Abstract
DNA topoisomerase II (topo II) regulates the topological state of DNA and is necessary for DNA replication, transcription, and chromosome segregation. Topo II has essential functions in cell proliferation and therefore is a critical target of anticancer drugs. In this study, using Phos-tag SDS-PAGE analysis in fission yeast (Schizosaccharomyces pombe), we identified casein kinase II (Cka1/CKII)–dependent phosphorylation at the C-terminal residues Ser1363 and Ser1364 in topo II. We found that this phosphorylation decreases the inhibitory effect of an anticancer catalytic inhibitor of topo II, ICRF-193, on mitosis. Consistent with the constitutive activity of Cka1/CKII, Ser1363 and Ser1364 phosphorylation of topo II was stably maintained throughout the cell cycle. We demonstrate that ICRF-193–induced chromosomal mis-segregation is further exacerbated in two temperature-sensitive mutants, cka1–372 and cka1/orb5-19, of the catalytic subunit of CKII or in the topo II nonphosphorylatable alanine double mutant top2-S1363A,S1364A but not in cells of the phosphomimetic glutamate double mutant top2-S1363E,S1364E. Our results suggest that Ser1363 and Ser1364 in topo II are targeted by Cka1/CKII kinase and that their phosphorylation facilitates topo II ATPase activity in the N-terminal region, which regulates protein turnover on chromosome DNA. Because CKII-mediated phosphorylation of the topo II C-terminal domain appears to be evolutionarily conserved, including in humans, we propose that attenuation of CKII-controlled topo II phosphorylation along with catalytic topo II inhibition may promote anticancer effects.
Highlights
DNA topoisomerase II regulates the topological state of DNA and is necessary for DNA replication, transcription, and chromosome segregation
We present evidence that phosphorylation at these sites diminishes the effect of an anticancer catalytic topoisomerase II (topo II) inhibitor, ICRF-193, on mitotic chromosome segregation
As we are aware, our results provide the first evidence that casein kinase II (CKII)-mediated phosphorylation of the topo II C-terminal domain (CTD) suppresses sensitivity to topo II catalytic inhibitors
Summary
Phos-tag analysis of S. pombe DNA Topoisomerase II (Top2) identified residues Ser1363 and Ser1364 as phosphorylation sites in the Top C-terminal region. Anti-Top phospho-Ser1363 and phospho-Ser1364 antibodies detected the rephosphorylated Top protein bands in an immunoblot assay, suggesting that CKII directly phosphorylates Ser1363 and Ser1364 residues (Fig. 2C). Bands of phospho-Ser1363 and phosphoSer1364 were almost constant throughout the cell cycle, phospho-Ser1363 and overall phosphorylation of Top2–3FLAG (Phos-tag gel) were slightly more intense at 30 and 45 min, which correspond to early mitosis (Fig. 2E). In the presence of an anticancer drug, ICRF-193, which inhibits the ATPase activity of topo II protein [15, 16], both WT and 2A mutant proteins lost much of their ability to hydrolyze ATP This result suggests that CKII-mediated Top phosphorylation at Ser1363 and Ser1364 facilitates ATP hydrolysis. Phosphorylation at Ser1363 and Ser1364 apparently increases the fidelity of chromosome segregation when Top function is impaired by ICRF-193
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