DARPP-32 is required for MAPK/ERK signaling in thyroid cells

Modulation of MAPK signaling duration by cAMP defines its physiological output by driving cells toward proliferation or differentiation. Understanding how the kinetics of MAPK signaling are integrated with other cellular signals is a key issue in development and cancer. Here we show that dopamine and cAMP-regulated neuronal phosphoprotein, 32 kDa (DARPP-32), a protein required for thyroid cell differentiation, determines whether MAPK/ERK activation is sustained or transient. Serum, a stimulus that activates MAPK signaling and does not independently increase DARPP-32 levels results in transient activation of the MAPK pathway. By contrast, TSH + (IGF-I) activate MAPK signaling but also independently increase DARPP-32 levels. Our results are consistent with a model in which maintenance of DARPP-32 expression by TSH + IGF-I leads to sustained MAPK signaling. Moreover, the sensitivity of MAPK/ERK signaling in thyroid cells is lost when de novo DARPP-32 expression is blocked by small interfering RNA. Because both DARPP-32 levels and function as inhibitor of protein phosphatase 1, a key inhibitor of MAPK kinase activity, are governed by cAMP/protein kinase A, the results may explain why in thyroid cells cAMP signaling downstream from TSH controls the duration of MAPK pathway activity. Thus, fine-tuning of DARPP-32 levels leads to changes in the kinetics or sensitivity of MAPK/ERK signaling. Given the implications of MAPK signaling in thyroid cancer and the loss of DARPP-32 in tumor and transformed thyroid cells, DARPP-32 may represent a key therapeutic target.     

Distinct Changes in cAMP and Extracellular Signal-Regulated Protein Kinase Signalling in L-DOPA-Induced Dyskinesia

Background

In rodents, the development of dyskinesia produced by L-DOPA in the dopamine-depleted striatum occurs in response to increased dopamine D1 receptor-mediated activation of the cAMP - protein kinase A and of the Ras-extracellular signal-regulated kinase (ERK) signalling pathways. However, very little is known, in non-human primates, about the regulation of these signalling cascades and their association with the induction, manifestation and/or maintenance of dyskinesia.

Methodology/Results

We here studied, in the gold-standard non-human primate model of Parkinson''s disease, the changes in PKA-dependent phosphorylation of DARPP-32 and GluR1 AMPA receptor, as well as in ERK and ribosomal protein S6 (S6) phosphorylation, associated to acute and chronic administration of L-DOPA. Increased phosphorylation of DARPP-32 and GluR1 was observed in both L-DOPA first-ever exposed and chronically-treated dyskinetic parkinsonian monkeys. In contrast, phosphorylation of ERK and S6 was enhanced preferentially after acute L-DOPA administration and decreased during the course of chronic treatment.

Conclusion

Dysregulation of cAMP signalling is maintained during the course of chronic L-DOPA administration, while abnormal ERK signalling peaks during the initial phase of L-DOPA treatment and decreases following prolonged exposure. While cAMP signalling enhancement is associated with dyskinesia, abnormal ERK signalling is associated with priming.     

l-DOPA activates ERK signaling and phosphorylates histone H3 in the striatonigral medium spiny neurons of hemiparkinsonian mice

In the dopamine-depleted striatum, extracellular signal-regulated kinase (ERK) signaling is implicated in the development of l -DOPA-induced dyskinesia. To gain insights on its role in this disorder, we examined the effects of l -DOPA on the state of phosphorylation of ERK and downstream target proteins in striatopallidal and striatonigral medium spiny neurons (MSNs). For this purpose, we employed mice expressing enhanced green fluorescent protein (EGFP) under the control of the promoters for the dopamine D₂ receptor ( Drd2 -EGFP mice) or the dopamine D₁ receptor ( Drd1a -EGFP mice), which are expressed in striatopallidal and striatonigral MSNs, respectively. In 6-hydroxydopamine-lesioned Drd2 -EGFP mice, l -DOPA increased the phosphorylation of ERK, mitogen- and stress-activated kinase 1 and histone H3, selectively in EGFP-negative MSNs. Conversely, a complete co-localization between EGFP and these phosphoproteins was observed in Drd1a -EGFP mice. The effect of l -DOPA was prevented by blockade of dopamine D₁ receptors. The same pattern of activation of ERK signaling was observed in dyskinetic mice, after repeated administration of l -DOPA. Our results demonstrate that in the dopamine-depleted striatum, l -DOPA activates ERK signaling specifically in striatonigral MSNs. This regulation may result in ERK-dependent changes in striatal plasticity leading to dyskinesia.     

