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The role of type 4 phosphodiesterases in generating microdomains of cAMP: large scale stochastic simulations.

Oliveira RF, Terrin A, Di Benedetto G, Cannon RC, Koh W, Kim M, Zaccolo M, Blackwell KT - PLoS ONE (2010)

Bottom Line: Cyclic AMP (cAMP) and its main effector Protein Kinase A (PKA) are critical for several aspects of neuronal function including synaptic plasticity.Simulations further demonstrate that generation of the cAMP microdomain requires a pool of PDE4D anchored in the cytosol and also requires PKA-mediated phosphorylation of PDE4D which increases its activity.The microdomain does not require impeded diffusion of cAMP, confirming that barriers are not required for microdomains.

View Article: PubMed Central - PubMed

Affiliation: The Krasnow Institute for Advanced Study, George Mason University, Fairfax, Virginia, United States of America.

ABSTRACT
Cyclic AMP (cAMP) and its main effector Protein Kinase A (PKA) are critical for several aspects of neuronal function including synaptic plasticity. Specificity of synaptic plasticity requires that cAMP activates PKA in a highly localized manner despite the speed with which cAMP diffuses. Two mechanisms have been proposed to produce localized elevations in cAMP, known as microdomains: impeded diffusion, and high phosphodiesterase (PDE) activity. This paper investigates the mechanism of localized cAMP signaling using a computational model of the biochemical network in the HEK293 cell, which is a subset of pathways involved in PKA-dependent synaptic plasticity. This biochemical network includes cAMP production, PKA activation, and cAMP degradation by PDE activity. The model is implemented in NeuroRD: novel, computationally efficient, stochastic reaction-diffusion software, and is constrained by intracellular cAMP dynamics that were determined experimentally by real-time imaging using an Epac-based FRET sensor (H30). The model reproduces the high concentration cAMP microdomain in the submembrane region, distinct from the lower concentration of cAMP in the cytosol. Simulations further demonstrate that generation of the cAMP microdomain requires a pool of PDE4D anchored in the cytosol and also requires PKA-mediated phosphorylation of PDE4D which increases its activity. The microdomain does not require impeded diffusion of cAMP, confirming that barriers are not required for microdomains. The simulations reported here further demonstrate the utility of the new stochastic reaction-diffusion algorithm for exploring signaling pathways in spatially complex structures such as neurons.

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Propagation of cAMP microdomains to downstream targets.(A) The increase in the quantity of PKA with 4 cAMP molecules bound is greater in the submembrane region than in the cytosol. However, the percent increase is the same submembrane and cytosol. (B) PKA catalytic subunit (PKAc) concentration is the same in submembrane and cytosolic compartments. (C) The higher cAMP concentration observed submembrane translates into a larger fraction of phosphorylated PDE4s in the submembrane (pPDE4B) as compared to the cytosol (pPDE4D). A single representative trace is illustrated in each panel.
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pone-0011725-g006: Propagation of cAMP microdomains to downstream targets.(A) The increase in the quantity of PKA with 4 cAMP molecules bound is greater in the submembrane region than in the cytosol. However, the percent increase is the same submembrane and cytosol. (B) PKA catalytic subunit (PKAc) concentration is the same in submembrane and cytosolic compartments. (C) The higher cAMP concentration observed submembrane translates into a larger fraction of phosphorylated PDE4s in the submembrane (pPDE4B) as compared to the cytosol (pPDE4D). A single representative trace is illustrated in each panel.

Mentions: Fig. 6A shows that the increase in the quantity of PKA with 4 cAMP molecules bound is greater in the submembrane region than in the cytosol; however, the percent increase is the same submembrane and cytosol. The reason for the discrepancy between total increase and percent increase is that the initial percentage of fully bound PKA is higher submembrane than in the cytosol, because initial submembrane cAMP concentration is greater than the affinity of cAMP for PKA. Fig. 6B also shows the quantity of free PKA catalytic subunit. The concentration in the submembrane region equals that in the cytosol (Fig. 6B), both the initial value and after stimulation. Diffusion of the PKA catalytic subunit is not likely to explain the lack of a PKA microdomain because the diffusion constant of the PKA catalytic subunit is ten times smaller than that for cAMP. Instead, these results reinforce the importance of degradative mechanisms (e.g. PDE4s) for the production of microdomains: no microdomain of PKA catalytic subunit is observed because the model does not include mechanisms that directly consume the PKA catalytic subunit, as opposed to the situation with cAMP.


