RESUMEN
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.
Asunto(s)
Artefactos , ADN-Topoisomerasas de Tipo II/metabolismo , Proteínas de Unión al ADN/metabolismo , Isoenzimas/metabolismo , Tirosina/metabolismo , Anticuerpos Fosfo-Específicos/metabolismo , Biocatálisis , Western Blotting , Dicroismo Circular , Bromuro de Cianógeno/química , ADN/metabolismo , ADN-Topoisomerasas de Tipo II/genética , Proteínas de Unión al ADN/genética , Células HL-60 , Halogenación , Humanos , Isoenzimas/genética , Modelos Moleculares , Mutación , Fosforilación , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae , Serina/genética , Serina/metabolismo , Treonina/genética , Treonina/metabolismo , Tripsina/metabolismo , Tirosina/genéticaRESUMEN
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.
Asunto(s)
Antígenos de Neoplasias/metabolismo , Caseína Cinasa 1 épsilon/metabolismo , Quinasa Idelta de la Caseína/metabolismo , División del ADN , ADN-Topoisomerasas de Tipo II/metabolismo , Proteínas de Unión al ADN/metabolismo , Serina/metabolismo , Antígenos de Neoplasias/química , Caseína Cinasa 1 épsilon/antagonistas & inhibidores , Caseína Cinasa 1 épsilon/genética , Quinasa de la Caseína I/genética , Quinasa Idelta de la Caseína/antagonistas & inhibidores , ADN-Topoisomerasas de Tipo II/química , Proteínas de Unión al ADN/química , Regulación hacia Abajo , Etopósido/farmacología , Células HL-60 , Humanos , Péptidos/química , Péptidos/metabolismo , Fosforilación , Inhibidores de Proteínas Quinasas/farmacología , Interferencia de ARN , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Transformación GenéticaRESUMEN
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.