Luciferase Complementation Imaging Research Articles

Structural features important for the U12 snRNA binding and minor spliceosome assembly of Arabidopsis U11/U12-small nuclear ribonucleoproteins

ABSTRACTAlthough seven proteins unique to U12 intron-specific minor spliceosomes, denoted as U11/U12-65K, -59K, -48K, -35K, -31K, -25K, and -20K, have been identified in humans and the roles of some of them have been demonstrated, the functional role of most of these proteins in plants is not understood. A recent study demonstrated that Arabidopsis U11/U12-65K is essential for U12 intron splicing and normal plant development. However, the structural features and sequence motifs important for 65 K binding to U12 snRNA and other spliceosomal proteins remain unclear. Here, we demonstrated by domain-deletion analysis that the C-terminal region of the 65 K protein bound specifically to the stem-loop III of U12 snRNA, whereas the N-terminal region of the 65 K protein was responsible for interacting with the 59 K protein. Analysis of the interactions between each snRNP protein using yeast two-hybrid analysis and in planta bimolecular fluorescence complementation and luciferase complementation imaging assays demonstrated that the core interactions among the 65 K, 59 K, and 48 K proteins were conserved between plants and animals, and multiple interactions were observed among the U11/U12-snRNP proteins. Taken together, these results reveal that U11/U12-65K is an indispensible component of the minor spliceosome complex by binding to both U11/U12-59K and U12 snRNA, and that multiple interactions among the U11/U12-snRNP proteins are necessary for minor spliceosome assembly.

CXCR7 controls competition for recruitment of β-arrestin 2 in cells expressing both CXCR4 and CXCR7.

Chemokine CXCL12 promotes growth and metastasis of more than 20 different human cancers, as well as pathogenesis of other common diseases. CXCL12 binds two different receptors, CXCR4 and CXCR7, both of which recruit and signal through the cytosolic adapter protein β-arrestin 2. Differences in CXCL12-dependent recruitment of β-arrestin 2 in cells expressing one or both receptors remain poorly defined. To quantitatively investigate parameters controlling association of β-arrestin 2 with CXCR4 or CXCR7 in cells co-expressing both receptors, we used a systems biology approach combining real-time, multi-spectral luciferase complementation imaging with computational modeling. Cells expressing only CXCR4 maintain low basal association with β-arrestin 2, and CXCL12 induces a rapid, transient increase in this interaction. In contrast, cells expressing only CXCR7 have higher basal association with β-arrestin 2 and exhibit more gradual, prolonged recruitment of β-arrestin 2 in response to CXCL12. We developed and fit a data-driven computational model for association of either CXCR4 or CXCR7 with β-arrestin 2 in cells expressing only one type of receptor. We then experimentally validated model predictions that co-expression of CXCR4 and CXCR7 on the same cell substantially decreases both the magnitude and duration of CXCL12-regulated recruitment of β-arrestin 2 to CXCR4. Co-expression of both receptors on the same cell only minimally alters recruitment of β-arrestin 2 to CXCR7. In silico experiments also identified β-arrestin 2 as a limiting factor in cells expressing both receptors, establishing that CXCR7 wins the “competition” with CXCR4 for CXCL12 and recruitment of β-arrestin 2. These results reveal how competition for β-arrestin 2 controls integrated responses to CXCL12 in cells expressing both CXCR4 and CXCR7. These results advance understanding of normal and pathologic functions of CXCL12, which is critical for developing effective strategies to target these pathways therapeutically.

Monitoring Notch activation in cultured mammalian cells: luciferase complementation imaging assays.

Notch activation and cleavage releases the Notch intracellular domain (NICD), which translocates to the nucleus, where it associates with its DNA-binding partner CSL to recruit the coactivator MAML and additional cofactors to ultimately activate target gene expression. Taking advantage of the specific interaction between NICD and these factors, we have developed a luciferase complementation imaging (LCI)-based reporter system to quantitatively monitor Notch activation in real time in live cells. In this chapter, we describe the use of Notch LCI reporters for measuring protein interactions and performing detailed kinetic analyses of receptor activation and its responses to various stimuli.

