Research ArticleCell Biology

Context-specific flow through the MEK/ERK module produces cell- and ligand-specific patterns of ERK single and double phosphorylation

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Sci. Signal.  02 Feb 2016:
Vol. 9, Issue 413, pp. ra13
DOI: 10.1126/scisignal.aab1967

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ERK phosphorylation patterns

In the ERK pathway, the dual-specificity kinase MEK phosphorylates a threonine and a tyrosine residue in ERK, and this dual-phosphorylated form is the fully active kinase. Iwamoto et al. used mass spectrometry, quantitative Western blotting, and mathematical modeling to explore MEK-dependent phosphorylation dynamics of ERK in skin and liver cells exposed to either a cytokine, IL-6, or a growth factor, HGF. Not surprisingly, the different stimuli produced different dynamics of ERK phosphorylation, and skin and liver cells responded differently to the same ligand. The dynamics of the changes in the abundance of the phosphorylated forms of ERK (pT-ERK, pY-ERK, and pTpY-ERK) and the relative distributions of the single- and double-phosphorylated forms of ERK were different. Mathematical modeling indicated that distinct network structures with or without regulated feedback loops produced the different dynamics of ERK phosphorylation and distributions of phosphorylated ERK. This study provides biochemical insight into how a single pathway can produce distinct responses, such as differentiation or proliferation.

Abstract

The same pathway, such as the mitogen-activated protein kinase (MAPK) pathway, can produce different cellular responses, depending on stimulus or cell type. We examined the phosphorylation dynamics of the MAPK kinase MEK and its targets extracellular signal–regulated kinase 1 and 2 (ERK1/2) in primary hepatocytes and the transformed keratinocyte cell line HaCaT A5 exposed to either hepatocyte growth factor or interleukin-6. By combining quantitative mass spectrometry with dynamic modeling, we elucidated network structures for the reversible threonine and tyrosine phosphorylation of ERK in both cell types. In addition to differences in the phosphorylation and dephosphorylation reactions, the HaCaT network model required two feedback mechanisms, which, as the experimental data suggested, involved the induction of the dual-specificity phosphatase DUSP6 and the scaffold paxillin. We assayed and modeled the accumulation of the double-phosphorylated and active form of ERK1/2, as well as the dynamics of the changes in the monophosphorylated forms of ERK1/2. Modeling the differences in the dynamics of the changes in the distributions of the phosphorylated forms of ERK1/2 suggested that different amounts of MEK activity triggered context-specific responses, with primary hepatocytes favoring the formation of double-phosphorylated ERK1/2 and HaCaT A5 cells that produce both the threonine-phosphorylated and the double-phosphorylated form. These differences in phosphorylation distributions explained the threshold, sensitivity, and saturation of the ERK response. We extended the findings of differential ERK phosphorylation profiles to five additional cultured cell systems and matched liver tumor and normal tissue, which revealed context-specific patterns of the various forms of phosphorylated ERK.

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