Graded Mitogen-Activated Protein Kinase Activity Precedes Switch-Like c-Fos Induction in Mammalian Cells

Abstract

The mitogen-activated protein kinase (MAPK) pathway is an evolutionarily conserved signaling module that controls important cell fate decisions in a variety of physiological contexts. During Xenopus oocyte maturation, the MAPK cascade converts an increasing progesterone stimulus into a switch-like, all-or-nothing response. While the importance of such switch-like behavior is widely discussed in the literature, it is not known whether the MAPK pathway in mammalian cells exhibits a switch-like or graded response. For this study, we used flow cytometry and immunofluorescence to generate single-cell measurements of MAPK signaling in Swiss 3T3 fibroblasts. In contrast to the case in Xenopus oocytes, we found that ERK activation in individual mammalian cells is not ultrasensitive and shows a graded response to changes in agonist concentration. Thus, the conserved MAPK signaling module exhibits different systems-level properties in different cellular contexts. Furthermore, the graded ERK response was converted into a more switch-like behavior at the level of immediate-early gene induction and cell cycle progression. Thus, while MAPK signaling is involved in all-ornothing cell fate decisions for both Xenopus oocyte maturation and mammalian fibroblast proliferation, the underlying mechanisms responsible for the switch-like nature of the cellular responses are different in these two systems, with the mechanism appearing to lie downstream of the kinase cascade in mammalian fibroblasts.Ultrasensitive switch-like responses control cell fate decisions in many biological settings, and the regulation of kinase activity is one way in which ultrasensitive behavior can be initiated (9,14). Such behavior is characterized by an all-ornothing response to increasing concentrations of stimulus (Fig. 1A). An ultrasensitive switch-like response displays a near vertical sigmoidal stimulus response curve. In the case of extreme ultrasensitive responses, signaling molecules switch between two discontinuous stable states and intermediate responses are not observed; this is referred to as a bistable response. Bistability may result from multistep phosphorylation events, positive feedback loops, double-negative feedback loops, and the relative abundance/activity of kinases and phosphatases driving the signaling cascade. In contrast to ultrasensitive signaling, a graded response in protein kinase activity changes incrementally with the stimulus concentration (Fig. 1B). Given the irreversible, all-or-nothing nature of many cell behaviors, including differentiation and cell cycle control, significant efforts have been focused on identifying the cellular mechanisms underlying bistability in biological systems.Bistability in mitogen-activated kinase (MAPK) signaling was first demonstrated experimentally by the progesteroneinduced maturation of Xenopus laevis oocytes. In pools of oocytes incubated with increasing concentrations of progesterone, extracellular signal-regulated kinase (ERK) activity appears to increase in a graded manner. However, whe...

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