Regulation of Topoisomerase II-Drug-DNA Ternary Complex

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DESCRIPTION (provided by applicant): The overall goal of this application is to determine the functional role of phosphorylation of serine (S) 1106 in topoisomerase (topo) IIalpha and the upstream mechanisms regulating phosphorylation of this site. Topo IIalpha alters DNA topology and maintains cellular integrity. This enzyme is phosphorylated at specific sites in a cell cycle dependent manner. Recently we identified a phosphorylation site, Ser1106, in the catalytic domain, which regulates enzymatic activity and drug sensitivity. Preliminary studies suggest that Ser1106 is phosphorylated by CKI in a Ca2+dependent manner. Thus, our working hypothesis is that Ca 2+dependent phosphorylation of S1106 by CKI (and/or CKII) regulates the biologic activity of topo IIalpha. To test this hypothesis, in vitro and in vivo studies in S. cerevesiae (strains BJ201 or JN394) expressing recombinant topo IIalpha (wild-type or mutant) and the human leukemia HL-60 model systems will be employed. Topo IIalkpha function will be assessed by: a) decatenation activity b) etoposide (VP-16)-stabilized DNA cleavable complex formation in vitro, and c) sensitivity to VP-16 in vivo. The effect of mutation of: a) Ser1106 to glutamic acid or aspartic acid, and b) in vivo CKII phosphorylation sites, alone or in combination with S1106 to alanine, on topo IIa function will be evaluated. Cell cycle phase dependent expression and subcellular localization of Ser1106 phosphorylated topo IIa will be determined by mass spectrometry/2D phosphopeptide mapping and immunofluorescence staining. Phospho-S1106 specific antibodies will be generated and used for immunoblot analyses of synchronized G1, S, or G2 or M cells and for immunofluorescence staining. A role for CKI and/or CKII in phosphorylating Ser1106 will be tested in vitro and in vivo. For in vitro assays purified or recombinant CKI or CKII and purified wild type or mutant topo IIa or synthetic peptides containing Ser1106 will be employed. In vivo studies, performed in yeast and HL-60 cells, will examine topo IIa phosphorylation and function following: a) alteration of kinase consensus sequences around S1106, and b) depletion of CKI and/or CKII by pharmacological (inhibitors of CKI/CKII) or molecular (CKI/CKII antisense oligonucleotides or siRNA, or yeast CKI homolog knock out) approaches. Expression and activity of these kinases in cells exhibiting differential sensitivity to topo II poisons will be determined. In the long term, the proposed studies should provide salient information on the functional significance of topo IIalpha phosphorylation and the upstream events, i.e. specific kinase(s), that modulate phosphorylation.