ABSTRACT
Topoisomerase (topo) II catalyzes topological changes in DNA. Although both human isozymes, topo IIα and ß are phosphorylated, site-specific phosphorylation of topo IIß is poorly characterized. Using LC-MS/MS analysis of topo IIß, cleaved with trypsin, Arg C or cyanogen bromide (CNBr) plus trypsin, we detected four +80-Da modified sites: tyr656, ser1395, thr1426 and ser1545. Phosphorylation at ser1395, thr1426 and ser1545 was established based on neutral loss of H(3) PO(4) (-98 Da) in the CID spectra and on differences in 2-D-phosphopeptide maps of (32) P-labeled wild-type (WT) and S1395A or T1426A/S1545A mutant topo IIß. However, phosphorylation at tyr656 could not be verified by 2-D-phosphopeptide mapping of (32) P-labeled WT and Y656F mutant protein or by Western blotting with phosphotyrosine-specific antibodies. Since the +80-Da modification on tyr656 was observed exclusively during cleavage with CNBr and trypsin, this modification likely represented bromination, which occurred during CNBr cleavage. Re-evaluation of the CID spectra identified +78/+80-Da fragment ions in CID spectra of two peptides containing tyr656 and tyr711, confirming bromination. Interestingly, mutation of only tyr656, but not ser1395, thr1326 or ser1545, decreased topo IIß activity, suggesting a functional role for tyr656. These results, while identifying an important tyrosine in topo IIß, underscore the importance of careful interpretation of modifications having the same nominal mass.
Subject(s)
Artifacts , DNA Topoisomerases, Type II/metabolism , DNA-Binding Proteins/metabolism , Isoenzymes/metabolism , Tyrosine/metabolism , Antibodies, Phospho-Specific/metabolism , Biocatalysis , Blotting, Western , Circular Dichroism , Cyanogen Bromide/chemistry , DNA/metabolism , DNA Topoisomerases, Type II/genetics , DNA-Binding Proteins/genetics , HL-60 Cells , Halogenation , Humans , Isoenzymes/genetics , Models, Molecular , Mutation , Phosphorylation , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Saccharomyces cerevisiae , Serine/genetics , Serine/metabolism , Threonine/genetics , Threonine/metabolism , Trypsin/metabolism , Tyrosine/geneticsABSTRACT
We previously reported that phosphorylation of topoisomerase (topo) IIalpha at serine-1106 (Ser-1106) regulates enzyme activity and sensitivity to topo II-targeted drugs. In this study we demonstrate that phosphorylation of Ser-1106, which is flanked by acidic amino acids, is regulated in vivo by casein kinase (CK) Idelta and/or CKIepsilon, but not by CKII. The CKI inhibitors, CKI-7 and IC261, reduced Ser-1106 phosphorylation and decreased formation of etoposide-stabilized topo II-DNA cleavable complex. In contrast, the CKII inhibitor, 5,6-dichlorobenzimidazole riboside, did not affect etoposide-stabilized topo II-DNA cleavable complex formation. Since, IC261 specifically targets the Ca(2+)-regulated isozymes, CKIdelta and CKIepsilon, we examined the effect of down-regulating these enzymes on Ser-1106 phosphorylation. Down-regulation of these isozymes with targeted si-RNAs led to hypophosphorylation of the Ser-1106 containing peptide. However, si-RNA-mediated down-regulation of CKIIalpha and alpha' did not alter Ser-1106 phosphorylation. Furthermore, reduced phosphorylation of Ser-1106, observed in HRR25 (CKIdelta/epsilon homologous gene)-deleted Saccharomyces cerevisiae cells transformed with human topo IIalpha, was enhanced following expression of human CKIepsilon. Down-regulation of CKIdelta and CKIepsilon also led to reduced formation of etoposide stabilized topo II-DNA cleavable complex. These results provide strong support for an essential role of CKIdelta/epsilon in phosphorylating Ser-1106 in human topo IIalpha and in regulating enzyme function.
Subject(s)
Antigens, Neoplasm/metabolism , Casein Kinase 1 epsilon/metabolism , Casein Kinase Idelta/metabolism , DNA Cleavage , DNA Topoisomerases, Type II/metabolism , DNA-Binding Proteins/metabolism , Serine/metabolism , Antigens, Neoplasm/chemistry , Casein Kinase 1 epsilon/antagonists & inhibitors , Casein Kinase 1 epsilon/genetics , Casein Kinase I/genetics , Casein Kinase Idelta/antagonists & inhibitors , DNA Topoisomerases, Type II/chemistry , DNA-Binding Proteins/chemistry , Down-Regulation , Etoposide/pharmacology , HL-60 Cells , Humans , Peptides/chemistry , Peptides/metabolism , Phosphorylation , Protein Kinase Inhibitors/pharmacology , RNA Interference , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/genetics , Transformation, GeneticABSTRACT
A simple, rapid, and sensitive analytical method for the measurement of docetaxel in human plasma was developed and validated. The method is based on positive electrospray ionization tandem mass spectrometry (ESI+-MS-MS) with on-line sample extraction. It uses paclitaxel as internal standard for calibration. The on-line sample extraction minimizes sample handling and is readily adopted for automation. Quantitation of plasma docetaxel was done by the multiple reaction monitoring (MRM) mode. The method had a linear calibration range of 1.00-3000 ng/mL with a correlation coefficient >0.9999. The limit of quantitation (LOQ) for docetaxel in plasma was 1.00 ng/mL. The on-line extraction recovery of docetaxel was between 86.1-94.7%, with %CV < or = 6.1%. This method has high accuracy (90.1-96.3%), and excellent intra-assay (0.6-3.8%) and inter-assay (2.0-5.7%) precision. Its applicability to clinical samples was demonstrated by measuring patient plasma samples after treatment of weekly docetaxel at 25 mg/m2 as 60-min infusion.