Regulation of Abiotic Stress Signalling by Arabidopsis C-Terminal Domain Phosphatase-Like 1 Requires Interaction with a K-Homology Domain-Containing Protein

Arabidopsis thaliana CARBOXYL-TERMINAL DOMAIN (CTD) PHOSPHATASE-LIKE 1 (CPL1) regulates plant transcriptional responses to diverse stress signals. Unlike typical CTD phosphatases, CPL1 contains two double-stranded (ds) RNA binding motifs (dsRBMs) at its C-terminus. Some dsRBMs can bind to dsRNA and/or other proteins, but the function of the CPL1 dsRBMs has remained obscure. Here, we report identification of REGULATOR OF CBF GENE EXPRESSION 3 (RCF3) as a CPL1-interacting protein. RCF3 co-purified with tandem-affinity-tagged CPL1 from cultured Arabidopsis cells and contains multiple K-homology (KH) domains, which were predicted to be important for binding to single-stranded DNA/RNA. Yeast two-hybrid, luciferase complementation imaging, and bimolecular fluorescence complementation analyses established that CPL1 and RCF3 strongly associate in vivo, an interaction mediated by the dsRBM1 of CPL1 and the KH3/KH4 domains of RCF3. Mapping of functional regions of CPL1 indicated that CPL1 in vivo function requires the dsRBM1, catalytic activity, and nuclear targeting of CPL1. Gene expression profiles of rcf3 and cpl1 mutants were similar during iron deficiency, but were distinct during the cold response. These results suggest that tethering CPL1 to RCF3 via dsRBM1 is part of the mechanism that confers specificity to CPL1-mediated transcriptional regulation.

OsCYP2, a chaperone involved in degradation of auxin‐responsive proteins, plays crucial roles in rice lateral root initiation

Auxin plays a pivotal role in many facets of plant development. It acts by inducing the interaction between auxin-responsive [auxin (AUX)/indole-3-acetic acid (IAA)] proteins and the ubiquitin protein ligase SCF(TIR) to promote the degradation of the AUX/IAA proteins. Other cofactors and chaperones that participate in auxin signaling remain to be identified. Here, we characterized rice (Oryza sativa) plants with mutations in a cyclophilin gene (OsCYP2). cyp2 mutants showed defects in auxin responses and exhibited a variety of auxin-related growth defects in the root. In cyp2 mutants, lateral root initiation was blocked after nuclear migration but before the first anticlinal division of the pericycle cell. Yeast two-hybrid and in vitro pull-down results revealed an association between OsCYP2 and the co-chaperone Suppressor of G2 allele of skp1 (OsSGT1). Luciferase complementation imaging assays further supported this interaction. Similar to previous findings in an Arabidopsis thaliana SGT1 mutant (atsgt1b), degradation of AUX/IAA proteins was retarded in cyp2 mutants treated with exogenous 1-naphthylacetic acid. Our results suggest that OsCYP2 participates in auxin signal transduction by interacting with OsSGT1.

The TobaccoBLADE-ON-PETIOLE2Gene Mediates Differentiation of the Corolla Abscission Zone by Controlling Longitudinal Cell Expansion

