| Autor: |
Shin YC; Department of Biochemistry, School of Life Science, Chungbuk National University, Cheongju 28644, Republic of Korea.; bHLBIO, Cheongju 28119, Republic of Korea., Cho M; Department of Biochemistry, School of Life Science, Chungbuk National University, Cheongju 28644, Republic of Korea., Hwang JM; Center for Genomic Integrity, Institute for Basic Science (IBS), Ulsan 44919, Republic of Korea., Myung K; Center for Genomic Integrity, Institute for Basic Science (IBS), Ulsan 44919, Republic of Korea.; Department of Biomedical Engineering, Ulsan National Institute of Science and Technology (UNIST), Ulsan 44919, Republic of Korea., Kweon HS; Center for Bio-Imaging & Translational Research and Bioimaging Data Curation Center, Korea Basic Science Institute (KBSI), Cheongju 28119, Republic of Korea., Lee ZW; bHLBIO, Cheongju 28119, Republic of Korea., Seong HA; Department of Biochemistry, School of Life Science, Chungbuk National University, Cheongju 28644, Republic of Korea., Lee KB; Center for Bio-Imaging & Translational Research and Bioimaging Data Curation Center, Korea Basic Science Institute (KBSI), Cheongju 28119, Republic of Korea. |
| Abstrakt: |
Conventional biochemical methods for studying cellular signaling cascades have relied on destructive cell disruption. In contrast, the live cell imaging of fluorescent-tagged transfected proteins offers a non-invasive approach to understanding signal transduction events. One strategy involves monitoring the phosphorylation-dependent shuttling of a fluorescent-labeled kinase between the nucleus and cytoplasm using nuclear localization, export signals, or both. In this paper, we introduce a simple method to visualize intracellular signal transduction in live cells by exploring the translocation properties of PKC from the cytoplasm to the membrane. We fused bait protein to PKC, allowing the bait (RFP-labeled) and target (GFP-labeled) proteins to co-translocate from the cytoplasm to the membrane. However, in non-interacting protein pairs, only the bait protein was translocated to the plasma membrane. To verify our approach, we examined the Raf-MEK-ERK signaling cascade (ERK pathway). We successfully visualized direct Raf1/MEK2 interaction and the KSR1-containing ternary complex (Raf1/MEK2/KSR1). However, the interaction between MEK and ERK was dependent on the presence of the KSR1 scaffold protein under our experimental conditions. |