mTORC1 signaling in Parkinson disease and L-DOPA-induced dyskinesia: A sensitized matter

Parkinson's disease is caused by the progressive loss of dopamine innervation to the basal ganglia and is commonly treated with the dopamine precursor, L-DOPA. Prolonged administration of L-DOPA results in the development of severe motor complications, or dyskinesia, which seriously hamper its clinical use. Recent evidence indicates that L-DOPA-induced dyskinesia (LID) is associated with persistent activation of the mammalian target of rapamycin complex 1 (mTORC1) in the medium spiny neurons (MSNs) of the striatum, the main component of the basal ganglia. This phenomenon is secondary to the development of a strong sensitization at the level of dopamine D1 receptors, which are abundantly expressed in a subset of MSNs. Such sensitization confers to dopaminergic drugs (including L-DOPA) the ability to activate the extracellular signal-regulated protein kinases 1/2, which, in turn promote mTORC1 signaling. Using a mouse model of LID, we recently showed that administration of the allosteric mTORC1 inhibitor, rapamycin, reduces dyskinesia. This finding is discussed with respect to underlying mechanisms and potential significance for the development of future therapeutic interventions.     

Role of phosphatidylinositide 3-kinase in brain-derived neurotrophic factor-induced DARPP-32 expression in medium size spiny neurons in vitro

Brain-derived neurotrophic factor (BDNF) regulates several properties of striatal dopaminoceptive medium-sized spiny neurons (MSNs) in vivo and in vitro, including expression levels of DARPP-32 (dopamine and cyclic adenosine 3',5'-monophosphate-regulated phosphoprotein, 32 kDa). DARPP-32 is expressed in 96% of the MSNs, and is a key modulator of dopamine actions. We investigated the intracellular signal transduction pathways activated by BDNF in MSNs and via which BDNF induces DARPP-32 expression. We found that phosphorylation of the cyclic AMP response element binding protein (CREB) is only transiently increased following stimulation of MSNs by BDNF, whereas increased phosphorylation of the extracellular signal regulated kinases 1 and 2 (Erk1/2) and Akt is sustained for longer than 4 h. Treatment of cultures with inhibitors of mitogen-activated protein kinase kinase (MEK) or phosphatidylinositide 3-kinase (PI3K) showed that the majority of the BDNF-induced increase in DARPP-32 requires the PI3K pathway. We also found that inhibition of PI3K reduces BDNF-induced Erk phosphorylation, indicating that cross-talk between these pathways may play a prominent role in MSNs.     

Neurotensin regulates DARPP-32 thr34 phosphorylation in neostriatal neurons by activation of dopamine D1-type receptors

Neurotensin modulates dopaminergic transmission in the nigrostriatal system. DARPP-32, a dopamine- and cAMP-regulated phosphoprotein of Mr 32 kDa, is phosphorylated on Thr34 by cAMP-dependent protein kinase, resulting in its conversion into a potent inhibitor of protein phosphatase-1 (PP 1). Here, we examined the effect of neurotensin on DARPP-32 Thr34 phosphorylation using mouse neostriatal slices. Neurotensin stimulated DARPP-32 Thr34 phosphorylation by 4-7-fold with a K(0.5) of approximately 50 nM. The effect of neurotensin was antagonized by a combined neurotensin receptor type-1 (NTR1)/type-2 (NTR2) antagonist, SR142948. It was not antagonized by a NTR1 antagonist, SR48692 or by a NTR2 antagonist, levocabastine; neither was it antagonized by the two combined. Pretreatment with TTX or cobalt abolished the effect of neurotensin. The effect of neurotensin was antagonized by a dopamine D1 antagonist, SCH23390, and by ionotropic glutamate receptor antagonists, MK801 and CNQX. These results indicate that neurotensin stimulates the release of dopamine from nigrostriatal presynaptic terminals in an NMDA receptor- and AMPA receptor-dependent manner, leading to the increase in DARPP-32 Thr34 phosphorylation. Neurotensin stimulated the phosphorylation of Ser845 of the AMPA receptor GluR1 subunit in wild-type mice but not in DARPP-32 knockout mice. Thus, neurotensin, by stimulating the release of dopamine, activates the dopamine D1-receptor/cAMP/PKA/DARPP-32/PP 1 cascade.     

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

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 Ca²⁺ signals. Dynamic simulations allowed us to observe the temporal relationships between cAMP and Ca²⁺ 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 Ca²⁺ regulation of DARPP-32. Progressive inclusion of additional components should lead to a realistic model of signalling networks underlying the function of striatal neurons.     

