Mol. Cell, published online 5 December 2012; doi:10.1016/j.molcel.2012.11.002

The EGFR-ERK-MAPK pathway drives cell proliferation and is a major target for anticancer therapy. Although this pathway has been intensively investigated, most experiments have focused on acute responses to EGF treatment. Albeck et al. now evaluate the pathway (ERK activity, effector activation and cell proliferation) in more physiological steady-state conditions, with high-content immunofluorescence in single cells. ERK activity varied in frequency and duration in an EGF concentration–dependent manner, and the duration of time cells were in the ERKON state rather than the number of active pulses correlated with S phase entry. Inhibition of EGFR with the small molecule gefitinib completely inhibited ERK at high concentrations but yielded pulses of ERK activity at lower concentrations (frequency modulation). In contrast, inhibition of MEK with the small molecule PD0325901 revealed a concentration-dependent decrease in ERK output (amplitude modulation). Additional analysis revealed that pathway effectors responded to changes in the amplitude and frequency of ERK signaling and that steady-state effector activation was an excellent predictor of cell proliferation. The authors also noted that the relationship between ERK activity and proliferation was nonlinear: inhibition of up to 85% of ERK activity had less than a 2-fold effect on cell proliferation, but 95% inhibition reduced proliferation by a factor of ten. Thus, the amplitude and frequency of ERK signals affect cell proliferation, and quantitative approaches to assess pathway output in response to inhibitors will be necessary to model inhibitor efficacy for the clinic.