Phosphorylation of DNA topoisomerase I: Role in mammalian cell signal transduction.
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PublisherThe University of Arizona.
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AbstractThe tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) induces an altered program of gene expression, followed by DNA synthesis and cell division, in quiescent murine fibroblasts. The phosphorylation of DNA topoisomerase I, a nuclear enzyme involved in transcription and replication, was characterized in quiescent 3T3-L1 cells treated with TPA. An anti-DNA topoisomerase I antibody from sera of patients with the autoimmune disease diffuse scleroderma was used to isolate phosphorylated DNA topoisomerase I. Phosphorylation was stimulated slightly within 10 min and 6-fold by 2 h of TPA treatment; TPA at 100 ng/ml maximally enhanced phosphorylation. DNA topoisomerase I was modified primarily on serine, with a minor phosphothreonine component. The half-life of incorporated phosphate on DNA topoisomerase I was approximately 40 min in both TPA-treated and control cells, suggesting that stimulation of a kinase was the mechanism for increased phosphorylation. In addition, the phosphorylation of DNA topoisomerase I was enhanced in rat H-35 hepatoma cells treated with insulin as well as Swiss/3T3 fibroblasts treated with epidermal growth factor. DNA topoisomerase I phosphorylation in vitro by protein kinase C, the cellular receptor for TPA, was also characterized. DNA topoisomerase I from HeLa cells was purified to apparent homogeneity and was phosphorylated in a Ca²⁺ and phospholipid-dependent fashion by type III protein kinase C, purified 860-fold from mouse brain; the reaction was stimulated by TPA. Approximately 1.3 moles of phosphate were incorporated per mole of substrate, predominantly on a tryptic peptide that comigrated with the major in vivo phosphopeptide, although other sites were phosphorylated to a lesser extent. Serine was the primary amino acid modified, however phosphothreonine was also detected. The incorporation of phosphate into DNA topoisomerase I was linear in the range of time and protein kinase C examined. The apparent K(m) and V(max) for the reaction were 0.4 μM and 0.7 μmol phosphate per min per mg, respectively. Thus, phosphorylation, possibly mediated by protein kinase C, was postulated to be a physiologically significant means of regulating DNA topoisomerase I during mammalian cell proliferation.
Degree ProgramMolecular and Cellular Biology