DNA topoisomerase II enzymes regulate essential cellular processes by altering the topology of chromosomal DNA. These enzymes function by creating transient double-stranded breaks in the DNA molecule that allow the DNA strands to pass through each other and unwind or unknot tangled DNA. Because of the integral role of topoisomerases in regulating DNA metabolism, these enzymes are vital for cell survival. Several clinically active antitumor agents target these enzymes. Mammalian cells contain two topoisomerase II isozymes that are encoded by different genes: topoisomerase IIα and IIβ. Although, both isozymes are homologous and exhibit similar catalytic activity, they are differentially regulated and are involved in distinct biological functions. The topoisomerase IIα and topoisomerase IIβ enzymes are regulated by post-translational modifications, including sumoylation, ubiquitination and phosphorylation. These post-translational modifications influence the biologic and catalytic activity of the enzyme and affect sensitivity of cells to topoisomerase II-targeted drugs. In this review, we describe how the catalytic and biologic activity of the topoisomerase II enzyme is regulated and discuss the mechanisms by which chemotherapeutic agents that target these enzymes function. Given the potential importance of site-specific modifications, in particular phosphorylation, in regulating sensitivity to topoisomerase II-targeted drugs, we discuss the potential role of altered topoisomerase II phosphorylation in development of drug resistance, which is often a limiting factor in the treatment of cancer.
We previously reported that phosphorylation of topoisomerase (topo) IIα 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) Iδ and/or CKIɛ, 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 Ca2+-regulated isozymes, CKIδ and CKIɛ, 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 CKIIα and α′ did not alter Ser-1106 phosphorylation. Furthermore, reduced phosphorylation of Ser-1106, observed in HRR25 (CKIδ/ɛ homologous gene)-deleted Saccharomyces cerevisiae cells transformed with human topo IIα, was enhanced following expression of human CKIɛ. Down-regulation of CKIδ and CKIɛ also led to reduced formation of etoposide stabilized topo II–DNA cleavable complex. These results provide strong support for an essential role of CKIδ/ɛ in phosphorylating Ser-1106 in human topo IIα and in regulating enzyme function.
Topoisomerase (topo) II catalyzes topological changes in DNA. Although both human isozymes, topo IIα and β are phosphorylated, site-specific phosphorylation of topo IIβ is poorly characterized. Using LC-MS/MS analysis of topo IIβ, 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 H3PO4 (−98 Da) in the CID spectra and on differences in 2-D-phosphopeptide maps of 32P-labeled wild-type (WT) and S1395A or T1426A/S1545A mutant topo IIβ. However, phosphorylation at tyr656 could not be verified by 2-D-phosphopeptide mapping of 32P-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 IIβ activity, suggesting a functional role for tyr656. These results, while identifying an important tyrosine in topo IIβ, underscore the importance of careful interpretation of modifications having the same nominal mass.
Topoisomerase (topo) II catalyzes topological changes in DNA. Although, both human isozymes, topo IIα and β, are phosphorylated, site-specific phosphorylation of topo IIβ is poorly characterized. Using LC-MS/MS analysis of topo IIβ, cleaved with trypsin, Arg C or cyanogen bromide (CNBr) plus trypsin we detected four +80-Da modified sites, tyrosine 656, serine 1395, threonine 1426 and serine 1545. Phosphorylation at serine 1395, threonine 1426 and serine 1545 was established based on neutral loss of H3PO4 (−98 Da) in the collision-induced dissociation (CID) spectra and on differences in 2D-phosphopeptide maps of 32P-labeled wild type (WT) and serine1395alanine or threonine1426alanine/serine1545alanine mutant topo IIβ. However, phosphorylation at tyrosine 656 could not be verified by 2D-phosphopeptide mapping of 32P-labeled WT and tyrosine656phenylalanine mutant protein or by Western blotting with phosphotyrosine-specific antibodies. Since the +80-Da modification on tyrosine 656 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 (characteristic of bromination) in CID spectra of two peptides containing tyrosine 656 and tyrosine 711, confirming bromination. Interestingly, mutation of only tyrosine 656, but not serine 1395, threonine 1426 or serine 1545, decreased topo IIβ activity suggesting a functional role for tyrosine 656. Homology modeling of the region of topo IIβ (amino acids 449-1108) containing these two reactive tyrosine residues revealed that tyrosine 711 is buried, whereas tyrosine 656 is highly exposed on the surface. This region is within, or very close to the dimerization interface, which suggests that these amino acids may be critical for topo IIβ dimer formation. These results, while identifying an important tyrosine in topo IIβ, underscore the importance of careful interpretation of modifications having the same nominal mass. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2526. doi:10.1158/1538-7445.AM2011-2526
