Abstract
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.
Highlights
Thephosphorylationof DNA topoisomerase I in receptor for TPA (Niedel et al, 1983; Leach et al, 1983; quiescent murine3T3-Ll fibroblasts treatedwith the Kikkawa et al, 1983)and is thought to mediate the cellular tumorpromoter 12-0-tetradecanoylphorbol-13-ace-response to phorbol ester tumorpromoters (Nishizuka, 1984)
By which mitogenic signals are transducedto the nucleus, we have investigated the phosphorylation state of DNA topoisomeraseI in TPA-treatedmurine fibroblasts.We report that treatment of quiescent 3T3-Ll cells with TPA rapidly stimulates the phosphorylation of DNA topoisomerase I and that the in uiuo phosphorylation appears to be at the same major site phosphorylatedby protein kinase C in uitro
In Vivo Phosphorylation of DNA Topoisomerase I-The phosphorylation state of DNA topoisomerase I was examined in fibroblaststreatedwith TPA becausephosphorylation enhances the activity of DNA topoisomerase I in uitro, and we postulated that this post-translationalmodification might be a mechanism for regulating the enzyme during the early
Summary
From the $Department of Molecular and Cellular Bwlogy, University of Arizona, Tucson, Arizona85721 and the VDepartment of Molecular Biology, Keio University School of Medicine, 35 Shimnomachi, Shinjuku-ku, Tokyo 160, Japan. In phosphopeptides from DNA topoisomerase I in TPA- addition, many nuclear proteins are phosphorylated in vitro treated fibroblasts and phosphorylatebdy protein ki- by protein kinase C, including DNA topoisomerase I The half-life of incorporated phosphateon DNA topoisomerase I was 40 min in both TPA-treated and control cells These results suggest that phosphorylationis a mechanism for activating DNA topoisomerase I in fibroblasts treatedwith TPA and that proteinkinase C functions in the phosphorylation. Protein ki- calized to actively transcribed genes and replication forks and nase C, the Ca2+ and phospholipid-dependent serineand functions in RNA and DNA synthesis (Wang, 1987;Liu, 1990; threonine protein kinase, is directly activated by TPA (Cas- Kornberg and Baker, 1992).
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