Visualization of cofilin-actin and Ras-Raf interactions by bimolecular fluorescence complementation assays using a new pair of split Venus fragments.
The bimolecular fluorescence complementation (BiFC) assay is a method for visualizing protein-protein interactions in living cells. To visualize the cofilin-actin interaction in living cells, a series of combinations of the N- and C-terminal fragments of Venus fused upstream or downstream of cofilin and actin were screened systematically. A new pair of split Venus fragments, Venus (1-210) fused upstream of cofilin and Venus (210-238) fused downstream of actin, was the most effective combination for visualizing the specific interaction between cofilin and actin in living cells. This pair of Venus fragments was also effective for detecting the active Ras-dependent interaction between H-Ras and Raf1 and the Ca(2+)-dependent interaction between calmodulin and its target M13 peptide. In vitro BiFC assays using the pair of purified BiFC probes provided the means to detect the specific interactions between cofilin and actin and between H-Ras and Raf1. In vivo and in vitro BiFC assays using the newly identified pair of Venus fragments will serve as a useful tool for measuring protein-protein interactions with high specificity and low background fluorescence and could be applied to the screening of inhibitors that block protein-protein interactions.
Department of Biomolecular Sciences, Graduate School of Life Sciences, Tohoku University, Sendai, Miyagi, Japan.
Kazumasa
K
Ohashi
Tai
T
Kiuchi
Kazuyasu
K
Shoji
Kaori
K
Sampei
Kensaku
K
Mizuno
D051339:Actin Depolymerizing Factors / Q000235:genetics / Q000378:metabolism*; D000199:Actins / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002147:Calmodulin / Q000235:genetics / Q000378:metabolism; D002474:Cell-Free System; D005453:Fluorescence; D006367:HeLa Cells; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism*; D008967:Molecular Biology / Q000379:methods*; D009219:Myosin-Light-Chain Kinase / Q000235:genetics / Q000378:metabolism; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D060066:Protein Interaction Maps; D019908:Proto-Oncogene Proteins c-raf / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D012680:Sensitivity and Specificity; D018631:ras Proteins / Q000235:genetics / Q000378:metabolism*
D051339:Actin Depolymerizing Factors; D000199:Actins; D001426:Bacterial Proteins; D002147:Calmodulin; D008164:Luminescent Proteins; C107660:M13 protein (myosin light-chain kinase); D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; C054562:yellow fluorescent protein, Bacteria; D019908:Proto-Oncogene Proteins c-raf; D009219:Myosin-Light-Chain Kinase; D018631:ras Proteins