Pharmacological and genetic evaluation of proposed roles of mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK), extracellular signal-regulated kinase (ERK), and p90(RSK) in the control of mTORC1 protein signaling by phorbol esters

The mammalian target of rapamycin complex 1 (mTORC1) links the control of mRNA translation, cell growth, and metabolism to diverse stimuli. Inappropriate activation of mTORC1 can lead to cancer. Phorbol esters are naturally occurring products that act as potent tumor promoters. They activate isoforms of protein kinase C (PKCs) and stimulate the oncogenic MEK/ERK signaling cascade. They also activate mTORC1 signaling. Previous work indicated that mTORC1 activation by the phorbol ester PMA (phorbol 12-myristate 13-acetate) depends upon PKCs and may involve MEK. However, the precise mechanism(s) through which they activate mTORC1 remains unclear. Recent studies have implicated both the ERKs and the ERK-activated 90-kDa ribosomal S6 kinases (p90(RSK)) in activating mTORC1 signaling via phosphorylation of TSC2 (a regulator of mTORC1) and/or the mTORC1 component raptor. However, the relative importance of each of these kinases and phosphorylation events for the activation of mTORC1 signaling is unknown. The recent availability of MEK (PD184352) and p90(RSK) (BI-D1870) inhibitors of improved specificity allowed us to address the roles of these protein kinases in controlling mTORC1 in a variety of human and rodent cell types. In parallel, we used specific shRNAs against p90(RSK1) and p90(RSK2) to further test their roles in regulating mTORC1 signaling. Our data indicate that p90(RSKs) are dispensable for the activation of mTORC1 signaling by phorbol esters in all cell types tested. Our data also reveal striking diversity in the requirements for MEK/ERK in the control of mTORC1 between different cell types, pointing to additional signaling connections between phorbol esters and mTORC1, which do not involve MEK/ERK. This study provides important information for the design of efficient strategies to combat the hyperactivation of mTORC1 signaling by oncogenic pathways.     

Downregulation of the cAMP/PKA Pathway in PC12 Cells Overexpressing NCS-1

It is well known that dopamine imbalances are associated with many psychiatric disorders and that the dopaminergic receptor D₂ is the main target of antipsychotics. Recently it was shown that levels of two proteins implicated in dopaminergic signaling, Neuronal calcium sensor-1 (NCS-1) and DARPP-32, are altered in the prefrontal cortex (PFC) of both schizophrenic and bipolar disorder patients. NCS-1, which inhibits D₂ internalization, is upregulated in the PFC of both patients. DARPP-32, which is a downstream effector of dopamine signaling, integrates the pathways of several neurotransmitters and is downregulated in the PFC of both patients. Here, we used PC12 cells stably overexpressing NCS-1 (PC12-NCS-1 cells) to address the function of this protein in DARPP-32 signaling pathway in vitro. PC12-NCS-1 cells displayed downregulation of the cAMP/PKA pathway, with decreased levels of cAMP and phosphorylation of CREB at Ser133. We also observed decreased levels of total and phosphorylated DARPP-32 at Thr34. However, these cells did not show alterations in the levels of D₂ and phosphorylation of DARPP-32 at Thr75. These results indicate that NCS-1 modulates PKA/cAMP signaling pathway. Identification of the cellular mechanisms linking NCS-1 and DARPP-32 may help in the understanding the signaling machinery with potential to be turned into targets for the treatment of schizophrenia and other debilitating psychiatric disorders.     

DARPP—32的结构,功能及其调节机制

DARPP-32是一种多巴胺（DA）和cAMP调节的磷蛋白，存在于所有接受DA能投射的神经元中，在中枢神经系统的分布与DAD1受体的分布非常一致。DA通过D1受体使DARPP-32第34位苏氨酸磷酸化，磷酸化DARPP-32成为蛋白磷酸酶1（PP-1）的强效抑制剂，在两个不同位点与PP-1相互作用，从而抑制PP-1活性。DARPP-32／PP-1级联反应在调节，如钙通道、电压依赖性钠通道、Na＋,K＋-ATPase和NMDANR1受体的功能等神经元兴奋性过程中起重要作用。DA对DARPP—32的磷酸化状态有双向调节作用，其他许多神经递质亦可调节其磷酸化状态。     

Regulation of DARPP-32 dephosphorylation at PKA- and Cdk5-sites by NMDA and AMPA receptors: distinct roles of calcineurin and protein phosphatase-2A

