Data-driven modeling reconciles kinetics of ERK phosphorylation, localization, and activity states

The extracellular signal-regulated kinase (ERK) signaling pathway controls cell proliferation and differentiation in metazoans. Two hallmarks of its dynamics are adaptation of ERK phosphorylation, which has been linked to negative feedback, and nucleocytoplasmic shuttling, which allows active ERK to phosphorylate protein substrates in the nucleus and cytosol. To integrate these complex features, we acquired quantitative biochemical and live-cell microscopy data to reconcile phosphorylation, localization, and activity states of ERK. While maximal growth factor stimulation elicits transient ERK phosphorylation and nuclear translocation responses, ERK activities available to phosphorylate substrates in the cytosol and nuclei show relatively little or no adaptation. Free ERK activity in the nucleus temporally lags the peak in nuclear translocation, indicating a slow process. Additional experiments, guided by kinetic modeling, show that this process is consistent with ERK's modification of and release from nuclear substrate anchors. Thus, adaptation of whole-cell ERK phosphorylation is a by-product of transient protection from phosphatases. Consistent with this interpretation, predictions concerning the dose-dependence of the pathway response and its interruption by inhibition of MEK were experimentally confirmed. Synopsis image Quantitative measurements reveal distinct kinetics of ERK phosphorylation, nuclear translocation and compartmentalized kinase activities. Experiments guided by kinetic simulations suggest that interactions with substrates transiently buffer activated ERK in the nucleus. Growth factor-stimulated phosphorylation and nuclear translocation of ERK show dramatic adaptation, whereas the kinase activities of ERK in the cytosol and nucleus do not. The rate-controlling process affecting ERK dynamics is linked to the availability of free, active ERK in the nucleus. A kinetic model including ERK interactions with cytosolic and nuclear substrates reconciles the diverse data. Model predictions emphasizing the transient buffering of double phosphorylated ERK by its substrates were confirmed experimentally.