The BLADE-ON-PETIOLE (BOP) genes of Arabidopsis (Arabidopsis thaliana) have been shown to play an essential role in floral abscission by specializing the abscission zone (AZ) anatomy. However, the molecular and cellular mechanisms that underlie differentiation of the AZ are largely unknown. In this study, we identified a tobacco (Nicotiana tabacum) homolog of BOP (designated NtBOP2) and characterized its cellular function. In tobacco plants, the NtBOP2 gene is predominantly expressed at the base of the corolla in an ethylene-independent manner. Both antisense suppression of NtBOP genes and overexpression of NtBOP2 in tobacco plants caused a failure in corolla shedding. Histological analysis revealed that the differentiation of the corolla AZ was blocked in the transgenic flowers. This blockage was due to uncontrolled cell elongation at the region corresponding to wild-type AZ. The role of NtBOP2 in regulating cell elongation was further demonstrated in Bright Yellow 2 single cells: perturbation of NtBOP2 function by a dominant negative strategy led to the formation of abnormally elongated cells. Subcellular localization analysis showed that NtBOP2-green fluorescent protein fusion proteins were targeted to both the nucleus and cytoplasm. Yeast two-hybrid, firefly luciferase complementation imaging, and in vitro pull-down assays demonstrated that NtBOP2 proteins interacted with TGA transcription factors. Taken together, these results indicated that NtBOP2 mediated the differentiation of AZ architecture by controlling longitudinal cell growth. Furthermore, NtBOP2 may achieve this outcome through interaction with the TGA transcription factors and via an ethylene-independent signaling pathway.

ROP11 GTPase Negatively Regulates ABA Signaling by Protecting ABI1 Phosphatase Activity from Inhibition by the ABA Receptor RCAR1/PYL9 in Arabidopsis

The phytohormone abscisic acid (ABA) regulates many key processes in plants, such as seed germination, seedling growth, and abiotic stress tolerance. In recent years, a minimal set of core components of a major ABA signaling pathway has been discovered. These components include a RCAR/PYR/PYL family of ABA receptors, a group of PP2C phosphatases, and three SnRK2 kinases. However, how the interactions between the receptors and their targets are regulated by other proteins remains largely unknown. In a companion paper published in this issue, we showed that ROP11, a member of the plant-specific Rho-like small GTPase family, negatively regulates multiple ABA responses in Arabidopsis. The current work demonstrated that the constitutively active ROP11 (CA-ROP11) can modulate the RCAR1/PYL9-mediated ABA signaling pathway based on reconstitution assays in Arabidopsis thaliana protoplasts. Furthermore, using luciferase complementation imaging, yeast two-hybrid assays, co-immunoprecipitation assays in Nicotiana benthamiana and bimolecular fluorescence complementation assays, we demonstrated that CA-ROP11 directly interacts with ABI1, a signaling component downstream of RCAR1/PYL9. Finally, we provided biochemical evidence that CA-ROP11 protects ABI1 phosphatase activity from inhibition by RCAR1/PYL9 and thus negatively regulates ABA signaling in plant cells. A model of how ROP11 acts to negatively regulate ABA signaling is presented.

Noninvasive Imaging Reveals Inhibition of Ovarian Cancer by Targeting CXCL12-CXCR4

Patients with metastatic ovarian cancer continue to have a dismal prognosis, emphasizing the need for new strategies to identify and develop new molecular targets for therapy. Chemokine CXCL12 and its receptor CXCR4 are upregulated in metastatic ovarian cancer cells and the intraperitoneal tumor microenvironment. CXCL12-CXCR4 signaling promotes multiple steps in proliferation and dissemination of ovarian cancer cells, suggesting that targeted inhibition of this pathway will limit tumor progression. To investigate CXCL12-CXCR4 signaling in ovarian cancer and establish effects of inhibiting this pathway on tumor progression and survival, we designed a Gaussia luciferase complementation imaging reporter system to detect CXCL12 binding to CXCR4 in ovarian cancer cells. In cell-based assays, we established that the complementation imaging reporter could detect CXCL12 binding to CXCR4 and quantify specific inhibition of ligand-receptor interaction. We monitored CXCL12-CXCR4 binding and inhibition in a mouse xenograft model of metastatic human ovarian cancer by imaging Gaussia luciferase complementation and assessed tumor progression with firefly luciferase. Bioluminescence imaging studies in living mice showed that treatment with AMD3100, a clinically approved inhibitor of CXCL12-CXCR4, blocked ligand-receptor binding and reduced growth of ovarian cancer cells. Treatment with AMD3100 also modestly improved overall survival of mice with metastatic ovarian cancer. The Gaussia luciferase complementation imaging reporter system will facilitate further preclinical development and optimization of CXCL12-CXCR4 targeted compounds for treatment of ovarian cancer. Our research supports clinical translation of existing CXCR4 inhibitors for molecular therapy for ovarian cancer.

