Establishing a Split Luciferase Assay for Proteinkinase G (PKG) Interaction Studies
AbstractNitric oxide (NO/cyclic guanosine monophosphate (cGMP)-regulated cellular mechanisms are involved in a variety of (patho-) physiological processes. One of the main effector molecules in this system, proteinkinase G (PKG), serves as a molecular switch by phosphorylating different target proteins and thereby turning them on or off. To date, only a few interaction partners of PKG have been described although the identification of protein–protein interactions (PPI) is indispensable for the understanding of cellular processes and diseases. Conventionally used methods to detect PPIs exhibit several disadvantages, e.g., co-immunoprecipitations, which depend on suitable high-affinity antibodies. Therefore, we established a cell-based protein-fragment complementation assay (PCA) for the identification of PKG target proteins. Here, a reporter protein (click beetle luciferase) is split into two fragments and fused to two different possible interaction partners. If interaction occurs, the reporter protein is functionally complemented and the catalyzed reaction can then be quantitatively measured. By using this technique, we confirmed the regulator of G-Protein signaling 2 (RGS2) as an interaction partner of PKGIα (a PKG-isoform) following stimulation with 8-Br-cGMP and 8-pCPT-cGMP. Hence, our results support the conclusion that the established approach could serve as a novel tool for the rapid, easy and cost-efficient detection of novel PKG target proteins. View Full-Text
- Supplementary File 1:
Supplementary (PDF, 476 KB)
Share & Cite This Article
Schramm, A.; Mueller-Thuemen, P.; Littmann, T.; Harloff, M.; Ozawa, T.; Schlossmann, J. Establishing a Split Luciferase Assay for Proteinkinase G (PKG) Interaction Studies. Int. J. Mol. Sci. 2018, 19, 1180.
Schramm A, Mueller-Thuemen P, Littmann T, Harloff M, Ozawa T, Schlossmann J. Establishing a Split Luciferase Assay for Proteinkinase G (PKG) Interaction Studies. International Journal of Molecular Sciences. 2018; 19(4):1180.Chicago/Turabian Style
Schramm, Andrea; Mueller-Thuemen, Philip; Littmann, Timo; Harloff, Manuela; Ozawa, Takeaki; Schlossmann, Jens. 2018. "Establishing a Split Luciferase Assay for Proteinkinase G (PKG) Interaction Studies." Int. J. Mol. Sci. 19, no. 4: 1180.
Note that from the first issue of 2016, MDPI journals use article numbers instead of page numbers. See further details here.