To ensure proper conversion of a specific environmental input into a distinct cellular output, cells exploit a number of molecular mechanisms to tightly regulate signal transduction in space and time. In PC12 cells, specific controls of the duration of the activity of extracellular signal-regulated kinase (ERK), a canonical mitogen-activated protein kinase (MAPK), are believed to help determine distinct cell fates (32
). Specifically, activation of epidermal growth factor receptor (EGFR) by epidermal growth factor (EGF) leads to transient ERK activity and cell proliferation, whereas nerve growth factor (NGF) binding to and activating its receptor, TrkA, leads to sustained ERK activity and signals the cells to differentiate (23
). An accepted model for the growth factor (GF) signaling specificity in these cells involves the activation of specific GTPases capable of activating the Raf family of kinases, which activate MEK, the upstream activator of ERK. In particular, while both EGF and NGF can transiently activate the GTPase Ras to recruit Raf to the plasma membrane, where it can be activated, only NGF activates Rap1, a cyclic AMP (cAMP)-regulated GTPase also capable of activating Raf (23
). Since this NGF-induced Rap1 activation is sustained, it is suggested that the selective activation of Rap1 by NGF but not EGF leads to the sustained phase of ERK activity and the initiation of neurite outgrowth. Furthermore, EGF-stimulated ERK negatively regulates Raf activity, whereas NGF-stimulated ERK exerts positive feedback on Raf activity, further contributing to the transient and sustained duration of ERK activity as a result of the respective stimuli (11
As an added level of complexity in signal transduction regulation, many canonical signaling cascades are subject to cross talk, in which the molecular players of one pathway alter the state of another. For example, it is known that the ERK pathway and the cAMP-mediated signaling pathway are intricately connected (28
). While the precise regulation that these pathways have on one another is complex and cell type specific (28
), it is widely accepted that in PC12 cells, two cAMP effectors, namely, cAMP-dependent protein kinase (PKA) and exchange protein directly activated by cAMP (Epac), can indirectly activate the Raf/MEK/ERK cascade (4
Intracellular cAMP is enzymatically produced from ATP by adenylyl cyclases, either transmembrane adenylyl cyclase (tmAC) or soluble adenylyl cyclase (sAC) (10
), and degraded by phosphodiesterases (PDEs), of which there are 11 known isoforms (2
). In PC12 cells, NGF binds to TrkA, which activates sAC to produce cAMP (26
). Subsequently, activated PKA and Epac converge to activate Rap1 (26
), the aforementioned mediator of sustained ERK activity (36
). In contrast, EGF was not known to increase cAMP or activate PKA in PC12 cells (14
). Interestingly, a number of studies have shown that when EGF is used in conjunction with cAMP-elevating agents, neurite outgrowth can be induced in PC12 cells (9
). These studies suggest that cAMP-mediated signaling may play a role in GF signaling specificity in PC12 cells and point to a simple model showing that lack of cAMP-mediated cross-regulation specifies the transient ERK activity stimulated by EGF. However, GF-stimulated cAMP increases and PKA activities have not been characterized in this system, and this model remains to be tested.
Characterization of cAMP-mediated signaling in a number of cell systems has revealed complex spatiotemporal regulation that is often achieved by the formation of localized signaling complexes containing the key regulatory and effector molecules involved in cAMP signaling. One critical component of these complexes, which are frequently formed with the help of scaffolding proteins known as A-kinase anchoring proteins (AKAPs), are PDEs (1
). Localization of PDEs to distinct intracellular locales results in the formation of discrete cAMP gradients which are able to control the signaling of PKA and Epac with high specificity (1
). Proper compartmentalization of PKA- and Epac-mediated signaling is necessary for normal cellular function and regulates processes such as excitation and contraction of cardiomyocytes and insulin secretion in pancreatic β cells (13
). In hippocampal neurons, PDEs play an important role in establishing localized pools of cyclic nucleotides in undifferentiated neurites, which ultimately regulates the process of axon and dendrite formation (24
). Although cAMP-mediated signaling contributes to specifying the cell fates of PC12 cells as discussed above, the spatiotemporal regulation of cAMP signaling and its functional roles in this cell system have not been fully characterized.
In this study we investigated the spatiotemporal regulation of GF-mediated PKA activity in PC12 cells by employing genetically encoded fluorescence resonance energy transfer (FRET)-based biosensors targeted to distinct subcellular locales. In doing so, we observed that both NGF and EGF stimulate PKA activity at the plasma membrane with discrete temporal activity patterns, but both stimuli fail to activate the cytosolic pool of PKA. Using pharmacological perturbations, we identify PDE3 to be a critical regulator of this GF-stimulated compartmentalized PKA activity. Moreover, PKA and PDE3 are shown to regulate both the onset and duration of GF-stimulated ERK activity. When PDE3 is inhibited and the spatiotemporal regulation of PKA is disrupted, PC12 cells treated with EGF display heightened durations of nuclear ERK activity. Together these findings reveal the distinct NGF- and EGF-induced spatiotemporal patterns of PKA activity as a novel level of regulation underlying the precise GF signaling specificity in PC12 cells.