Real-Time Imaging of Notch Activation with a Luciferase Complementation-Based Reporter

Notch signaling regulates many cellular processes during development and adult tissue renewal. Upon ligand binding, Notch receptors undergo ectodomain shedding followed by γ-secretase-mediated release of the Notch intracellular domain (NICD), which translocates to the nucleus and associates with the DNA binding protein CSL [CBF1/RBPjκ/Su(H)/Lag1] to activate gene expression. Mammalian cells contain four Notch receptors that can have both redundant and specific activities. To monitor activation of specific Notch paralogs in live cells and in real time, we developed luciferase complementation imaging (LCI) reporters for NICD-CSL association and validated them as a specific, robust, and sensitive assay system that enables structure-function and pharmacodynamic analyses. Detailed kinetic analyses of various mechanistic aspects of Notch signaling, including nuclear translocation and inhibition of the activities of γ-secretase and ADAM metalloproteases, as well as agonist- and ligand-dependent activation, were conducted in live cells. These experiments showed that Notch-LCI is an effective approach for characterizing modulators that target Notch signaling and for studying pathway dynamics in normal and disease contexts.

Quantitative analysis of dynamic protein–protein interactions in planta by a floated‐leaf luciferase complementation imaging (FLuCI) assay using binary Gateway vectors

Dynamic protein-protein interactions are essential in all cellular and developmental processes. Protein-fragment complementation assays allow such protein-protein interactions to be investigated in vivo. In contrast to other protein-fragment complementation assays, the split-luciferase (split-LUC) complementation approach facilitates dynamic and quantitative in vivo analysis of protein interactions, as the restoration of luciferase activity upon protein-protein interaction of investigated proteins is reversible. Here, we describe the development of a floated-leaf luciferase complementation imaging (FLuCI) assay that enables rapid and quantitative in vivo analyses of protein interactions in leaf discs floating on a luciferin infiltration solution after transient expression of split-LUC-labelled interacting proteins in Nicotiana benthamiana. We generated a set of eight Gateway-compatible split-LUC destination vectors, enabling fast, and almost fail-safe cloning of candidate proteins to the LUC termini in all possible constellations. We demonstrate their functionality by visualizing the well-established homodimerization of the 14-3-3 regulator proteins. Quantitative interaction analyses of the molybdenum co-factor biosynthesis proteins CNX6 and CNX7 show that the luciferase-based protein-fragment complementation assay allows direct real-time monitoring of absolute values of protein complex assembly. Furthermore, the split-LUC assay is established as valuable tool to investigate the dynamics of protein interactions by monitoring the disassembly of actin filaments in planta. The new Gateway-compatible split-LUC destination vector system, in combination with the FLuCI assay, provides a useful means to facilitate quantitative analyses of interactions between large numbers of proteins constituting interaction networks in plant cells.

Functional analysis of the structural domain of ARF proteins in rice (Oryza sativa L.)

