Inducible translocation trap: a system for detecting inducible nuclear translocation.
Here we report a general system, inducible translocation trap (ITT), for identification of proteins that translocate into the nucleus following signal transduction from cell surface receptors. ITT consists of a retroviral cDNA expression library of fusion proteins consisting of a LexA DNA binding domain, the transactivation domain of a transcriptional activator, and proteins encoded by cDNA inserts. The retroviral library is then transduced into cell lines containing a reporter gene with LexA binding sites in its promoter. Cells expressing the reporter gene by extracellular stimuli are then selected by flow-cytometric sorting. By using ITT, we identified cDNA encoding Stat1 in a screen of proteins which translocate into the nucleus by IFNgamma, indicating that this system can be used for isolation of nuclear translocating proteins induced by extracellular stimuli. ITT may be a useful tool for dissecting dynamic translocation in various biological systems.
Hoshino
Akemi
A
Department of Pathology, New York University School of Medicine, 550 First Avenue, New York, NY 10016, USA.
Matsumura
Satoko
S
Kondo
Kimie
K
Hirst
John A
JA
Fujii
Hodaka
H
D021581:Active Transport, Cell Nucleus / Q000235:genetics*; D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D002460:Cell Line; D018076:DNA, Complementary / Q000032:analysis* / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics; D005434:Flow Cytometry; D017930:Genes, Reporter / Q000235:genetics*; D005822:Genetic Vectors / Q000235:genetics* / Q000378:metabolism; D015698:Genomic Library; D007371:Interferon-gamma / Q000235:genetics / Q000378:metabolism; D051379:Mice; D011401:Promoter Regions, Genetic / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D021381:Protein Transport / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000378:metabolism; D015203:Reproducibility of Results; D012190:Retroviridae / Q000235:genetics; D050794:STAT1 Transcription Factor; D012697:Serine Endopeptidases / Q000235:genetics; D015534:Trans-Activators / Q000235:genetics; D014161:Transduction, Genetic / Q000379:methods*; D000099160:LexA Repressor Protein
D001426:Bacterial Proteins; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D007371:Interferon-gamma; D011993:Recombinant Fusion Proteins; D050794:STAT1 Transcription Factor; D012697:Serine Endopeptidases; D015534:Trans-Activators; D000099160:LexA Repressor Protein; C494080:Stat1 protein, mouse