Receptor Level Mechanisms Are Required for Epidermal Growth Factor (EGF)-stimulated Extracellular Signal-regulated Kinase (ERK) Activity Pulses

Abstract

In both physiological and cell culture systems, EGF-stimulated ERK activity occurs in discrete pulses within individual cells. Many feedback loops are present in the EGF receptor (EGFR)-ERK network, but the mechanisms driving pulsatile ERK kinetics are unknown. Here, we find that in cells that respond to EGF with frequency-modulated pulsatile ERK activity, stimulation through a heterologous TrkA receptor system results in non-pulsatile, amplitude-modulated activation of ERK. We further dissect the kinetics of pulse activity using a combination of FRET- and translocation-based reporters and find that EGFR activity is required to maintain ERK activity throughout the 10-20-minute lifetime of pulses. Together, these data indicate that feedbacks operating within the core Ras-Raf-MEK-ERK cascade are insufficient to drive discrete pulses of ERK activity and instead implicate mechanisms acting at the level of EGFR.

Keywords: EKAR; ERKTR; TRK1-transforming tyrosine kinase protein (Trk-A); epidermal growth factor receptor (EGFR); extracellular-signal-regulated kinase (ERK); fluorescence resonance energy transfer (FRET); mitogen-activated protein kinase (MAPK); nerve growth factor (NGF); signal transduction.

FIGURE 1.

Encoding of extracellular signal strength by the ERK network. A, schematic diagram of EGFR-ERK signaling system and reporter readouts. Green arrows indicate potential positive feedback loops. B, conceptual comparison of amplitude- and frequency-modulated signal responses.

FIGURE 2.

EKAR3, an improved FRET reporter for detection of ERK activity. A, ratiometric CFP/YFP images of MCF10A-EKAR3 cells before and after EGF (20 ng/ml) addition. Scale bars, 50 μm. B, comparison of EKAR3 and EKAR-EV reporter responses to saturating EGF (20 ng/ml) stimulation followed by MEK inhibition. CV_on, CV_off, and CVΔ indicate the coefficient of variation for cells in the EGF-stimulated state, cells in the MEK inhibited state, and the individual cell difference between the on and off state, respectively. Data shown were collected in MCF10A FOSL1::mCherry cells; mCherry fluorescence (not shown) does not interfere with the FRET signals. C, ERK response to different levels of EGF stimulation, as measured by EKAR3. MCF10A-EKAR3 cells were cultured in serum-free, growth factor-free imaging medium and then treated at the indicated time. Reporter data are displayed in three ways: two representative individual cells (top), 50 cell traces overlaid with the population mean in red and the 25th and 75th percentile values in blue (middle), and a heat map in which each row represents one cell and EKAR3 signal is indicated by color (bottom).

FIGURE 3.

Expression of TrkA confers NGF sensitivity to MCF10A cells. MCF10A cells carrying an inducible TrkA gene were cultured in serum-free, growth factor-free medium in the absence or presence of 50 ng/ml doxycycline and then treated at the indicated time with 1 ng/ml EGF, 25 ng/ml NGF, or 1 μm gefitinib. EKAR3 responses are plotted as heat maps depicting a representative set of 20 cells for each condition.

FIGURE 4.

Amplitude-modulated ERK signaling induced by NGF-TrkA. MCF10A-EKAR3 cells expressing TrkA were cultured in serum-free, growth factor-free medium in the presence of 100 ng/ml doxycycline and then treated with the indicated concentrations of NGF at the times indicated by arrowheads. Data are plotted as in Fig. 2C.

FIGURE 5.

Kinetic differences between EKAR3 and ERKTR. A, comparison of EKAR3 and ERKTR responses to EGF stimulation (left) or EGFR inhibition (right). MCF10A cells stably expressing both reporters were imaged in the absence of growth factors and serum and then treated with 20 ng/ml EGF at time 0 (left) or cultured in 20 ng/ml EGF and treated with 1 μm gefitinib at time 0 (right). For each reporter, the mean signal of >200 cells, normalized to its minimum and maximum, is shown as a line plot; shaded regions indicate one S.D. The inset in the right panel highlights the more rapid decrease in ERKTR signal during the initial period of inhibition. B, comparison of EKAR3 and ERKTR-mCherry signals in two representative MCF10A cells expressing both reporters during spontaneous pulsing (under continuous treatment with 0.05 ng/ml EGF). Each reporter was normalized to its maximum and minimum value in each cell. C, detailed view of a single spontaneous ERK activity pulse detected by EKAR3 and ERKTR-mCherry. MCF10A-EKAR3-ERKTR cells were cultured continuously in the presence of 0.1 ng/ml EGF, and one cell with an isolated representative pulse was chosen for display. Top panel, reporter signals, normalized to the minimum and maximum for each signal. Bottom panels, frame-to-frame difference for each reporter signal. D, signature of pulse continuation in dual ERK reporter cells exposed continuously to 0.05 ng/ml EGF. Distributions represent the value of ΔERKTR at an arbitrarily chosen time point for all cells (gray) or for cells in which ΔEKAR3 was greater than the threshold value for pulse activity at the previous time point (orange). Frequency is normalized to its peak value.

FIGURE 6.

Requirement of continuous EGFR activity during ERK activity pulses. A, kinetics of ERK inhibition by gefitinib. Top, heat maps of MCF10A-EKAR3 cells exposed to high or low EGF and treated with 1 μm gefitinib at the indicated time. Bottom, average EKAR3 signal for 500 cells following gefitinib treatment in the presence of 20 ng/ml EGF. The point marked t₁ indicates the first time point following gefitinib application, whereas t₂ indicates the first time point at which a reduction in EKAR3 signal is detectable, indicating sufficient binding of gefitinib to inhibit EGFR. B, representative EKAR3 and ERKTR responses in an individual cell treated with 1 μm gefitinib during a spontaneous ERK activity pulse (stimulated by 0.02 ng/ml EGF). C, pulse continuation signatures in the presence of gefitinib. Cells cultured in the presence of 0.1, 0.05, or 0.02 ng/ml EGF were imaged and treated with gefitinib as in A, or mock-treated. Distributions of ΔERKTR at t₂ are shown for all cells (gray) or for cells with above-threshold ΔEKAR3 at t₁ (orange). Data shown were pooled from all three EGF concentrations.