Glutamatergic inputs from corticostriatal and thalamostriatal pathways have been shown to modulate dopaminergic signaling in neostriatal neurons. DARPP-32 (dopamine- and cAMP-regulated phosphoprotein of M (r) 32 kDa) is a signal transduction molecule that regulates the efficacy of dopamine signaling in neostriatal neurons. Dopamine signaling is mediated in part through phosphorylation of DARPP-32 at Thr34 by cAMP-dependent protein kinase, and antagonized by phosphorylation of DARPP-32 at Thr75 by cyclin-dependent protein kinase 5. We have now investigated the effects of the ionotropic glutamate NMDA and AMPA receptors on DARPP-32 phosphorylation in neostriatal slices. Activation of NMDA and AMPA receptors decreased the state of phosphorylation of DARPP-32 at Thr34 and Thr75. The decrease in Thr34 phosphorylation was mediated through Ca(2+) -dependent activation of the Ca(2+) -/calmodulin-dependent phosphatase, calcineurin. In contrast, the decrease in Thr75 phosphorylation was mediated through Ca(2+) -dependent activation of dephosphorylation by protein phosphatase-2A. The results provide support for a complex effect of glutamate on dopaminergic signaling through the regulation of dephosphorylation of different sites of DARPP-32 by different protein phosphatases.     

Regulation of DARPP-32 phosphorylation by three distinct dopamine D1-like receptor signaling pathways in the neostriatum

Dopamine D(1)-like receptors play a key role in dopaminergic signaling. In addition to G(s/olf)/adenylyl cyclase (AC)-coupled D(1) receptors, the presence of D(1)-like receptors coupled to G(q)/phospholipase C (PLC) has been proposed. Benzazepine D(1) receptor agonists are known to differentially activate G(s/olf)/AC and G(q)/PLC signaling. By utilizing SKF83959 and SKF83822, we investigated the D(1)-like receptor signaling cascades, which regulate DARPP-32 phosphorylation at Thr34 (the PKA-site) in mouse neostriatal slices. Treatment with SKF83959 or SKF83822 increased DARPP-32 phosphorylation. The SKF83959- and SKF83822-induced increase in DARPP-32 phosphorylation was largely, but partially, antagonized by a D(1) receptor antagonist, SCH23390, and the residual SCH23390-insensitive increase was abolished by an adenosine A(2A) receptor antagonist. In addition, the SKF83959-induced, SCH23390-sensitive increase in DARPP-32 phosphorylation was enhanced by a PLC inhibitor. Analysis in slices from D(1)R/D(2)R-DARPP-32 mice revealed that both D(1) receptor agonists regulate DARPP-32 phosphorylation in striatonigral, but not in striatopallidal, neurons. Thus, dopamine D(1)-like receptors are coupled to three signaling cascades in striatonigral neurons: (i) SCH23390-sensitive G(s/olf)/AC/PKA, (ii) adenosine A(2A) receptor-dependent G(s/olf)/AC/PKA, and (iii) G(q)/PLC signaling. Interestingly, G(q)/PLC signaling interacts with SCH23390-sensitive G(s/olf)/AC/PKA signaling, resulting in its inhibition. Three signaling cascades activated by D(1)-like receptors likely play a distinct role in dopaminergic regulation of psychomotor functions.     

ERK and Akt signaling pathways function through parallel mechanisms to promote mTORC1 signaling

The mammalian target of rapamycin (mTOR) is a protein kinase that, when present in a complex referred to as mTOR complex 1 (mTORC1), acts as an important regulator of growth and metabolism. The activity of the complex is regulated through multiple upstream signaling pathways, including those involving Akt and the extracellular-regulated kinase (ERK). Previous studies have shown that, in part, Akt and ERK promote mTORC1 signaling through phosphorylation of a GTPase activator protein (GAP), referred to as tuberous sclerosis complex 2 (TSC2), that acts as an upstream inhibitor of mTORC1. In the present study we extend the earlier studies to show that activation of the Akt and ERK pathways acts in a synergistic manner to promote mTORC1 signaling. Moreover, we provide evidence that the Akt and ERK signaling pathways converge on TSC2, and that Akt phosphorylates residues on TSC2 distinct from those phosphorylated by ERK. The results also suggest that leucine-induced stimulation of mTORC1 signaling occurs through a mechanism distinct from TSC2 and the Akt and ERK signaling pathways. Overall, the results are consistent with a model in which Akt and ERK phosphorylate distinct sites on TSC2, leading to greater repression of its GAP activity, and consequently a magnified stimulation of mTORC1 signaling, when compared with either input alone. The results further suggest that leucine acts through a mechanism distinct from TSC2 to stimulate mTORC1 signaling.     

