title: Bioluminescent imaging of MAPK function with intein-mediated reporter gene assay.
abstract: For nondestructive analysis of chemical processes in living mammalian cells, here we show a new reporter gene assay for detecting Ras-Raf-1 interactions based on protein splicing of transcription factors with DnaE inteins. The protein splicing induces connection of a DNA-binding protein (modified LexA; mLexA) with a transcription activation domain of a herpes simplex virus protein (VP16AD). Ras is connected with N-terminal DnaE and mLexA, while Raf-1 is connected with C-terminal DnaE and VP16AD. Upon stimulation with EGF, the interaction between Ras and Raf-1 triggers folding of the DnaEs, thereby inducing protein splicing to form mLexA-VP16AD fusion protein and transcription of a reporter gene, firefly luciferase. The extent of Ras-Raf-1 interaction is quantified by measuring the luciferase activity. By using the protein-splicing elements and the reporter gene, the Ras-Raf-1 interaction close to cell membranes can be evaluated.
authors.affiliation: Department of Chemistry, School of Science, The University of Tokyo, Tokyo, Japan.
authors.forename: Akira
authors.initials: A
authors.lastname: Kanno
authors.forename: Takeaki
authors.initials: T
authors.lastname: Ozawa
authors.forename: Yoshio
authors.initials: Y
authors.lastname: Umezawa
mesh_terms: D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D066246:ErbB Receptors / Q000235:genetics; D017930:Genes, Reporter; D047668:Inteins; D049449:Luminescence; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D015689:Oncogene Protein p21(ras) / Q000378:metabolism; D019908:Proto-Oncogene Proteins c-raf / Q000378:metabolism
chemical_list: D066246:ErbB Receptors; D019908:Proto-Oncogene Proteins c-raf; D020928:Mitogen-Activated Protein Kinases; D015689:Oncogene Protein p21(ras)