Abstract LB-137: Reversibility of click beetle luciferase heteroprotein fragment complementation systems for imaging cancer pathways in live cells
Authors: Dothager Robin S. , Villalobos Victor , Naik Snehal , Bruinsma Monique , Pan Mei-Hsiu , Samrakandi Mustapha , Piwnica-Worms David 
Published: February 2011

Abstract
Fragment complementation systems based on various luciferase enzymes have allowed for a more complete exploration of the protein interactome within the context of the cellular environment over biologically-relevant timescales. However, this strategy has been plagued by lingering questions regarding the reversibility of the systems that involve luciferase complementation fragments. To further address the need for tools to study the protein interactome, we have developed a novel set of multicolored heteroprotein fragment complementation systems based on various firefly and click beetle luciferases that utilize the same substrate, D-luciferin. Selected heteroprotein fragment complementation systems enabled simultaneous dual-color quantification of multiple protein interactions within live cells in realtime through use of spectral deconvolution analysis. Importantly, we have rigorously characterized the reversibility that is inherent in these systems. Reversibility was noted by the reproduction of association and dissociation constants when probing FK506 blockade of rapamycin-induced mTOR/FKBP interactions. The mean apparent Kd value upon rapamycin induction was 0.25 ± 0.05 nM, irrespective of the composition of the paired luciferases, spectral characteristics, total photon flux, or fold-inducibility. Competitive inhibition by FK506 with all the different pairs, when tested in the presence of 10 nM rapamycin, yielded a mean apparent Ki value of 1.6 ± 0.9 nM, consistent with conventional protein assays. Additionally, using split click-beetle green luciferase fused to the C-termini of EGFR, a more temporally dynamic system, we were able to monitor reversible protein conformational changes associated with the cytoplasmic domains of EGFR upon ligand-induced activation. Addition of EGF resulted in a biphasic response, characterized by an immediate drop in luciferase activity within the first minute after EGF addition followed by a rapid recovery of signal. The initial signal loss correlated with asymmetric movement of the cytosolic C-termini of EGFR dimers and adoption of a constrained conformation after phosphorylation. The subsequent recovery in luciferase activity corresponded to additional conformational changes that placed the relaxed C-terminal tails once again in close proximity. Addition of the MAPK inhibitor U0126 (10 µM) resulted in a blunted recovery of click-beetle green luciferase activity, again demonstrating the requirement for MAPK-mediated phosphorylation to induce post-activation rearrangements of the cytoplamic domain of the receptor. These dual-color reversible protein interaction reporters should enable dynamic analysis of a variety of protein networks in living cells.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-137.