The Nuclear Translocation Assay for Intracellular Protein-Protein Interactions and its Application to the Bcr Coiled-Coil Domain

Abstract

Protein interactions are critical for normal biological processes and molecular pathogenesis. While it is important to study these interactions, there are limited assays that are performed inside the cell, in the native cell environment, where the majority of protein-protein interactions take place. Here we present a method of studying protein interactions intracellularly using one protein of interest fused to a localization-controllable enhanced GFP (EGFP) construct and the other protein of interest fused to the red fluorescent protein, DsRed. Nuclear translocation of the EGFP construct is induced by addition of a ligand, and the difference in nuclear localization between the induced and noninduced states of the DsRed construct provides an indication of the interaction between the two proteins. This assay, the nuclear translocation assay (NTA), is introduced here as broadly applicable for studying protein interactions in the native environment inside cells and is demonstrated using forms of the coiled-coil domain from the breakpoint cluster region (Bcr) protein.

The Nuclear Option

Protein-protein interaction studies offer insight into the molecular mechanisms that govern nearly every normal cellular process as well as aberrant processes that lead to disease. Although the majority of these interactions occur inside cells, only a limited number of intracellular assays are available for detecting protein-protein interactions. Resonance energy transfer methods, such as Förster resonance energy transfer (FRET), are one option, but these depend on sensitive instruments to detect light emitted at specific wavelengths. Yeast and mammalian two-hybrid systems are also options, but these techniques require expression of at least three proteins, including bait, prey, and reporter—as well as any additional control proteins desired. Searching for a simpler approach for qualitative analysis of intracellular protein-protein interactions, A. Dixon and C. Lim from the University of Utah (Salt Lake City, UT) developed a novel nuclear translocation assay. The assay relies on the use of a protein switch containing a nuclear export signal (NES) and a nuclear localization signal (NLS) along with a dexamethasone binding domain. The NES retains the protein switch in the cytoplasm when ligand is absent, but when ligand binds, it induces a conformational change that exposes the NLS and directs the protein into the nucleus. This translocation is dose-dependent and can be reversed by removing the ligand. For the nuclear translocation assay, the authors fused a protein of interest to EFGP and the protein switch, rendering it both detectable by fluorescence microscopy and able to move into the nucleus when bound to the ligand. A potential interacting protein partner was then fused to DsRed. If the two proteins interact in the cytoplasm, both red and green signals would be detected in the nucleus upon addition of the ligand. Measurements of red signal prior to and after ligand addition enables differentiation of induced protein translocation from those proteins that partially localize to the nucleus under normal conditions. The authors validated the assay with wild-type and mutant forms of the coiled-coil domain from the breakpoint cluster region protein (Bcr), which is thought to contribute to oncogenesis during chronic myelogenous leukemia by mediating oligomerization of Bcr and Abelson kinase protein (Abl). These experiments demonstrated that the nuclear translocation assay exhibited the ability to quantify the amount of protein present in each cell and detect variations in binding and specificity of the mutant coiled coil.

See “The nuclear translocation assay for intracellular protein-protein interactions and its application to the Bcr coiled-coil domain” on page 519.

Keywords: :

• protein-protein interactions
• protein switch
• nuclear translocation assay
• Bcr
• EGFP
• DsRed

Acknowledgments

Funding was provided by the National Institutes of Health (NIH; grant no. R01 CA129528). We acknowledge the use of DNA/Peptide Core supported by the National Cancer Institute (NCI) Cancer Center Support Grant no. P30 CA042014, awarded to Huntsman Cancer Institute. We would like to thank Thomas Cheatham, Scott Pendley, Benjamin Bruno, Jonathan Constance, Rian Davis, Mohanad Mossalam, and David Woessner for scientific input. This paper is subject to the NIH Public Access Policy.

Competing interests

The authors declare no competing interests.

Supplementary data

To view the supplementary data that accompany this paper please visit the journal website at: www.tandfonline.com/doi/suppl/10.2144/000113450

Additional information

Funding

Funding was provided by the National Institutes of Health (NIH; grant no. R01 CA129528). We acknowledge the use of DNA/Peptide Core supported by the National Cancer Institute (NCI) Cancer Center Support Grant no. P30 CA042014, awarded to Huntsman Cancer Institute. We would like to thank Thomas Cheatham, Scott Pendley, Benjamin Bruno, Jonathan Constance, Rian Davis, Mohanad Mossalam, and David Woessner for scientific input. This paper is subject to the NIH Public Access Policy