Quantitative determination of protein nuclear transport induced by phosphorylation or by proteolysis.
Nucleocytoplasmic transport of proteins in eukaryotic cells is a fundamental process for gene expression. The transport is regulated by posttranslational modifications of the proteins, such as ligand-binding, phosphorylation, and proteolysis. For monitoring the nuclear transport of proteins induced by a ligand binding, we have recently developed a genetically encoded bioluminescent indicator based on reconstitution of split fragments of Renilla reniformis (RLuc) by protein splicing with DnaE inteins. We herein describe that the technique is used for detecting phosphorylation- or proteolysis-induced nuclear transports of a target protein. Two model proteins, signal transducer and activator of transcription 3 (STAT3) and sterol-regulatory element binding protein-2 (SREBP-2), were exemplified as phosphorylation- and proteolysis-induced nuclear transport, respectively. Each STAT3 or SREBP-2 is connected with C-terminal halves of RLuc and DnaE. If the protein translocates into the nucleus, the C-terminal fragment of RLuc meets the N-terminal fragment of RLuc, and full-length RLuc is reconstituted by protein splicing in the nucleus. The indicator with SREBP-2 enabled us to quantify the intracellular concentrations of cholesterol. The indicator with STAT3 quantified the extent of the nuclear transport induced by representative cytokines. This simple assay based on protein nuclear transports allows the selection of suitable drugs among candidates and has significant potential for risk assessments, such as carcinogenic chemical screening in vitro and in vivo.
Kim
Sung Bae
SB
Department of Chemistry, School of Science, University of Tokyo, Bunkyo-Ku, Tokyo 113-0033, Japan.
Takao
Ryohei
R
Ozawa
Takeaki
T
Umezawa
Yoshio
Y
D021581:Active Transport, Cell Nucleus / Q000502:physiology*; D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D006801:Humans; D049410:Luciferases, Renilla / Q000737:chemistry; D010766:Phosphorylation; D015202:Protein Engineering; D019154:Protein Splicing; D021381:Protein Transport / Q000502:physiology*; D050796:STAT3 Transcription Factor / Q000737:chemistry / Q000378:metabolism*; D051782:Sterol Regulatory Element Binding Protein 2 / Q000737:chemistry / Q000378:metabolism*
D050796:STAT3 Transcription Factor; D051782:Sterol Regulatory Element Binding Protein 2; D049410:Luciferases, Renilla