Auxin response factors (ARFs) are key regulators of plant growth and development. Through interaction with auxin/indole acetic acid (Aux/IAA) proteins, they influence the expression of auxin response genes. An ARF gene family has been predicted in rice, but the functions of the individual structural domains of the OsARFs remain obscure. Bioinformatics was used to analyse the position of the DNA-binding domain (DBD), middle region (MR), and C-terminal dimerization domain (CTD) of OsARFs, and experimentally confirmed the presence of a classical monopartite nuclear localization signal (NLS) in the DBD. The DBD was shown to contribute to nuclear localization of OsARF proteins in addition to its known DNA-binding function. Interactions between 14 integrated OsARFs and 15 OsIAA proteins were tested using yeast two-hybrid assays. It was found that eight OsARF activators interacted with the 15 OsIAA proteins, while six OsARF repressors did not. The interactions between the MR+CTD or CTD of 10 OsARFs and 15 OsIAA proteins were also tested and the results were consistent with those of each intact OsARF, although some slight differences in interaction intensity were observed by α-galactosidase quantitative assays. The truncated CTD of OsARF11 did not interact with any OsIAA, implying that the CTD is required for ARF–IAA dimerization, and that the MR influences the interaction intensity in yeast. A subset of the interactions in yeast were also observed in tobacco plants using firefly luciferase complementation imaging assays, indicating that these interactions are specific in plants, and might have a special role in the auxin signalling response. This study provides new insight into the structure of OsARF proteins and ARF–Aux/IAA interactions.

Abstract 2672: Characterization of Hsp90/Cdc37 interaction and their critical residues using luciferase fragment complementation imaging

Abstract The purpose of this study is to investigate Hsp90/Cdc37 interaction and identify residues critical for the interaction using real-time luciferase complementation imaging and computational modeling. Molecular imaging using a split Renilla luciferase fragment complementation (SRL-FC) was established to monitor real-time Hsp90/Cdc37interactions in living cells. Computational modeling and molecular dynamics simulations were used to evaluate the interaction interface of Hsp90/Cdc37 complex and critical residues in the interaction interface were identified for mutagenesis. Western Blotting and co-immunoprecipitation were used to determine protein expression and complex formation. The molecular imaging in living cells showed that Hsp90 and Cdc37 helped the complementation of N-terminus of Renilla luciferase (fused with Hsp90; N-RL-Hsp90) and C-terminus of Renilla luciferase (fused with Cdc37, Cdc37-C-RL) with 170-fold higher luciferase activity than controls without Hsp90/Cdc37. This suggests that the SRL-FC is sensitive and specific to monitor the interaction of full length human Hsp90 and Cdc37. Molecular dynamics simulations revealed that the interface was constituted by a series of residues interacting through hydrophobic or polar interactions. Mutagenesis confirmed that mutations in Hsp90 (A121N, Q133A, F134A) and mutations in Cdc37 (M164K, R167A) led to the loss of complementation of N-RL-Hsp90 and Cdc37-C-RL (5 to 10-fold lower luciferase activity compared to wild-type), indicating these residues are critical in Hsp90/Cdc37 interactions. In comparison, mutations in Hsp90 (E47A, S113A) and mutations in Cdc37 (A204E) only decreased 50% of the complementation of N-RL-Hsp90 and Cdc37-C-RL, which suggests that these residues contribute less to Hsp90/Cdc37 interaction. In contrast, mutations of Hsp90 (R46A, S50A) and mutation in Cdc37 (L165H) did not change the complementation of N-RL-Hsp90 and Cdc37-C-RL, indicating that these residues did not contribute to Hsp90/Cdc37 interaction. The data suggests that molecular imaging using split Renilla luciferase fragment complementation (SRL-FC) can be used to identify the critical residues in Hsp90/Cdc37 interaction. Although Hsp90/Cdc37 interactions are through both hydrophobic and polar interactions, mutation in a single amino acid residue, including Ala121, Gln133, Phe134 in human Hsp90 and Met164, Arg167 in human Cdc37, is sufficient to disrupt Hsp90/Cdc37 interaction. 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 2672.

