Spatiotemporally Regulated Protein Kinase A Activity Is a Critical Regulator of Growth Factor-Stimulated Extracellular Signal-Regulated Kinase Signaling in PC12 Cells

Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
Molecular and Cellular Biology (Impact Factor: 4.78). 08/2011; 31(19):4063-75. DOI: 10.1128/MCB.05459-11
Source: PubMed


PC12 cells exhibit precise temporal control of growth factor signaling in which stimulation with epidermal growth factor (EGF) leads to transient extracellular signal-regulated kinase (ERK) activity and cell proliferation, whereas nerve growth factor (NGF) stimulation leads to sustained ERK activity and differentiation. While cyclic AMP (cAMP)-mediated signaling has been shown to be important in conferring the sustained ERK activity achieved by NGF, little is known about the regulation of cAMP and cAMP-dependent protein kinase (PKA) in these cells. Using fluorescence resonance energy transfer (FRET)-based biosensors localized to discrete subcellular locations, we showed that both NGF and EGF potently activate PKA at the plasma membrane, although they generate temporally distinct activity patterns. We further show that both stimuli fail to induce cytosolic PKA activity and identify phosphodiesterase 3 (PDE3) as a critical regulator in maintaining this spatial compartmentalization. Importantly, inhibition of PDE3, and thus perturbation of the spatiotemporal regulation of PKA activity, dramatically increases the duration of EGF-stimulated nuclear ERK activity in a PKA-dependent manner. Together, these findings identify EGF and NGF as potent activators of PKA activity specifically at the plasma membrane and reveal a novel regulatory mechanism contributing to the growth factor signaling specificity achieved by NGF and EGF in PC12 cells.

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    • "A good example is the Raf/MEK/ERK signaling pathway, which plays a vital role in cell proliferation, differentiation, and apoptosis [7], [8], [9], [10]. Extensive studies of the Raf/MEK/ERK pathway have suggested that its functional outcome depends on its activation kinetics [11], [12]. For instance, although EGF and NGF trigger similar sets of signaling pathways in PC12 cells including Raf/MEK/ERK [7], [8], PI3K/AKT [13], and PLCγ pathways [14], [15], EGF induces cell proliferation while NGF induces cell differentiation accompanied by cell cycle arrest [3], [16], [17], [18], [19]. "
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    ABSTRACT: It has been proposed that differential activation kinetics allows cells to use a common set of signaling pathways to specify distinct cellular outcomes. For example, nerve growth factor (NGF) and epidermal growth factor (EGF) induce different activation kinetics of the Raf/MEK/ERK signaling pathway and result in differentiation and proliferation, respectively. However, a direct and quantitative linkage between the temporal profile of Raf/MEK/ERK activation and the cellular outputs has not been established due to a lack of means to precisely perturb its signaling kinetics. Here, we construct a light-gated protein-protein interaction system to regulate the activation pattern of the Raf/MEK/ERK signaling pathway. Light-induced activation of the Raf/MEK/ERK cascade leads to significant neurite outgrowth in rat PC12 pheochromocytoma cell lines in the absence of growth factors. Compared with NGF stimulation, light stimulation induces longer but fewer neurites. Intermittent on/off illumination reveals that cells achieve maximum neurite outgrowth if the off-time duration per cycle is shorter than 45 min. Overall, light-mediated kinetic control enables precise dissection of the temporal dimension within the intracellular signal transduction network.
    PLoS ONE 03/2014; 9(3):e92917. DOI:10.1371/journal.pone.0092917 · 3.23 Impact Factor
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    • "Therefore, qualitative, context‐dependent differences in observable kinetics should be expected across cell lineages and culture conditions that affect the expression levels of ERK substrates and phosphatases. Indeed, in EGF‐stimulated PC12 cells, free ERK activities in the cytosol and nucleus were found to be transient, with no apparent lag between the cytosolic and nuclear responses (Herbst et al, 2011). "
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    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.
    Molecular Systems Biology 01/2014; 10(1):718. DOI:10.1002/msb.134708 · 10.87 Impact Factor
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    ABSTRACT: Arguably, one of the foremost distinctions between life and non-living matter is the ability to sense environmental changes and respond appropriately--an ability that is invested in every living cell. Within a single cell, this function is largely carried out by networks of signaling molecules. However, the details of how signaling networks help cells make complicated decisions are still not clear. For instance, how do cells read graded, analog stress signals but convert them into digital live-or-die responses? The answer to such questions may originate from the fact that signaling molecules are not static but dynamic entities, changing in numbers and activity over time and space. In the past two decades, researchers have been able to experimentally monitor signaling dynamics and use mathematical techniques to quantify and abstract general principles of how cells process information. In this review, the authors first introduce and discuss various experimental and computational methodologies that have been used to study signaling dynamics. The authors then discuss the different types of temporal dynamics such as oscillations and bistability that can be exhibited by signaling systems and highlight studies that have investigated such dynamics in physiological settings. Finally, the authors illustrate the role of spatial compartmentalization in regulating cellular responses with examples of second-messenger signaling in cardiac myocytes.
    Protein Science 07/2012; 21(7):918-28. DOI:10.1002/pro.2089 · 2.85 Impact Factor
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