Sumoylation of topoisomerase I is involved in its partitioning between nucleoli and nucleoplasm and its clearing from nucleoli in response to camptothecin.
Previous studies identified a small fraction of putatively sumoylated topoisomerase I (TOP1) under basal conditions ( approximately 1%), and anticancer camptothecins that trap the TOP1-DNA covalent intermediate markedly increase the sumoylation of TOP1 (<or=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 (PsiKXE, where Psi 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 NH2-terminal region, showed a substantial reduction ( approximately 5-fold) in basal and camptothecin-induced sumoylation; thus, Lys-117 appears to be the major sumoylation site. A triple mutant having the PsiKXE 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 NH2-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.
Rallabhandi
Prasad
P
Department of Biochemistry and Molecular Biology, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814-4799, USA.
Hashimoto
Keiko
K
Mo
Yin-Yuan
YY
Beck
William T
WT
Moitra
Prasun K
PK
D'Arpa
Peter
P
D000595:Amino Acid Sequence; D000818:Animals; D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology; D001120:Arginine / Q000737:chemistry; D001665:Binding Sites; D016466:CHO Cells; D002166:Camptothecin / Q000494:pharmacology; D002460:Cell Line; D002466:Cell Nucleolus / Q000201:enzymology*; D002467:Cell Nucleus / Q000201:enzymology* / Q000378:metabolism; D006224:Cricetinae; D003593:Cytoplasm / Q000201:enzymology; D004264:DNA Topoisomerases, Type I / Q000737:chemistry* / Q000378:metabolism; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D008164:Luminescent Proteins / Q000378:metabolism; D008239:Lysine / Q000737:chemistry; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D014162:Transfection
D000972:Antineoplastic Agents, Phytogenic; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D001120:Arginine; D004264:DNA Topoisomerases, Type I; D008239:Lysine; D002166:Camptothecin