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.

Full-text preview

Available from:
  • Source
    • "By virtue of a nuclear export sequence, EKAR2G localizes to, and specifically measures ERK activity in the cytosol (Fig 1B). Although this does not seem to be true for all cell types (Ahmed et al, 2014), we assumed that cytosolic and nuclear pools of ERK activity are in equilibrium, since, at least for EGF-stimulated PC-12 cells, there is no apparent time lag between nuclear and cytosolic ERK activation dynamics (Herbst et al, 2011). Biosensor expression levels were homogeneous and displayed only small standard deviation with respect to the population median (Appendix Fig S1A). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Transient versus sustained ERK MAP kinase (MAPK) activation dynamics induce proliferation versus differentiation in response to epidermal (EGF) or nerve (NGF) growth factors in PC-12 cells. Duration of ERK activation has therefore been proposed to specify cell fate decisions. Using a biosensor to measure ERK activation dynamics in single living cells reveals that sustained EGF/NGF application leads to a heterogeneous mix of transient and sustained ERK activation dynamics in distinct cells of the population, different than the population average. EGF biases toward transient, while NGF biases toward sustained ERK activation responses. In contrast, pulsed growth factor application can repeatedly and homogeneously trigger ERK activity transients across the cell population. These datasets enable mathematical modeling to reveal salient features inherent to the MAPK network. Ultimately, this predicts pulsed growth factor stimulation regimes that can bypass the typical feedback activation to rewire the system toward cell differentiation irrespective of growth factor identity.
    Full-text · Article · Nov 2015 · Molecular Systems Biology
  • Source
    • "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]. "
    [Show abstract] [Hide abstract]
    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.
    Full-text · Article · Mar 2014 · PLoS ONE
    • "Recently, many papers have shown that the biological response is dictated by the duration, magnitude and subcellular compartmentalization of enzymatic activity [37] [38] [39]. Specifically, the response of the cell is based on the modulation of the spatiotemporal dynamics of kinase activity. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Cell death contributes to the maintenance of homeostasis, but mounting evidence has confirmed the involvement of programmed cell death in some diseases. The concept of programmed cell death, which was coined several decades ago to refer to apoptosis, now also encompasses necroptosis, a newly characterized cell death program. Research on programmed cell death has become essential for the development of some new therapies. To study cell death signaling and its molecular mechanisms, new biochemical and fluorogenic approaches have been devised. Here, we first provide an overview of programmed cell death modes and the importance of dynamic cell death studies. Next, we focus on both apoptotic and necroptotic signaling and their mechanisms by providing a systematic review of all the methods and approaches that have been used. We emphasize the contribution of advanced approaches based on fluorescent probes, reporters, and Förster resonance energy transfer (FRET)-based biosensors for studying programmed cell death. Because apoptosis and necroptosis signaling pathways share some effectors molecules, we discuss how these new tools could be used to discriminate between apoptosis and necroptosis. We also describe how we developed specific FRET-based biosensors for detecting necroptosis. Finally, we touch on how dynamic measurement of biomolecules in living models will play a role in personalized prognosis and therapy.
    No preview · Article · Feb 2014 · Biotechnology Journal
Show more