CK2-mediated hyperphosphorylation of topoisomerase I targets serine 506, enhances topoisomerase I-DNA binding, and increases cellular camptothecin sensitivity

Abstract

Topoisomerase I is the target for a potent class of chemotherapeutic drugs derived from the plant alkaloid camptothecin that includes irinotecan and topotecan. In this study we have identified a novel site of CK2-mediated topoisomerase I (topo I) phosphorylation at serine 506 (PS506) that is relevant to topo I function and to cellular responses to these topo I-targeted drugs. CK2 treatment induced hyperphosphorylation of recombinant topo I and expression of the PS506 epitope, and resulted in increased binding of topo I to supercoiled plasmid DNA. Hyperphosphorylated topo I was approximately three times more effective than the basal phosphorylated enzyme at relaxing plasmid supercoils but had similar DNA cleavage activity once bound to DNA. The PS506 epitope was expressed in cancer cell lines with elevated CK2 activity, hyperphosphorylated topo I, and increased sensitivity to camptothecin. In contrast, PS506 was not detected in normal cells or cancer cell lines with lower levels of CK2 activity. By experimentally manipulating CK2 activity in cancer cell lines, we demonstrate a cause and effect relationship between CK2 activity, PS506 expression, camptothecin-induced cellular DNA damage, and cellular camptothecin sensitivity. Our results show that the PS506 epitope is an indicator of dysregulated, hyperphosphorylated topo I in cancer cells, and may thus serve as a diagnostic or prognostic biomarker and predict tumor responsiveness to widely used topo I-targeted therapies.

Figure 1. Western analysis of CK2-mediated phosphorylation of recombinant topo I.

(A) Recombinant, baculovirus-expressed topo I was analyzed before or after treatment with CK2. Membranes were probed with antibodies to total phosphoserine (P-ser), PS506, S506, or total topo I. Each lane contains 0.5 µg protein. (B) His-tagged wild-type (wt) or ser506ala (A506) topo I gene products were analyzed with antibodies to PS506, S506, or total topo I. Gene products were selected on cobalt agarose from lysates of H358 cells 2 days after transduction. Each lane represents 2×10⁶ cell equivalents.

Figure 2. Effects of hyperphosphorylation on topo I binding, relaxation, and nicking activity.

(A) Agarose gel electrophoresis of products of a supercoiled plasmid DNA relaxation assay carried out with basal or hyperphosphorylated R-topo I. Reactions contained 5, 7.5, or 10 ng of basal phosphorylated R-topo I, or 1, 2, or 3 ng of hyperphosphorylated R-topo I. C = untreated plasmid control, s = supercoiled DNA, r = relaxed DNA. (B) (Top) Western analysis of PS506 and FLAG expression in cobalt agarose-selected A506 (“A”) and wild-type (S506, “S”) gene products. Nuclear extracts of transduced SW480 cells were collected before or 2 days after treatment with the CK2 activator 1-ethyl-4,5-dicarbamoyl imidazole. Each lane represents 75 µg. (Bottom) Agarose gel showing results of a plasmid relaxation assay carried out with the same cobalt agarose-selected proteins. C = untreated control plasmid. (C) Schematic of the steps in topo I-mediated relaxation of DNA supercoils, involving non-covalent association of topo I with DNA (intermediate ) followed by catalytic single-strand nicking (intermediate ). (D) Time course of non-covalent association of 0.3 pmol basal (•) or hyperphosphorylated (○) R-topo I to 0.03 pmol of radiolabeled plasmid DNA. Topo I–DNA complexes were recovered and DNA quantified by scintillation counting. Results show the % of input DNA present in DNA–topo I complexes. (E) Catalytic rate of basal (•) and hyperphosphorylated (○) R-topo I on a synthetic suicide substrate following the formation of non-covalent complexes at 4°C. (F) Diagram of hairpin structure of suicide substrate showing topo I cleavage sequence (↓) and blocked 5′-end carrying a phosphate group (•). Diagram adapted from reference with permission from Oxford University Press.

Figure 3. Modulation of CK2 activity and effects on topo I and cellular camptothecin responses.

(A) CK2 activity measured in OVCAR-3 and SKOV-3 ovarian cancer cell lysates, before and after experimental modulation of CK2 levels. CK2 level in the control (C) Hs27a stromal cell line is shown for comparison. (B) Topo I serine phosphorylation status in OVCAR-3 and SKOV-3 cells before and after modulation of CK2 levels. Cell lysates (175 µg) were immunoprecipitated with goat anti-topo I C-terminal followed by Western analysis of phosphoserine (P-ser) or total topo I. (C) Western analysis of topo I PS506, S506, total topo I, and actin in lysates of OVCAR-3 and SKOV-3 cells before and after modulation of CK2 levels. Each lane represents 75 µg. (D) Topo I activity assayed by conversion of supercoiled plasmid DNA to the relaxed form by nuclear extracts prepared from OVCAR-3 or SKOV-3 cells before and after modulation of CK2 levels. First lane shows control, untreated plasmid. (E) Western analysis of γ-H2A.X expression as a marker of DNA damage in lysates of OVCAR-3 or SKOV-3 cells before or after modulation of CK2 levels, and following a 1 h exposure to 10 µM camptothecin (CPT). (F) Viability of OVCAR-3 or SKOV-3 cells on day 3 with or without experimental modulation of CK2, and following 18 h exposure to the indicated doses of camptothecin (CPT).

Figure 4. Correlation of CK2 activity, PS506 expression, and camptothecin sensitivity in cancer cell lines.

(A) CK2 activity in cell lysates of the indicated cell lines. (B) Western analysis of PS506, total topo I, and actin in lysates of the indicated cell lines. Each lane contains 75 µg of cell lysate. Numbers below lanes refer to the intensities of the PS506 band relative to H358, as determined digitally. (C) Day 3 viability assays of the indicated cell lines following exposure to the indicated doses of camptothecin (CPT) for the first 18 h of the incubation.