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First published online 1 June 2004
doi: 10.1242/jcs.01147

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Human topoisomerase II nuclear export is mediated by two CRM-1-dependent nuclear export signals

Joel G. Turner^1,*, Roxanne Engel^1,*, Jennifer A. Derderian¹, Richard Jove² and Daniel M. Sullivan^1,

¹ Experimental Therapeutics, Departments of Interdisciplinary Oncology and Biochemistry and Molecular Biology, H. Lee Moffitt Cancer Center and Research Institute, University of South Florida, 12902 Magnolia Drive, Tampa, FL 33612, USA
² Molecular Oncology Programs, Departments of Interdisciplinary Oncology and Biochemistry and Molecular Biology, H. Lee Moffitt Cancer Center and Research Institute, University of South Florida, 12902 Magnolia Drive, Tampa, FL 33612, USA

View larger version (30K): [in a new window]   Fig. 1 Site-directed mutagenesis. The figure compares nucleotide sequences from the wild-type topo II gene and the mutated sequences used in this study. Hydrophobic amino acid residues thought to be necessary for nuclear export were mutated to alanine using site-directed mutagenesis (gray boxes). Putative NES amino acid sequences are in boxes below their corresponding nucleotide sequences.

View larger version (70K): [in a new window]   Fig. 2. HeLa cells microinjected with either wild-type (left column) or mutated (right column) peptide-BSA FITC conjugates (green), and then counterstained with DAPI (blue). A total of 20-50 cells were successfully microinjected per peptide and similar results were seen in all cells. The bottom of the figure presents HeLa cells that were microinjected with wild-type peptide-BSA-FITC conjugates in the presence of 2 ng/ml LMB (leptomycin B). Panels showing mutated peptide BSA-NES 1054-66 are at a higher magnification than the other micrographs.

View larger version (74K): [in a new window]   Fig. 3. Western blot of full-length FLAG-topo II protein. HeLa cells were transfected with plasmid containing FLAG-topo II plasmid via cationic lipid, and harvested after 20 hours. Protein extracts from 2x105 cells per lane were separated on a SDS-PAGE gel, blotted onto nitrocellulose and probed with FLAG M2 antibody. Lane A is protein from cells transfected with non-mutated FLAG-topo II plasmid, lane B is FLAG-topo II plasmid 1054-1066, lane C transfected with FLAG-topo II plasmid 1017-1028, and lane D is a non-transfected control.

View larger version (85K): [in a new window]   Fig. 4. FLAG-topo II immunofluorescence. Human multiple myeloma H929 cells were transfected by electroporation with full-length wild-type and mutated topo II and plated for 20 hours at log and plateau cell densities. Cytospins containing fixed cells were stained with FITC-labeled anti-FLAG M2 antibody (green), counterstained with DAPI for nuclear staining, and assayed by immunofluorescent microscopy.

View larger version (46K): [in a new window]   Fig. 5. Nuclear export of wild-type and mutant FLAG-topo II plasmids. Human myeloma H929 transfected cells (n=20) stained with anti-FLAG M2 monoclonal antibody-FITC conjugate were assayed for nuclear and cytoplasmic immunofluorescence. Quantitation of FITC fluorescence was performed using Adobe Photoshop 7.0 program. Wild-type FLAG-topo II was exported to the cytoplasm in cells at plateau density (P=0.00001), whereas topo II mutated at the putative export sites, 1016-1027 and 1053-1066, did not demonstrate statistically significant levels of export to the cytoplasm.

View larger version (80K): [in a new window]   Fig. 6. The complete amino acid sequence of human DNA topo IIaccession number NP 001058). The active-site tyrosine residue (805) is indicated (*); NES1017-1028, C1017DILRDFFELRLK1028 and NES1054-1066, C1054FILEKIDGKIIIE1066 are shaded; bipartite NLS is single underlined; Ser/Thr CK2 phosphorylation sites are italicized; the predicted coiled-coil region is boxed.

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