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DARPP-32 is a robust integrator of dopamine and glutamate signals.

Fernandez E, Schiappa R, Girault JA, Le Novère N - PLoS Comput. Biol. (2006)

Bottom Line: We confirmed that the proposed regulation of protein phosphatase-2A (PP2A) by calcium can account for the observed decrease of Threonine 75 phosphorylation upon glutamate receptor activation.This integration did not depend on the concentration of DARPP-32, while the absolute effect on PP1 varied linearly.This work is a first attempt to better understand the complex interactions between cAMP and Ca(2+) regulation of DARPP-32.

View Article: PubMed Central - PubMed

Affiliation: EMBL-EBI, Wellcome-Trust Genome Campus, Hinxton, United Kingdom.

ABSTRACT
Integration of neurotransmitter and neuromodulator signals in the striatum plays a central role in the functions and dysfunctions of the basal ganglia. DARPP-32 is a key actor of this integration in the GABAergic medium-size spiny neurons, in particular in response to dopamine and glutamate. When phosphorylated by cAMP-dependent protein kinase (PKA), DARPP-32 inhibits protein phosphatase-1 (PP1), whereas when phosphorylated by cyclin-dependent kinase 5 (CDK5) it inhibits PKA. DARPP-32 is also regulated by casein kinases and by several protein phosphatases. These complex and intricate regulations make simple predictions of DARPP-32 dynamic behaviour virtually impossible. We used detailed quantitative modelling of the regulation of DARPP-32 phosphorylation to improve our understanding of its function. The models included all the combinations of the three best-characterized phosphorylation sites of DARPP-32, their regulation by kinases and phosphatases, and the regulation of those enzymes by cAMP and Ca(2+) signals. Dynamic simulations allowed us to observe the temporal relationships between cAMP and Ca(2+) signals. We confirmed that the proposed regulation of protein phosphatase-2A (PP2A) by calcium can account for the observed decrease of Threonine 75 phosphorylation upon glutamate receptor activation. DARPP-32 is not simply a switch between PP1-inhibiting and PKA-inhibiting states. Sensitivity analysis showed that CDK5 activity is a major regulator of the response, as previously suggested. Conversely, the strength of the regulation of PP2A by PKA or by calcium had little effect on the PP1-inhibiting function of DARPP-32 in these conditions. The simulations showed that DARPP-32 is not only a robust signal integrator, but that its response also depends on the delay between cAMP and calcium signals affecting the response to the latter. This integration did not depend on the concentration of DARPP-32, while the absolute effect on PP1 varied linearly. In silico mutants showed that Ser137 phosphorylation affects the influence of the delay between dopamine and glutamate, and that constitutive phosphorylation in Ser137 transforms DARPP-32 in a quasi-irreversible switch. This work is a first attempt to better understand the complex interactions between cAMP and Ca(2+) regulation of DARPP-32. Progressive inclusion of additional components should lead to a realistic model of signalling networks underlying the function of striatal neurons.

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Effects of a Train of Ca2+ Spikes on DARPP-32 PhosphorylationTime-course of DARPP-32 isoforms triggered by a train of Ca2+ spikes. Bordeaux line represents the number of calcium ions in the dendritic spine. Colour code of DARPP-32 isoforms is the same as for Figure 3. In the absence of a cAMP signal, the phosphorylation on Thr34 remains . (A) model A; (B) model B.
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pcbi-0020176-g004: Effects of a Train of Ca2+ Spikes on DARPP-32 PhosphorylationTime-course of DARPP-32 isoforms triggered by a train of Ca2+ spikes. Bordeaux line represents the number of calcium ions in the dendritic spine. Colour code of DARPP-32 isoforms is the same as for Figure 3. In the absence of a cAMP signal, the phosphorylation on Thr34 remains . (A) model A; (B) model B.

Mentions: Activation of the Ca2+ pathway was performed by repeatedly increasing the influx rate of Ca2+ into the system, from 2.5 × 10−8 Ms−1 to 6.6 × 10−6 Ms−1 , every 4 s for 2 s. This triggered the formation of a series of spikes during which Ca2+ transiently reached a concentration of 4 × 10−6 M, then decayed to its basal level. The two models A and B exhibited different behaviours upon activation of the Ca2+ pathway. In model A, without Ca2+ activation of PP2A, Ca2+ spikes increased Ser137 phosphorylation, up to ∼32% (Figure 4). The increase of this phosphorylation was performed by consuming unphosphorylated DARPP-32 (which dropped to 42%) or by phosphorylating D75. The total level of phosphorylation on Thr75 was found almost not modified. This result is not consistent with in vivo experiments on rats that showed a dephosphorylation of DARPP-32 on Thr75 triggered by the activation NMDA receptors. We therefore tested model B, which included the activation of PP2A by Ca2+ suggested by Nishi et al. [16]. Phosphorylation of DARPP-32 on Ser137 upon activation of the Ca2+ pathway was observed at the same rate as in model A. However, we observed a decrease of D75*, as observed in vivo. The levels of unphosphorylated DARPP-32 dropped as in model A, due to the increased phosphorylation on Ser137. However, during the calcium spikes, this decrease was counteracted by the increased dephosphorylation of D75. Overall, D137 was produced mainly from unphosphorylated DARPP-32 rather than from D75, resulting in lower D75:137 double phosphorylations (unpublished data).


