Abstract
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.
Highlights
Signal transduction networks mediate diverse cellular processes by modulating the cell’s gene-regulatory and cytoskeletal systems
In the signaling networks accessed by growth factor and cytokine receptors, the extracellular signal-regulated kinase (ERK) pathway is a principal mode of controlling cell proliferation and other responses, and its aberrant activation contributes to uncontrolled proliferation in the majority of human cancers (Dhillon et al, 2007; Roberts & Der, 2007)
ERK1 and ERK2 are among the mammalian mitogen-activated protein kinases (MAPKs), which are canonically activated in a three-tiered protein kinase cascade
Summary
Signal transduction networks mediate diverse cellular processes by modulating the cell’s gene-regulatory and cytoskeletal systems. In the signaling networks accessed by growth factor and cytokine receptors, the extracellular signal-regulated kinase (ERK) pathway is a principal mode of controlling cell proliferation and other responses, and its aberrant activation contributes to uncontrolled proliferation in the majority of human cancers (Dhillon et al, 2007; Roberts & Der, 2007). ERK cascade as a pathway motif belies a rich complexity in the regulation of ERK signaling, which has largely emerged from quantitative studies combining experimental measurements and kinetic modeling (Bhalla et al, 2002; Schoeberl et al, 2002; Sasagawa et al, 2005; Fujioka et al, 2006; Birtwistle et al, 2007; Chen et al, 2009; Schilling et al, 2009; Wang et al, 2009; Cirit et al, 2010; Cirit & Haugh, 2012). One important mode of regulation is adaptation of the pathway by ERK-dependent negative feedback, which desensitizes the activity of Raf and/or other upstream components (McKay & Morrison, 2007). The kinetics of ERK activation and adaptation have been quantitatively characterized (Cirit et al, 2010; Sturm et al, 2010; Fritsche-Guenther et al, 2011) and have proven to be important for proliferation and cell-fate decisions (Marshall, 1995; von Kriegsheim et al, 2009; Chung et al, 2010; Albeck et al, 2013)
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