Targeted therapy with multiple receptor tyrosine kinase inhibitors (RTKI) has led to a substantial improvement in the standard of care for patients with advanced or metastatic clear cell renal cell carcinoma (CCRCC). We have previously reported that using a phosphoproteomics approach, Ras and Rab interactor 1 (RIN1) was identified as a potential target modulating the antiproliferative effects of sorafenib and sunitinib in a panel of human CCRCC (Proc. AACR 51: Abst. #1626, 2010). While both 10 μM sorafenib and 7.5 μM sunitinib were effective in down-regulating RIN1 protein levels in CCRCC following treatment for 48h, during subsequent recovery for 48h in drug free medium, recovery of RIN1 protein levels was observed in sorafenib but not sunitinib treated cells. mRNA expression profiling revealed that: (a) VEGF-A and HIF-1α levels were unaffected during 48h treatment with sorafenib or sunitinib or subsequent recovery; and (b) while HIF-2α levels were increased >2-fold following 48h exposure to sorafenib or sunitinib, during subsequent recovery in drug-free medium, the increased levels of HIF-2α persisted in cells treated with sorafenib but not sunitinib. Compared to CCRCC cells that were growth inhibited in vitro at concentrations of 5–10 μM sorafenib or sunitinib, in 27 patient CCRCC samples evaluated, the median mRNA levels of RIN1 was 7.2-fold lower in 22 samples (Range 1.6–22.3) and 2.2-fold higher in 5 samples (Range 1.2–27.1). Stable over-expression and down-regulation of RIN1 in CCRCC led to resistance or sensitivity respectively, to the anti-proliferative effects of sorafenib and sunitinib but not the mTOR inhibitor temsirolimus. In contrast, CCRCC cells adapted to proliferate in 4 μM everolimus exhibited a >3-fold increase in IC50, and cross-resistance to temsirolimus but not sorafenib or sunitinib. Evaluation in vitro of other targeted agents, demonstrated dose-dependent anti-proliferative effects with the PI3K/mTOR dual inhibitor BEZ235 (>80% growth inhibition at 10nM) but not the pan-PI3K inhibitor BKM120 (<30% growth inhibition at 10–500 nM) with CCRCC cells. The observed anti-proliferative effects of BEZ235 or BKM120 were comparable in CCRCC cells harboring the wild type or mutant von Hippel-Lindau gene. While BEZ235 (1–5 nM) or everolimus (100 nM) alone led to <40% growth inhibition, the combination significantly enhanced anti-proliferative effects (>85% growth inhibition) in CCRCC cells expressing resistance to everolimus. These results suggest the PI3K/mTOR dual inhibitor BEZ235 may possibly improve the treatment efficacy of mTOR inhibitors for CCRCC. The findings suggest RTKI and mTOR inhibitors have distinct mechanisms of resistance, and combinations may be useful to improve disease control of metastatic CCRCC.
We have recently characterized a pentapeptide, DYDYQ, from coagulation factor V (Beck et. al. 2004J. Biol. Chem.279, 3084) that inhibits both factor V activation and prothrombinase function. The pentapeptide does not interfere with the active site of thrombin but rather interferes with substrate attachment. Our aim was to characterize at the molecular level the binding site of DYDYQ on thrombin. First we used computational methods (blind and focused docking) to propose a hypothetical binding site. Blind docking of the pentapeptide (structure obtained from a 20 ns molecular dynamics simulation) on thrombin was performed using a docking grid with large spacing. This approach provided us a favorable site (−4.8 kcal/mol) that was further investigated using a smaller spaced docking grid. Hydrogen bonding was analyzed between thrombin and DYDYQ. The final free energy of binding was −9.69 kcal/mol. Amino acids Y76R77I79I82 from thrombin anion binding exosite I (ABE-I), were identified to participate in the interaction of the enzyme with DYDYQ. We next investigated the Thrombin-DYDYQ interaction following cross-linking with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide(EDC) and mass spectrometry. In these experiments purified thrombin was inhibited in the active site with a chloromethyl ketone, and treated with the DYDYQ peptide in the presence of EDC. Two bands were observed, one corresponding to thrombin cross linked to the peptide (CT) and another band corresponding to free thrombin (T). The proteins were either (i) stained with Coomassie blue for further digestion with trypsin or (ii) transferred to nitrocellulose membranes following by staining with Coomassie blue for treatment with cyanogen bromide (CNBr). Stained bands were isolated from the gel and subjected to trypsin digestion and liquid chromatography/mass spectrometry (LC-MS). Following trypsin digestion thrombin presence in both, T and CT samples was confirmed and the peptides identified in both samples covered approximately 63% of the entire thrombin sequence. The only difference observed between the sets of peptides obtained from T and CT following digestion with trypsin, was the peptide N78IEKISM*LEK87 (M* = oxidized Methionine), which was present in the T sample but was absent in the CT sample. These results suggest that the binding site of DYDYQ to thrombin is localized in the area of the above mentioned peptide protecting it from hydrolysis by trypsin. We next analyzed T and CT by LC-MS following CNBr digestion. Three important bands (peptide products from CNBr digestion) were detected in the sample containing the T, having an approximate molecular weight of ~4,500, ~7,500, and ~9,000. CNBr digestion products of CT lacked the median band (peptide mass: ~7,500). This band corresponded to the sequence L33…Y76ERN78IEKISM84 - as confirmed by ESI-ion trap mass spectrometry amino acid sequence of its first eleven amino acid residues. The difference between the two in gel CNBr digest profiles of T and CT, confirms the conclusion drawn from the MS analysis of the triptic digests which in turn was predicted by our computational analyses. Overall our data demonstrate that 1) amino acid residues Y76R77I79I82 from thrombin provide an interactive site for DYDYQ, and 2) our results from computational methods that identify protein-peptide interaction are valid and can be confirmed by mass spectrometry.
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