DARPP-32 is a robust integrator of dopamine and glutamate signals.

Fernandez E, Schiappa R, Girault JA, Le Novère N - PLoS Comput. Biol. (2006)

Effects of a Train of Ca2+ Spikes on DARPP-32 PhosphorylationTime-course of DARPP-32 isoforms triggered by a train of Ca2+ spikes. Bordeaux line represents the number of calcium ions in the dendritic spine. Colour code of DARPP-32 isoforms is the same as for Figure 3. In the absence of a cAMP signal, the phosphorylation on Thr34 remains . (A) model A; (B) model B.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1761654&req=5

pcbi-0020176-g004: Effects of a Train of Ca2+ Spikes on DARPP-32 PhosphorylationTime-course of DARPP-32 isoforms triggered by a train of Ca2+ spikes. Bordeaux line represents the number of calcium ions in the dendritic spine. Colour code of DARPP-32 isoforms is the same as for Figure 3. In the absence of a cAMP signal, the phosphorylation on Thr34 remains . (A) model A; (B) model B.
Mentions: Activation of the Ca2+ pathway was performed by repeatedly increasing the influx rate of Ca2+ into the system, from 2.5 × 10−8 Ms−1 to 6.6 × 10−6 Ms−1 , every 4 s for 2 s. This triggered the formation of a series of spikes during which Ca2+ transiently reached a concentration of 4 × 10−6 M, then decayed to its basal level. The two models A and B exhibited different behaviours upon activation of the Ca2+ pathway. In model A, without Ca2+ activation of PP2A, Ca2+ spikes increased Ser137 phosphorylation, up to ∼32% (Figure 4). The increase of this phosphorylation was performed by consuming unphosphorylated DARPP-32 (which dropped to 42%) or by phosphorylating D75. The total level of phosphorylation on Thr75 was found almost not modified. This result is not consistent with in vivo experiments on rats that showed a dephosphorylation of DARPP-32 on Thr75 triggered by the activation NMDA receptors. We therefore tested model B, which included the activation of PP2A by Ca2+ suggested by Nishi et al. [16]. Phosphorylation of DARPP-32 on Ser137 upon activation of the Ca2+ pathway was observed at the same rate as in model A. However, we observed a decrease of D75*, as observed in vivo. The levels of unphosphorylated DARPP-32 dropped as in model A, due to the increased phosphorylation on Ser137. However, during the calcium spikes, this decrease was counteracted by the increased dephosphorylation of D75. Overall, D137 was produced mainly from unphosphorylated DARPP-32 rather than from D75, resulting in lower D75:137 double phosphorylations (unpublished data).

Bottom Line: We confirmed that the proposed regulation of protein phosphatase-2A (PP2A) by calcium can account for the observed decrease of Threonine 75 phosphorylation upon glutamate receptor activation.This integration did not depend on the concentration of DARPP-32, while the absolute effect on PP1 varied linearly.This work is a first attempt to better understand the complex interactions between cAMP and Ca(2+) regulation of DARPP-32.

View Article: PubMed Central - PubMed

Affiliation: EMBL-EBI, Wellcome-Trust Genome Campus, Hinxton, United Kingdom.

ABSTRACT
Integration of neurotransmitter and neuromodulator signals in the striatum plays a central role in the functions and dysfunctions of the basal ganglia. DARPP-32 is a key actor of this integration in the GABAergic medium-size spiny neurons, in particular in response to dopamine and glutamate. When phosphorylated by cAMP-dependent protein kinase (PKA), DARPP-32 inhibits protein phosphatase-1 (PP1), whereas when phosphorylated by cyclin-dependent kinase 5 (CDK5) it inhibits PKA. DARPP-32 is also regulated by casein kinases and by several protein phosphatases. These complex and intricate regulations make simple predictions of DARPP-32 dynamic behaviour virtually impossible. We used detailed quantitative modelling of the regulation of DARPP-32 phosphorylation to improve our understanding of its function. The models included all the combinations of the three best-characterized phosphorylation sites of DARPP-32, their regulation by kinases and phosphatases, and the regulation of those enzymes by cAMP and Ca(2+) signals. Dynamic simulations allowed us to observe the temporal relationships between cAMP and Ca(2+) signals. We confirmed that the proposed regulation of protein phosphatase-2A (PP2A) by calcium can account for the observed decrease of Threonine 75 phosphorylation upon glutamate receptor activation. DARPP-32 is not simply a switch between PP1-inhibiting and PKA-inhibiting states. Sensitivity analysis showed that CDK5 activity is a major regulator of the response, as previously suggested. Conversely, the strength of the regulation of PP2A by PKA or by calcium had little effect on the PP1-inhibiting function of DARPP-32 in these conditions. The simulations showed that DARPP-32 is not only a robust signal integrator, but that its response also depends on the delay between cAMP and calcium signals affecting the response to the latter. This integration did not depend on the concentration of DARPP-32, while the absolute effect on PP1 varied linearly. In silico mutants showed that Ser137 phosphorylation affects the influence of the delay between dopamine and glutamate, and that constitutive phosphorylation in Ser137 transforms DARPP-32 in a quasi-irreversible switch. This work is a first attempt to better understand the complex interactions between cAMP and Ca(2+) regulation of DARPP-32. Progressive inclusion of additional components should lead to a realistic model of signalling networks underlying the function of striatal neurons.

Show MeSH
Related in: MedlinePlus