左旋千金藤啶碱D1激动作用增加6-OHDA损毁大鼠的DARPP-32磷酸化效应

为了进一步阐明SPD对大鼠纹状体突触后D1受体的激动作用特性,本文应用反磷酸化在体内测定及放射配体结合方法,分别观察SPD对6-OHDA损毁大鼠纹状体DARPP-32体内磷酸化作用及突触后D1受体密度的影响.结果表明:皮下给予SPD(20,40 mg/kg,21 d),损毁侧纹状体DARPP-32体外[32P]的掺入量较健侧下降50%(P<0.01).换言之,损毁侧纹状体内DARPP-32的磷酸化程度增加了.然而,SPD使损毁导致D1受体上调的作用减弱(Bmax 从385.0±26.1 fmol/mg 降至319.7±20.1 fmol/mg水平).因此,SPD激动D1受体,使6-OHDA损毁大鼠纹状体内DARPP-32磷酸化作用加强,而受体密度减少.这是SPD调节脑内D1受体信号转导功能的重要机制.     

Segregation and Crosstalk of D1 Receptor-Mediated Activation of ERK in Striatal Medium Spiny Neurons upon Acute Administration of Psychostimulants

The convergence of corticostriatal glutamate and dopamine from the midbrain in the striatal medium spiny neurons (MSN) triggers synaptic plasticity that underlies reinforcement learning and pathological conditions such as psychostimulant addiction. The increase in striatal dopamine produced by the acute administration of psychostimulants has been found to activate not only effectors of the AC5/cAMP/PKA signaling cascade such as GluR1, but also effectors of the NMDAR/Ca²⁺/RAS cascade such as ERK. The dopamine-triggered effects on both these cascades are mediated by D1R coupled to Golf but while the phosphorylation of GluR1 is affected by reductions in the available amount of Golf but not of D1R, the activation of ERK follows the opposite pattern. This segregation is puzzling considering that D1R-induced Golf activation monotonically increases with DA and that there is crosstalk from the AC5/cAMP/PKA cascade to the NMDAR/Ca²⁺/RAS cascade via a STEP (a tyrosine phosphatase). In this work, we developed a signaling model which accounts for this segregation based on the assumption that a common pool of D1R and Golf is distributed in two D1R/Golf signaling compartments. This model integrates a relatively large amount of experimental data for neurons in vivo and in vitro. We used it to explore the crosstalk topologies under which the sensitivities of the AC5/cAMP/PKA signaling cascade to reductions in D1R or Golf are transferred or not to the activation of ERK. We found that the sequestration of STEP by its substrate ERK together with the insensitivity of STEP activity on targets upstream of ERK (i.e. Fyn and NR2B) to PKA phosphorylation are able to explain the experimentally observed segregation. This model provides a quantitative framework for simulation based experiments to study signaling required for long term potentiation in MSNs.     

Phosphorylation of DARPP-32, a dopamine- and cAMP-regulated phosphoprotein, by casein kinase II

DARPP-32 (dopamine- and cAMP-regulated phosphorprotein, Mr = 32,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) is an inhibitor of protein phosphatase-1 and is enriched in dopaminoceptive neurons possessing the D1 dopamine receptor. Purified bovine DARPP-32 was phosphorylated in vitro by casein kinase II to a stoichiometry greater than 2 mol of phosphate/mol of protein whereas two structurally and functionally related proteins, protein phosphatase inhibitor-1 and G-substrate, were poor substrates for this enzyme. Sequencing of chymotryptic and thermolytic phosphopeptides from bovine DARPP-32 phosphorylated by casein kinase II suggested that the main phosphorylated residues were Ser45 and Ser102. In the case of rat DARPP-32, the identification of these phosphorylation sites was confirmed by manual Edman degradation. The phosphorylated residues are located NH2-terminal to acidic amino acid residues, a characteristic of casein kinase II phosphorylation sites. Casein kinase II phosphorylated DARPP-32 with an apparent Km value of 3.4 microM and a kcat value of 0.32 s-1. The kcat value for phosphorylation of Ser102 was 5-6 times greater than that for Ser45. Studies employing synthetic peptides encompassing each phosphorylation site confirmed this difference between the kcat values for phosphorylation of the two sites. In slices of rat caudate-putamen prelabeled with [32P]phosphate, DARPP-32 was phosphorylated on seryl residues under basal conditions. Comparison of thermolytic phosphopeptide maps and determination of the phosphorylated residue by manual Edman degradation identified the main phosphorylation site in intact cells as Ser102. In vitro, DARPP-32 phosphorylated by casein kinase II was dephosphorylated by protein phosphatases-1 and -2A. Phosphorylation by casein kinase II did not affect the potency of DARPP-32 as an inhibitor of protein phosphatase-1, which depended only on phosphorylation of Thr34 by cAMP-dependent protein kinase. However, phosphorylation of DARPP-32 by casein kinase II facilitated phosphorylation of Thr34 by cAMP-dependent protein kinase with a 2.2-fold increase in the Vmax and a 1.4-fold increase in the apparent Km. Phosphorylation of DARPP-32 by casein kinase II in intact cells may therefore modulate its phosphorylation in response to increased levels of cAMP.     

