosine links covalently to a 3 phosphate at the break site as a transient intermediate. The antitumor drug camptothecin (CPT) and its analogs inhibit the rejoining step of the breakage/rejoining reaction, which traps the enzyme in covalent linkage with DNA (the cleavable complex). Little is known about the fate of cellular TOP1 trapped in the cleavable complex. We have analyzed TOP1 in mammalian cell lines treated with CPT. When CPT-treated cells were lysed with either SDS or alkali and analyzed by Western blotting, greater than 90% of the TOP1 was linked to DNA. Nuclease treatment of the cell lysate to remove the covalently linked DNA from TOP1 revealed a distinct ladder of higher molecular weight bands having properties indicative of multi-ubiquitin (Ub) conjugates of TOP1. Approximately 5-10% of TOP1 was present as these conjugates within minutes of CPT treatment. Consistent with ubiquitination, TOP1 was not modified in ts85 cells at the restrictive temperature for its thermolabile ubiquitin-activating enzyme (E1). Because conjugation with ubiquitin can mark proteins for destruction by the 26S proteasome, we analyzed TOP1 protein levels during prolonged CPT treatment. TOP1 protein levels were reduced to about 25% during CPT treatments of 2-4 h resulting from increased destruction, with the half-life dropping from 10 -16 h down to 1-2 h. The destruction of TOP1, like the formation of Ub-TOP1 conjugates, was not observed in ts85 cells at the restrictive temperature. The destruction of TOP1 was also prevented in cells treated with MG-132 and lactacystin, specific inhibitors of the 26S proteasome. Finally, the multi-Ub conjugates of TOP1 were observed whether or not aphidicolin was included in cotreatment with CPT, indicating that replication fork activity was not involved in making TOP1 a substrate for ubiquitination. These results demonstrate that independent of DNA replication, the TOP1 cleavable complex is ubiquitinated and destroyed in cells treated with antitumor drugs that block the religation step of the TOP1 reaction. DNA topoisomerase I (TOP1)1 is a vital DNA metabolic enzyme as well as a molecular target of antitumor drugs. TOP1 relaxes DNA supercoils by cleaving a single strand of duplex DNA and passing the complimentary DNA strand though the cleaved strand before religation (1). In a transient reaction intermediate, termed the cleavable complex, the enzyme links covalently to DNA through tyrosine 723 (human TOP1) leaving a DNA break having a free 5Ј hydroxyl end. Stopping the TOP1 reaction with strong protein denaturants produces a very low yield of TOP1 linked covalently to DNA. The rarity of this product is a reflection of the transient nature of the "cleaved" reaction intermediate. Antitumor drugs that act on TOP1 inhibit the DNA religation step through a reversible mechanism. The inhibition of religation can be observed as an increase in the yield of the enzyme-DNA covalent complex pro...

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Phosphotyrosyl protein phosphatase from the liver ofTilapia mossambica: Activating the Src-phosphorylated DNA topoisomerase I

Liu

Chuang

1997

J. Exp. Zool.

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Phosphotyrosyl protein phosphatase was purified from the liver of the Tilapia mossambica. Specific activity of the final purified preparation was 5,095 U/mg of protein with the 32P‐peptide 1142–1153 of the insulin receptor as substrate. SDS‐polyacrylamide gel electrophoresis revealed that monomers of the phosphotyrosyl protein phosphatase have relative masses of 67,000 ± 500 and 60,000 ± 500. Since the active phosphotyrosyl protein phosphatase was found to have a relative mass of 62,000 ± 1,000, the purified enzyme was concluded to be monomeric. The phosphotyrosyl protein phosphatase had an isoelectric point of 5.79 ± 0.02 and an optimal pH of 7.0. Incubation of the purified enzyme fraction with Src‐phosphorylated DNA topoisomerase I resulted in an increase activity of DNA topoisomerase I in a dose‐dependent manner. Activity of phosphotyrosyl protein phosphatase is inhibited by MnCl2 and vanadate at μmolar concentrations, but less inhibition was observed with CaCl2 and MgCl2. J. Exp. Zool. 277:14–22, 1997. © 1997 Wiley‐Liss, Inc.

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Cell Cycle-Specific and Transcription-Related Phosphorylation of Mammalian Topoisomerase I

Cited by 29 publications

References 0 publications

Mitotic Phosphorylation Stimulates DNA Relaxation Activity of Human Topoisomerase I

Mitotic Phosphorylation Stimulates DNA Relaxation Activity of Human Topoisomerase I

Ubiquitin-dependent Destruction of Topoisomerase I Is Stimulated by the Antitumor Drug Camptothecin

Phosphotyrosyl protein phosphatase from the liver ofTilapia mossambica: Activating the Src-phosphorylated DNA topoisomerase I

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