DNA topoisomerase I phosphorylation in murine fibroblasts treated with 12-O-tetradecanoylphorbol-13-acetate and in vitro by protein kinase.
The phosphorylation of DNA topoisomerase I in quiescent murine 3T3-L1 fibroblasts treated with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) was characterized by in vivo labeling with [32P] orthophosphate and immunoprecipitation with a scleroderma anti-DNA topoisomerase I autoantibody. DNA topoisomerase I phosphorylation was stimulated 4-fold by 2 h of TPA treatment (TPA at 100 ng/ml maximally enhanced phosphorylation). Purified DNA topoisomerase I was phosphorylated in vitro in a Ca2+ and phospholipid-dependent fashion by types I, II, and III protein kinase C. The phosphorylation reaction was stimulated by TPA and had an apparent K(m) of 0.4 microM. DNA topoisomerase I was phosphorylated in vivo and in vitro predominantly at serine. The major tryptic phosphopeptides from DNA topoisomerase I in TPA-treated fibroblasts and phosphorylated by protein kinase C comigrated in thin-layer electrophoresis. The half-life of incorporated phosphate on DNA topoisomerase I was 40 min in both TPA-treated and control cells. These results suggest that phosphorylation is a mechanism for activating DNA topoisomerase I in fibroblasts treated with TPA and that protein kinase C functions in the phosphorylation.
Samuels
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Department of Molecular and Cellular Biology, University of Arizona, Tucson 85721.
Shimizu
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D016475:3T3 Cells; D000818:Animals; D004264:DNA Topoisomerases, Type I / Q000378:metabolism*; D015180:Electrophoresis, Gel, Two-Dimensional; D005347:Fibroblasts / Q000187:drug effects / Q000201:enzymology; D007700:Kinetics; D051379:Mice; D010449:Peptide Mapping; D010766:Phosphorylation; D011233:Precipitin Tests; D011493:Protein Kinase C / Q000494:pharmacology*; D015398:Signal Transduction; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D014357:Trypsin
D011493:Protein Kinase C; D014357:Trypsin; D004264:DNA Topoisomerases, Type I; D013755:Tetradecanoylphorbol Acetate