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Imaging the Raf-MEK-ERK Signaling Cascade in Living Cells.
- Source :
-
International journal of molecular sciences [Int J Mol Sci] 2024 Oct 01; Vol. 25 (19). Date of Electronic Publication: 2024 Oct 01. - Publication Year :
- 2024
-
Abstract
- 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.
- Subjects :
- Humans
Cell Membrane metabolism
MAP Kinase Kinase 2 metabolism
raf Kinases metabolism
Protein Kinase C metabolism
HeLa Cells
Phosphorylation
Animals
Protein Transport
Cytoplasm metabolism
Green Fluorescent Proteins metabolism
Green Fluorescent Proteins genetics
Protein Kinases
MAP Kinase Signaling System
Proto-Oncogene Proteins c-raf metabolism
Subjects
Details
- Language :
- English
- ISSN :
- 1422-0067
- Volume :
- 25
- Issue :
- 19
- Database :
- MEDLINE
- Journal :
- International journal of molecular sciences
- Publication Type :
- Academic Journal
- Accession number :
- 39408915
- Full Text :
- https://doi.org/10.3390/ijms251910587