Multiple sources of signal amplification within the B-cell Ras/MAPK pathway

The Ras-Map kinase (MAPK) cascade underlies functional decisions in a wide range of cell types and organisms. In B-cells, positive feedback-driven Ras activation is the proposed source of the digital (all or none) MAPK responses following antigen stimulation. However, an inability to measure endogenous Ras activity in living cells has hampered our ability to test this model directly. Here we leverage biosensors of endogenous Ras and ERK activity to revisit this question. We find that B-cell receptor (BCR) ligation drives switch-like Ras activation and that lower BCR signaling output is required for the maintenance versus the initiation of Ras activation. Surprisingly, digital ERK responses persist in the absence of positive feedback-mediated Ras activation, and digital ERK is observed at a threshold level of Ras activation. These data suggest an independent analogue-to-digital switch downstream of Ras activation and reveal that multiple sources of signal amplification exist within the Ras-ERK module of the BCR pathway.