Variable Thresholds for Phosphorylation Targets of the ERK Signaling Pathway

ABSTRACT: Cell fates regulated by ERK/MAP kinases respond to different thresholds of signaling strength. In mammalian cells, conditions that activate ERK to submaximal levels are sufficient to sustain proliferation, survival and transformation, while stimuli that activate ERK to very high levels often lead to cell death or cell cycle arrest. But while this “Goldilocks effect” is well known, the mechanisms have never been fully explained. In particular, threshold responses have been shown at the level of transcription and cell state changes, but whether phosphorylation responses upstream of these events also respond to thresholds is unknown. Here we used mass spectrometry-based phosphoproteomics to ask if molecular events in the ERK pathway respond to different thresholds of signaling strength, by quantifying changes in phosphorylation of pathway targets against the occupancy of the two activating phosphosites in ERK. The results show that most phosphorylation events track ERK activation faithfully, responding linearly with increasing 2P-ERK occupancy. But some sites respond nonlinearly, reaching maximal phosphorylation when 2P-ERK exceeds lower thresholds (10-40%), or increasing substantially after 2P-ERK exceeds higher thresholds (>60%). Low threshold sites are found on transcriptional repressors that facilitate proliferation when inactivated by ERK/RSK phosphorylation. By contrast, high threshold sites are found on proteins that are recruited to dsDNA breaks and mediate DNA repair. Measurement of phosphorylation occupancies also revealed unexpected differences between cell states not apparent from inhibitor fold-changes. Our findings demonstrate that signaling thresholds exist at the level of the phosphoproteome, providing potential mechanisms for regulating cellular responses to pathway strength.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Malignant Cell, Cell Culture

DISEASE(S): Melanoma

SUBMITTER: Kristyn Hayashi  

LAB HEAD: Natalie Ahn

PROVIDER: PXD071110 | Pride | 2026-02-25

REPOSITORIES: Pride