Identification of hNopp140 as a binding partner for doxorubicin with a phage display cloning method.
Doxorubicin is a widely used anti-cancer drug. It is assumed to act by inhibiting DNA replication or transcription, although its precise targets and mechanism of cytotoxicity remain unresolved. A T7 phage library expressing human liver cDNA was screened against immobilized doxorubicin to isolate doxorubicin binding proteins. The selected phage contained the C-terminal region of nucleolar phosphoprotein hNopp140, an important factor in the biogenesis of the nucleolus. When the cloned sequence was expressed in E. coli, the recombinant protein was phosphorylated by casein kinase II and oligomerized in the presence of magnesium and fluoride ions, as occurs in vivo. Doxorubicin bound to the expressed protein with a dissociation constant of 4.5 x 10(-6) M, and this interaction was inhibited by the phosphorylation of hNopp140. These results suggested that doxorubicin might disrupt the cellular function of hNopp140.
Jin
Youngnam
Y
Structural Biology Center, Korea Institute of Science and Technology, P.O. Box 131, Cheongryang, Seoul 130-650, South Korea.
Yu
Jaehoon
J
Yu
Yeon Gyu
YG
D000429:Alcohol Oxidoreductases; D000970:Antineoplastic Agents / Q000378:metabolism* / Q000494:pharmacology; D001665:Binding Sites; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D004317:Doxorubicin / Q000378:metabolism* / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D008099:Liver / Q000166:cytology; D009687:Nuclear Proteins / Q000819:agonists* / Q000235:genetics; D019151:Peptide Library; D010750:Phosphoproteins / Q000819:agonists* / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000235:genetics; D013050:Spectrometry, Fluorescence
D000970:Antineoplastic Agents; D004268:DNA-Binding Proteins; C075564:NOLC1 protein, human; D009687:Nuclear Proteins; D019151:Peptide Library; D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins; D004317:Doxorubicin; D000429:Alcohol Oxidoreductases; C096794:C-terminal binding protein