Dopamine D(1) and D(3) receptors oppositely regulate NMDA- and cocaine-induced MAPK signaling via NMDA receptor phosphorylation

Development of drug addiction involves complex molecular changes in the CNS. The mitogen-activated protein kinase (MAPK) signaling pathway plays a key role in mediating neuronal activation induced by dopamine, glutamate, and drugs of abuse. We previously showed that dopamine D(1) and D(3) receptors play different roles in regulating cocaine-induced MAPK activation. Although there are functional and physical interactions between dopamine and glutamate receptors, little is known regarding the involvement of D(1) and D(3) receptors in modulating glutamate-induced MAPK activation and underlying mechanisms. In this study, we show that D(1) and D(3) receptors play opposite roles in regulating N-methyl-d-aspartate (NMDA) -induced activation of extracellular signal-regulated kinase (ERK) in the caudate putamen (CPu). D(3) receptors also inhibit NMDA-induced activation of the c-Jun N-terminal kinase and p38 kinase in the CPu. NMDA-induced activation of the NMDA-receptor R1 subunit (NR1), Ca(2+)/calmodulin-dependent protein kinase II and the cAMP-response element binding protein (CREB), and cocaine-induced CREB activation in the CPu are also oppositely regulated by dopamine D(1) and D(3) receptors. Finally, the blockade of NMDA-receptor reduces cocaine-induced ERK activation, and inhibits phosphorylation of NR1, Ca(2+)/calmodulin-dependent protein kinase II, and CREB, while inhibiting ERK activation attenuates cocaine-induced CREB phosphorylation in the CPu. These results suggest that dopamine D(1) and D(3) receptors oppositely regulate NMDA- and cocaine-induced MAPK signaling via phosphorylation of NR1.     

Glucagon transiently stimulates mTORC1 by activation of an EPAC/Rap1 signaling axis

Activation of the protein kinase mechanistic target of rapamycin (mTOR) in both complexes 1 and 2 (mTORC1/2) in the liver is repressed during fasting and rapidly stimulated in response to a meal. The effect of feeding on hepatic mTORC1/2 is attributed to an increase in plasma levels of nutrients, such as amino acids, and insulin. By contrast, fasting is associated with elevated plasma levels of glucagon, which is conventionally viewed as having a counter-regulatory role to insulin. More recently an expanded role for glucagon action in post-prandial metabolism has been demonstrated. Herein we investigated the impact of insulin and glucagon on mTORC1/2 activation. In H4IIE and HepG2 cultures, insulin enhanced phosphorylation of the mTORC1 substrates S6K1 and 4E-BP1. Surprisingly, the effect of glucagon on mTORC1 was biphasic, wherein there was an acute increase in phosphorylation of S6K1 and 4E-BP1 over the first hour of exposure, followed by latent suppression. The transient stimulatory effect of glucagon on mTORC1 was not additive with insulin, suggesting convergent signaling. Glucagon enhanced cAMP levels and mTORC1 stimulation required activation of the glucagon receptor, PI3K/Akt, and exchange protein activated by cAMP (EPAC). EPAC acts as the guanine nucleotide exchange factor for the small GTPase Rap1. Rap1 expression enhanced S6K1 phosphorylation and glucagon addition to culture medium promoted Rap1-GTP loading. Signaling through mTORC1 acts to regulate protein synthesis and we found that glucagon promoted an EPAC-dependent increase in protein synthesis. Overall, the findings support that glucagon elicits acute activation of mTORC1/2 by an EPAC-dependent increase in Rap1-GTP.