Imaging the Raf-MEK-ERK Signaling Cascade in Living Cells

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.

Keywords: ERK pathway; Raf–MEK–ERK signaling cascade; cell-based assay; scaffold protein; visualizing protein interaction.

Figure 1

Confocal images of the Raf–MEK–ERK signaling cascade in living cells without exogenous expression of scaffold proteins. HEK-293T cells were co-transfected with PKCδ–mRFP–MEK2 (bait) and either eGFP–Raf1 or eGFP–ERK2 (prey). Transiently co-transfected cells were serum-starved for 16–18 h in serum-free DMEM before stimulation with EGF. After induction with 100 ng/mL of EGF for 5 min, the cells were treated with PMA (final concentration 1 μM) in serum-free DMEM. Raf1 was co-translocated with MEK2 to the plasma membrane due to the translocation property of PKCδ (top row); in comparison, only MEK2 protein from the MEK2/ERK2 pair was translocated to the plasma membrane (bottom row). The images were acquired 3–5 min after PMA treatment. The scale bar is 10 μm.

Figure 2

A schematic representation of the Raf–MEK–ERK signaling cascade, including the KSR scaffold protein in cells. In quiescent cells, the KSR scaffold protein is typically cytoplasmic and constitutively bound to MEK and 14–3-3. Upon receptor tyrosine kinase (RTK) stimulation, signaling cells initially utilize KSR to enhance signal transmission from Raf to MEK by facilitating Raf/MEK interaction. Subsequently, KSR attenuates signaling by docking activated ERK. Overall, KSR regulates the intensity and duration of Raf–MEK–ERK signaling cascade activation.

Figure 3

Confocal images of the KSR1/MEK2/14-3-3ζ ternary protein complex in quiescent cells. (A) HEK-293T cells were transiently co-transfected with the following protein pairs: PKCδ–mRFP–KSR1/eGFP–MEK2, PKCδ–mRFP–KSR1/eGFP–14-3-3ζ, and PKCδ–mRFP–MEK2/eGFP–14-3-3ζ. After PMA treatment, KSR1 and MEK2 were co-translocated to the plasma membrane, with evidence of constitutive interaction between them (top row); KSR1 and 14-3-3ζ were also co-translocated to the plasma membrane, with their interaction shown (middle row); and MEK2 was translocated alone to the plasma membrane, showing no interaction with 14-3-3ζ (bottom row). (B) HEK-293T cells were transiently co-transfected with PKCδ–mRFP–MEK2, TagBFP–KSR1, and eGFP–14-3-3ζ. The ternary protein complex, MEK2/KSR1/14-3-3ζ, was co-translocated to the plasma membrane after PMA treatment. The images were acquired 3-5 min after PMA treatment. The scale bar is 10 μm.

Figure 4

Confocal images of the Raf–MEK–ERK signaling cascade in living cells with KSR1 scaffold protein. (A) HEK-293T cells were transiently co-transfected with PKCδ–mRFP–KSR1 and each of eGFP–Raf1, eGFP–MEK2, and eGFP–ERK2. The cells were then serum-starved, stimulated with EGF, and treated with PMA. KSR1 translocation to the plasma membrane was observed, with or without interaction with Raf1, MEK2, or ERK2, as indicated (top, middle, and bottom rows, respectively). (B) HEK-293T cells were transiently co-transfected with either PKCδ–mRFP–MEK2/TagBFP–KSR1/eGFP–Raf1 or PKCδ–mRFP–MEK2/TagBFP–KSR1/eGFP–ERK2. The cells were then serum-starved, stimulated with EGF, and treated with PMA. The ternary protein complexes, both Raf1/KSR1/MEK2 and MEK2/KSR1/ERK2, were co-translocated to the plasma membrane. The images were acquired 3–5 min after PMA treatment. The scale bar is 10 μm.