Split luciferase as an optical probe for detecting protein-protein interactions in mammalian cells based on protein splicing

We describe a new method for detecting protein-protein interactions in inta ct mammalian cells; the approach is based on protein splicing-induced compl ementation of rationally designed fragments of firefly Luciferase. The prot ein splicing is a posttranslational protein modification through which inte ins (internal proteins) are excised out from a precursor fusion protein, li gating the flanking exteins (external proteins) into a contiguous polypepti de. As the intein, naturally split DnaE from Synechocystis sp, PCC6803 was used: The N- and C-terminal DnaE, each fused respectively to N- and C-termi nal fragments of split luciferase, were connected to proteins of interest. In this approach, protein-protein interactions trigger the folding of DnaE intein, wherein the protein splicing occurs and thereby the extein of Ligat ed luciferase recovers its enzymatic activity. To test the applicability of this split luciferase complementation, we used insulin-induced interaction between known binding partners, phosphorylated insulin receptor substrate 1 (IRS-1) and its target N-terminal SH2 domain of PI 3-kinase. Enzymatic lu ciferase activity triggered by insulin sen-ed to monitor the interaction be m een IRS-1 and the SH2 domain in an insulin dose-dependent manner, of whic h amount was assessed by the luminescent intensity, This provides a conveni ent method to study phosphorylation of any protein or interactions of integ ral membrane proteins, a class of molecules that has been difficult to stud y by existing biochemical or genetic methods. High-throughput drug screenin g and quantitative analysis for a specific pathway in tyrosine phosphorylat ion of IRS-1 in insulin signaling are also made possible in this system.