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

Abstract

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.

Multiplexing PKA and ERK1&2 kinases FRET biosensors in living cells using single excitation wavelength dual colour FLIM

“…From the biological point of view, the interplay between cAMP/PKA and MAPK/ERK1&2 signalling pathways has long been established (for review11). Both cascades modulate common cellular processes, and multiple levels of crosstalk depending on the cellular type and the cellular context have been described1011153435. Because ERK1&2 is one of the cAMP targets associated with cell proliferation, emphasis has been put on hormone-mediated cAMP/PKA effect on ERK1&2 activation.…”

“…Because ERK1&2 is one of the cAMP targets associated with cell proliferation, emphasis has been put on hormone-mediated cAMP/PKA effect on ERK1&2 activation. So, until recently35, the Growth Factor (GF)-induced PKA activation effect has been less studied. From a biotechnological standpoint, we know from our own work36 and that of others2837 that modifying FRET pairs has an impact on the performance of KARs.…”

“…Compelling evidences demonstrated that the ERK1&2 cascade is regulated by cAMP/PKA pathway (for review10). This crosstalk was reported to modulate the duration and the strength of ERK1/2 activity35, mainly in a Raf-isoform-dependent manner11.…”

“…Because the study of the intricate relationship between cAMP/PKA and MAPK/ERK1&2 signal transduction pathways regularly provides new mechanistic insights151835, we opted for it as a structured and defined playground for application purposes.…”

“…In addition, scaffolds proteins such as AKAP-Lbc and KSR-1, that were reported to favour discrete localizations of both PKA and ERK1&2 generating pockets of concentrated enzyme activities, were shown to provide platforms for mitogenic signal amplification15. Actually, compartmentalized GF-induced PKA activation modulating the features of ERK1&2 kinase activity was recently illustrated in an elegant study from Zhang’s group35. While monitoring of compartmentalized PKA and ERK1&2 enzyme activities was beyond the scope of our study, it could now be performed in a multiplex manner thus maximizing crosstalk relevance at the single living cell level.…”