Multiple receptor complexes assembled for transmitting CLV3 signaling in Arabidopsis

In Arabidopsis, the feedback regulatory loop between CLAVATA3 (CLV3) signaling pathway and transcription factor, WUSCHEL (WUS) plays a significant role in shoot apical meristems (SAM) maintenance. Previously, CLV1/CLV2 heterodimers were supposed to perceive and transmit CLV3 signaling. Recent genetic analysis isolated a novel receptor kinase, CORYNE (CRN), which was found to be involved in the CLV3 pathway. Therefore, new hypothesis was put forward that CRN probably acts with CLV2 to transmit CLV3 in parallel with CLV1 based on genetic analysis. In our recent work, we took advantage of firefly luciferase complementation imaging (LCI) assay to analyze the interactions among CLV1, CLV2 and CRN in both Arabidopsis thaliana protoplasts and Nicotiana benthamiana leaves. We identified the physical interaction between CLV2 and CRN in the absence of CLV3 and found some interesting phenomenon such as CLV1, CLV2, and CRN may form a complex, and that CRN was able to form homodimers. These new observations make the relationships among these three proteins more complex than that indicated in two-parallel pathway model. Combining current genetic and our new biochemical evidence, a more possible and detailed model for CLV3 pathway was developed.

Analysis of interactions among the CLAVATA3 receptors reveals a direct interaction between CLAVATA2 and CORYNE in Arabidopsis

In Arabidopsis, CORYNE (CRN), a new member of the receptor kinase family, was recently isolated as a key player involved in the CLAVATA3 (CLV3) signaling pathway, thereby playing an important role in regulating the development of shoot and root apical meristems. However, the precise relationships among CLAVATA1 (CLV1), CLAVATA2 (CLV2), and CRN receptors remain unclear. Here, we demonstrate the subcellular localization of CRN and analyze the interactions among CLV1, CLV2, and CRN using firefly luciferase complementation imaging (LCI) assays in both Arabidopsis mesophyll protoplasts and Nicotiana benthamiana leaves. Fluorescence targeting showed that CRN was localized to the plasma membrane. The LCI assays coupled with co-immunoprecipitation assays demonstrated that CLV2 can directly interact with CRN in the absence of CLV3. Additional LCI assays showed that CLV1 did not interact with CLV2, but can interact weakly with CRN. We also found that CLV1 can interact with CLV2-CRN heterodimers, implying that these three proteins may form a complex. Moreover, CRN, rather than CLV1 and CLV2, was able to form homodimers without CLV3 stimulation. Taken together, our results add direct evidence to the newly proposed two-parallel receptor pathways model and therefore provide new insights into the CLV3 signaling pathway.

Luciferase Fragment Complementation Imaging of Conformational Changes in the Epidermal Growth Factor Receptor

Crystal structures of the epidermal growth factor (EGF) receptor suggest that its activation is associated with extensive conformational changes in both the extracellular and intracellular domains. However, evidence of these structural dynamics in intact cells has been lacking. Here we use luciferase complementation imaging to follow EGF-induced conformational changes in its receptor in real time in live cells. When the luciferase fragments are fused to the C terminus of an EGF receptor lacking the cytoplasmic domain, EGF stimulates a rapid increase in luciferase activity, consistent with ligand-induced receptor dimerization. However, when the luciferase fragments are fused to the C terminus of the full-length receptor, EGF induces a rapid but transient decrease in luciferase activity. The decrease requires tyrosine kinase activity, whereas the subsequent recovery requires MAP kinase activity. Our data demonstrate the utility of the luciferase system for in vivo imaging changes in EGF receptor dimerization and conformation. They also identify two sequential ligand-induced conformational changes in the EGF receptor.

