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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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Schematic representation of the biochemical signaling pathway modeled.(A) GαGTP binds to and activates adenylate cyclase, which then produces cAMP from ATP. cAMP activates PKA, a heterotetramer with two regulatory and two catalytic subunits. After binding 4 molecules of cAMP, the two catalytic subunits (PKAc) dissociate from the regulatory subunit dimer (PKAr) and become active [26], [27]. cAMP is degraded by phosphodiesterase, type 4B (PDE4B) and type 4D (PDE4D). AC, GαGTP, PKA and PDE4B are anchored at the submembrane while PKA and PDE4D are distributed throughout the cytosol. cAMP, ATP, AMP and PKAc freely diffuse. (B) Confocal image showing the localization of the membrane-targeted version of the unimolecular Epac-based sensor for cAMP (mpH30) in HEK293 cells. Confocal images were acquired 24 hours after transfection by using the broadband confocal Leica TCS SP5 system (Leica Microsystems) and a HCX PL APO 63x1.4NA oil-immersion objective (scale bar 10 µm). The representation superimposed on the micrograph corresponds to the grid in C. (C) Schematic representation of the spatial structure of the HEK293 cell model, light gray compartments correspond to the cytosol while dark gray compartments correspond to the submembrane region in a slice of the 3-dimensional cell.
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pone-0011725-g001: Schematic representation of the biochemical signaling pathway modeled.(A) GαGTP binds to and activates adenylate cyclase, which then produces cAMP from ATP. cAMP activates PKA, a heterotetramer with two regulatory and two catalytic subunits. After binding 4 molecules of cAMP, the two catalytic subunits (PKAc) dissociate from the regulatory subunit dimer (PKAr) and become active [26], [27]. cAMP is degraded by phosphodiesterase, type 4B (PDE4B) and type 4D (PDE4D). AC, GαGTP, PKA and PDE4B are anchored at the submembrane while PKA and PDE4D are distributed throughout the cytosol. cAMP, ATP, AMP and PKAc freely diffuse. (B) Confocal image showing the localization of the membrane-targeted version of the unimolecular Epac-based sensor for cAMP (mpH30) in HEK293 cells. Confocal images were acquired 24 hours after transfection by using the broadband confocal Leica TCS SP5 system (Leica Microsystems) and a HCX PL APO 63x1.4NA oil-immersion objective (scale bar 10 µm). The representation superimposed on the micrograph corresponds to the grid in C. (C) Schematic representation of the spatial structure of the HEK293 cell model, light gray compartments correspond to the cytosol while dark gray compartments correspond to the submembrane region in a slice of the 3-dimensional cell.

Mentions: A computational model of cAMP production and degradation is employed to explore the generation of cAMP microdomains, which are important for synaptic specificity. Because this set of cAMP signaling pathways is widespread, we explore mechanisms underlying cAMP microdomains in a HEK293 cell (Fig. 1A), for which experimental measures of these microdomains provide model constraints. In this model, cAMP is produced from ATP by adenylate cyclase, which is activated by GαGTP binding. ATP is regenerated by a first order reaction AMP→ATP to prevent depletion. cAMP activates PKA, a heterotetramer with two regulatory and two catalytic subunits. After binding 4 molecules of cAMP, the two catalytic subunits (PKAc) dissociate from the regulatory subunit dimer (PKAr) and become active [26], [27]. As described below, to compare with FRET imaging data, the model also includes the Epac-based FRET sensor H30, which binds a single cAMP molecule.


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)

Schematic representation of the biochemical signaling pathway modeled.(A) GαGTP binds to and activates adenylate cyclase, which then produces cAMP from ATP. cAMP activates PKA, a heterotetramer with two regulatory and two catalytic subunits. After binding 4 molecules of cAMP, the two catalytic subunits (PKAc) dissociate from the regulatory subunit dimer (PKAr) and become active [26], [27]. cAMP is degraded by phosphodiesterase, type 4B (PDE4B) and type 4D (PDE4D). AC, GαGTP, PKA and PDE4B are anchored at the submembrane while PKA and PDE4D are distributed throughout the cytosol. cAMP, ATP, AMP and PKAc freely diffuse. (B) Confocal image showing the localization of the membrane-targeted version of the unimolecular Epac-based sensor for cAMP (mpH30) in HEK293 cells. Confocal images were acquired 24 hours after transfection by using the broadband confocal Leica TCS SP5 system (Leica Microsystems) and a HCX PL APO 63x1.4NA oil-immersion objective (scale bar 10 µm). The representation superimposed on the micrograph corresponds to the grid in C. (C) Schematic representation of the spatial structure of the HEK293 cell model, light gray compartments correspond to the cytosol while dark gray compartments correspond to the submembrane region in a slice of the 3-dimensional cell.
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Related In: Results  -  Collection

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pone-0011725-g001: Schematic representation of the biochemical signaling pathway modeled.(A) GαGTP binds to and activates adenylate cyclase, which then produces cAMP from ATP. cAMP activates PKA, a heterotetramer with two regulatory and two catalytic subunits. After binding 4 molecules of cAMP, the two catalytic subunits (PKAc) dissociate from the regulatory subunit dimer (PKAr) and become active [26], [27]. cAMP is degraded by phosphodiesterase, type 4B (PDE4B) and type 4D (PDE4D). AC, GαGTP, PKA and PDE4B are anchored at the submembrane while PKA and PDE4D are distributed throughout the cytosol. cAMP, ATP, AMP and PKAc freely diffuse. (B) Confocal image showing the localization of the membrane-targeted version of the unimolecular Epac-based sensor for cAMP (mpH30) in HEK293 cells. Confocal images were acquired 24 hours after transfection by using the broadband confocal Leica TCS SP5 system (Leica Microsystems) and a HCX PL APO 63x1.4NA oil-immersion objective (scale bar 10 µm). The representation superimposed on the micrograph corresponds to the grid in C. (C) Schematic representation of the spatial structure of the HEK293 cell model, light gray compartments correspond to the cytosol while dark gray compartments correspond to the submembrane region in a slice of the 3-dimensional cell.
Mentions: A computational model of cAMP production and degradation is employed to explore the generation of cAMP microdomains, which are important for synaptic specificity. Because this set of cAMP signaling pathways is widespread, we explore mechanisms underlying cAMP microdomains in a HEK293 cell (Fig. 1A), for which experimental measures of these microdomains provide model constraints. In this model, cAMP is produced from ATP by adenylate cyclase, which is activated by GαGTP binding. ATP is regenerated by a first order reaction AMP→ATP to prevent depletion. cAMP activates PKA, a heterotetramer with two regulatory and two catalytic subunits. After binding 4 molecules of cAMP, the two catalytic subunits (PKAc) dissociate from the regulatory subunit dimer (PKAr) and become active [26], [27]. As described below, to compare with FRET imaging data, the model also includes the Epac-based FRET sensor H30, which binds a single cAMP molecule.

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