Sumoylation of Topoisomerase I Is Involved in Its Partitioning between Nucleoli and Nucleoplasm and Its Clearing from Nucleoli in Response to Camptothecin

• Published in: The Journal of biological chemistry Vol. 277; no. 42; pp. 40020 - 40026
• Main Authors: Rallabhandi, Prasad, Hashimoto, Keiko, Mo, Yin-Yuan, Beck, William T, Moitra, Prasun K, D'Arpa, Peter
• Format: Journal Article
• Language: English
• Published: United States American Society for Biochemistry and Molecular Biology 18.10.2002
• Subjects: Amino Acid Sequence; Animals; Antineoplastic Agents, Phytogenic - pharmacology; Arginine - chemistry; Binding Sites; Camptothecin - pharmacology; Cell Line; Cell Nucleolus - enzymology; Cell Nucleus - enzymology; Cell Nucleus - metabolism; CHO Cells; Cricetinae; Cytoplasm - enzymology; DNA Topoisomerases, Type I - chemistry; DNA Topoisomerases, Type I - metabolism; Green Fluorescent Proteins; HeLa Cells; Humans; Luminescent Proteins - metabolism; Lysine - chemistry; Molecular Sequence Data; Mutation; Plasmids - metabolism; Protein Binding; Protein Structure, Tertiary; Transfection
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M200388200

Previous studies identified a small fraction of putatively sumoylated topoisomerase I (TOP1) under basal conditions (∼1%), and anticancer camptothecins that trap the TOP1-DNA covalent intermediate markedly increase the sumoylation of TOP1 (≤10%). To study the role of the sumoylation of TOP1, we mutated sites on green fluorescent protein (GFP)-TOP1 corresponding to the consensus sequence for protein sumoylation (ΨK X E, where Ψ is a hydrophobic residue) and assayed the mutants for basal and camptothecin-induced sumoylation. Only one of the eight mutants, K117R, located in the highly charged NH 2 -terminal region, showed a substantial reduction (∼5-fold) in basal and camptothecin-induced sumoylation; thus, Lys-117 appears to be the major sumoylation site. A triple mutant having the ΨK X E sequences flanking K117R additionally mutated (K103R/K117R/K153R) showed little if any sumoylation, but was degraded like wild-type GFP-TOP1 during camptothecin treatment. However, K103R/K117R/K153R-GFP-TOP1 was markedly concentrated within nucleoli, depleted from the remainder of nucleus, and failed to be cleared from nucleoli in response to camptothecin treatment. These data are consistent with a model wherein basal transient sumoylation of the NH 2 -terminal, highly charged, disordered region prevents TOP1 binding to sites in nucleoli, thus driving it to bind in the nucleoplasm; and camptothecin treatment, which increases TOP1 sumoylation, further shifts the binding resulting in delocalization of TOP1 from nucleoli to nucleoplasm.