P3-330: Luciferase complementation imaging as a real-time reporter for the gamma-secretase cleavage of notch

P3-330: Luciferase complementation imaging as a real-time reporter for the gamma-secretase cleavage of notch

Firefly Luciferase Complementation Imaging Assay for Protein-Protein Interactions in Plants

The development of sensitive and versatile techniques to detect protein-protein interactions in vivo is important for understanding protein functions. The previously described techniques, fluorescence resonance energy transfer and bimolecular fluorescence complementation, which are used widely for protein-protein interaction studies in plants, require extensive instrumentation. To facilitate protein-protein interaction studies in plants, we adopted the luciferase complementation imaging assay. The amino-terminal and carboxyl-terminal halves of the firefly luciferase reconstitute active luciferase enzyme only when fused to two interacting proteins, and that can be visualized with a low-light imaging system. A series of plasmid constructs were made to enable the transient expression of fusion proteins or generation of stable transgenic plants. We tested nine pairs of proteins known to interact in plants, including Pseudomonas syringae bacterial effector proteins and their protein targets in the plant, proteins of the SKP1-Cullin-F-box protein E3 ligase complex, the HSP90 chaperone complex, components of disease resistance protein complex, and transcription factors. In each case, strong luciferase complementation was observed for positive interactions. Mutants that are known to compromise protein-protein interactions showed little or much reduced luciferase activity. Thus, the assay is simple, reliable, and quantitative in detection of protein-protein interactions in plants.

Kinetics of regulated protein-protein interactions revealed with firefly luciferase complementation imaging in cells and living animals.

Signaling pathways regulating proliferation, differentiation, and apoptosis are commonly mediated through protein-protein interactions as well as reversible phosphorylation of proteins. To facilitate the study of regulated protein-protein interactions in cells and living animals, we optimized firefly luciferase protein fragment complementation by screening incremental truncation libraries of N- and C-terminal fragments of luciferase. Fused to the rapamycin-binding domain (FRB) of the kinase mammalian target of rapamycin and FK506-binding protein 12 (FKBP), respectively, the optimized FRB-N-terminal luciferase fragment (NLuc)/C-terminal luciferase fragment (CLuc)-FKBP luciferase complementation imaging (LCI) pair reconstituted luciferase activity in cells upon single-site binding of rapamycin in an FK506-competitive manner. LCI was used in three independent applications. In mice bearing implants of cells expressing the FRB-NLuc/CLuc-FKBP LCI pair, dose- and time-dependent luciferase activity allowed target-specific pharmacodynamic analysis of rapamycin-induced protein-protein interactions in vivo. In cells expressing a Cdc25C-NLuc/CLuc-14-3-3epsilon LCI pair, drug-mediated disruption of cell cycle regulated protein-protein interactions was demonstrated with the protein kinase inhibitor UCN-01 in a phosphoserine-dependent manner. When applied to IFN-gamma-dependent activation of Janus kinase/signal transducer and activator of transcription 1 (STAT1), LCI revealed, in the absence of ligand-induced phosphorylation, STAT1 proteins existing in live cells as preformed dimers. Thus, optimized LCI provides a platform for near real-time detection and characterization of regulated and small molecule-induced protein-protein interactions in intact cells and living animals and should enable a wide range of novel applications in drug discovery, chemical genetics, and proteomics research.

Optimizing Luciferase Protein Fragment Complementation for Bioluminescent Imaging of Protein–Protein Interactions in Live Cells and Animals

Publisher Summary This chapter discusses the optimization of luciferase protein fragment complementation for bioluminescent imaging of protein–protein interactions in live cells and animals. Protein–protein interactions regulate a variety of cellular functions, including cell cycle progression, signal transduction, and metabolic pathways. The strategies for detecting protein-binding partners using conventional cell biology assays with the intent of identifying properties that might be exploited for imaging applications are presented. Two-hybrid systems exploit the modular nature of transcription factors, many of which can be separated into discrete DNA-binding and activation domains. Protein–fragment complementation assays depend on the division of a monomeric reporter enzyme into two separate inactive components that can reconstitute function upon association. Luciferase complementation imaging of protein interactions in cells and small animal models has been developed to permit the rapid and repetitive measurement of protein pairs of interest. It is found that optimized luciferase complementation, quantified by imaging with a charge coupled device camera, equipped with appropriate software, can provide an accurate measure of relative protein association in animals and quantitative measurement of protein association in live cells.