The role of type 4 phosphodiesterases in generating microdomains of cAMP: large scale stochastic simulations.

Oliveira RF, Terrin A, Di Benedetto G, Cannon RC, Koh W, Kim M, Zaccolo M, Blackwell KT - PLoS ONE (2010)

Propagation of cAMP microdomains to downstream targets.(A) The increase in the quantity of PKA with 4 cAMP molecules bound is greater in the submembrane region than in the cytosol. However, the percent increase is the same submembrane and cytosol. (B) PKA catalytic subunit (PKAc) concentration is the same in submembrane and cytosolic compartments. (C) The higher cAMP concentration observed submembrane translates into a larger fraction of phosphorylated PDE4s in the submembrane (pPDE4B) as compared to the cytosol (pPDE4D). A single representative trace is illustrated in each panel.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2908681&req=5

pone-0011725-g006: Propagation of cAMP microdomains to downstream targets.(A) The increase in the quantity of PKA with 4 cAMP molecules bound is greater in the submembrane region than in the cytosol. However, the percent increase is the same submembrane and cytosol. (B) PKA catalytic subunit (PKAc) concentration is the same in submembrane and cytosolic compartments. (C) The higher cAMP concentration observed submembrane translates into a larger fraction of phosphorylated PDE4s in the submembrane (pPDE4B) as compared to the cytosol (pPDE4D). A single representative trace is illustrated in each panel.
Mentions: Fig. 6A shows that the increase in the quantity of PKA with 4 cAMP molecules bound is greater in the submembrane region than in the cytosol; however, the percent increase is the same submembrane and cytosol. The reason for the discrepancy between total increase and percent increase is that the initial percentage of fully bound PKA is higher submembrane than in the cytosol, because initial submembrane cAMP concentration is greater than the affinity of cAMP for PKA. Fig. 6B also shows the quantity of free PKA catalytic subunit. The concentration in the submembrane region equals that in the cytosol (Fig. 6B), both the initial value and after stimulation. Diffusion of the PKA catalytic subunit is not likely to explain the lack of a PKA microdomain because the diffusion constant of the PKA catalytic subunit is ten times smaller than that for cAMP. Instead, these results reinforce the importance of degradative mechanisms (e.g. PDE4s) for the production of microdomains: no microdomain of PKA catalytic subunit is observed because the model does not include mechanisms that directly consume the PKA catalytic subunit, as opposed to the situation with cAMP.

Bottom Line: Cyclic AMP (cAMP) and its main effector Protein Kinase A (PKA) are critical for several aspects of neuronal function including synaptic plasticity.Simulations further demonstrate that generation of the cAMP microdomain requires a pool of PDE4D anchored in the cytosol and also requires PKA-mediated phosphorylation of PDE4D which increases its activity.The microdomain does not require impeded diffusion of cAMP, confirming that barriers are not required for microdomains.

View Article: PubMed Central - PubMed

Affiliation: The Krasnow Institute for Advanced Study, George Mason University, Fairfax, Virginia, United States of America.

ABSTRACT
Cyclic AMP (cAMP) and its main effector Protein Kinase A (PKA) are critical for several aspects of neuronal function including synaptic plasticity. Specificity of synaptic plasticity requires that cAMP activates PKA in a highly localized manner despite the speed with which cAMP diffuses. Two mechanisms have been proposed to produce localized elevations in cAMP, known as microdomains: impeded diffusion, and high phosphodiesterase (PDE) activity. This paper investigates the mechanism of localized cAMP signaling using a computational model of the biochemical network in the HEK293 cell, which is a subset of pathways involved in PKA-dependent synaptic plasticity. This biochemical network includes cAMP production, PKA activation, and cAMP degradation by PDE activity. The model is implemented in NeuroRD: novel, computationally efficient, stochastic reaction-diffusion software, and is constrained by intracellular cAMP dynamics that were determined experimentally by real-time imaging using an Epac-based FRET sensor (H30). The model reproduces the high concentration cAMP microdomain in the submembrane region, distinct from the lower concentration of cAMP in the cytosol. Simulations further demonstrate that generation of the cAMP microdomain requires a pool of PDE4D anchored in the cytosol and also requires PKA-mediated phosphorylation of PDE4D which increases its activity. The microdomain does not require impeded diffusion of cAMP, confirming that barriers are not required for microdomains. The simulations reported here further demonstrate the utility of the new stochastic reaction-diffusion algorithm for exploring signaling pathways in spatially complex structures such as neurons.

Show MeSH