Abstract
In addition to its well-characterized function as a tumor suppressor, p14ARF (ARF) is a positive regulator of topoisomerase I (topo I), a central enzyme in DNA metabolism and a target for cancer therapy. We previously showed that topo I hyperphosphorylation, a cancer-associated event mediated by elevated levels of the protein kinase CK2, increases topo I activity and the cellular sensitivity to topo I-targeted drugs. Topo I hyperphosphorylation also increases its interaction with ARF. Because the ARF−topo I interaction could be highly relevant to DNA metabolism and cancer treatment, we identified the regions of topo I involved in ARF binding and characterized the effects of ARF binding on topo I function. Using a series of topo I deletion constructs, we found that ARF interacted with the topo I core domain, which encompasses most of the catalytic and DNA-interacting residues. ARF binding increased the DNA relaxation activity of hyperphosphorylated topo I by enhancing its association with DNA, but did not affect the topo I catalytic rate. In cells, ARF promoted the chromatin association of hyperphosphorylated, but not basal phosphorylated, topo I, and increased topo I-mediated DNA nicking under conditions of oxidative stress. The aberrant nicking was found to correlate with increased formation of DNA double-strand breaks, which are precursors of many genome destabilizing events. The results suggest that the convergent actions of oxidative stress and elevated CK2 and ARF levels, which are common features of cancer cells, lead to a dysregulation of topo I that may contribute both to the cellular response to topo I-targeted drugs and to genome instability.
Highlights
The p14 Alternate Reading Frame (ARF) protein is a cancerassociated protein that plays a well-characterized role in activating the p53 tumor suppressor pathway
Topo I consists of 4 domains: a poorly conserved N-terminal domain, a highly conserved DNA-binding core domain containing most of the catalytic residues, a poorly conserved linker domain, and a conserved C-terminal domain containing the catalytic residue tyrosine 723
The presence of the phosphorylation occurs on serine 506 (PS506) epitope was probed with pAb506-P, a rabbit polyclonal IgG raised to a PS506-containing topoisomerase I (topo I) peptide, which we previously showed recognizes PS506-containing topo I, but not the unphosphorylated or basal phosphorylated forms of topo I [15]
Summary
The p14 Alternate Reading Frame (ARF) protein is a cancerassociated protein that plays a well-characterized role in activating the p53 tumor suppressor pathway. ARF is present in normal cells at low to undetectable levels but accumulates in response to oncogene activation [1,2,3,4,5,6]. P53 is frequently inactivated in cancer, which disables the p53-dependent tumor suppressor activity of ARF but leaves ARF available to interact with a variety of other proteins, including topoisomerase I (topo I) [9,10], an essential enzyme that relaxes DNA supercoils during DNA synthesis [11,12]. We found that PS506-expressing hyperphosphorylated topo I displays both increased DNA binding and increased DNA relaxation activity compared with the basal phosphorylated topo I expressed in normal cells and in cancer cells without elevated CK2 [15]. Hyperphosphorylation of topo I is relevant to the cancer cell response to irinotecan and topotecan, two widely used camptothecin-derived chemotherapeutic drugs
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