Amplification of dopaminergic signaling by a positive feedback loop

Dopamine and cAMP-regulated phosphoprotein of M(r) 32,000 (DARPP-32) plays an obligatory role in most of the actions of dopamine. In resting neostriatal slices, cyclin-dependent kinase 5 (Cdk5) phosphorylates DARPP-32 at Thr-75, thereby reducing the efficacy of dopaminergic signaling. We report here that dopamine, in slices, and acute cocaine, in whole animals, decreases the state of phosphorylation of striatal DARPP-32 at Thr-75 and thereby removes this inhibitory constraint. This effect of dopamine is achieved through dopamine D1 receptor-mediated activation of cAMP-dependent protein kinase (PKA). The activated PKA, by decreasing the state of phosphorylation of DARPP-32-Thr-75, de-inhibits itself. Dopamine D2 receptor stimulation has the opposite effect. The ability of activated PKA to reduce the state of phosphorylation of DARPP-32-Thr-75 is apparently attributable to increased protein phosphatase-2A activity, with Cdk5 being unaffected. Together, these results indicate that via positive feedback mechanisms, Cdk5 signaling and PKA signaling are mutually antagonistic.     

Metabotropic mGlu5 receptors regulate adenosine A2A receptor signaling

Dopamine, by activating dopamine D1-type receptors, and adenosine, by activating adenosine A(2A) receptors, stimulate phosphorylation of DARPP-32 (dopamine- and cAMP-regulated phosphoprotein of M(r) 32,000) at Thr-34. In this study, we investigated the effect of metabotropic glutamate (mGlu) receptors on DARPP-32 phosphorylation at Thr-34 in neostriatal slices. A broad-spectrum mGlu receptor agonist, trans-ACPD, and a group I mGlu receptor agonist, DHPG, stimulated DARPP-32 phosphorylation at Thr-34. Studies with mGlu receptor antagonists revealed that the effects of trans-ACPD and DHPG were mediated through activation of mGlu5 receptors. The action of mGlu5 receptors required activation of adenosine A(2A) receptors by endogenous adenosine. Conversely, the action of adenosine A(2A) receptors required activation of mGlu5 receptors by endogenous glutamate. Coactivation of mGlu5 and adenosine A(2A) receptors by exogenous agonists synergistically increased DARPP-32 phosphorylation. mGlu5 receptors did not require activation of dopamine D1-type receptors by endogenous dopamine, nor did dopamine D1-type receptors require activation of mGlu5 receptors by endogenous glutamate. DHPG potentiated the effect of forskolin, but not that of 8-bromo-cAMP, and stimulated DARPP-32 phosphorylation in the presence of the phosphodiesterase inhibitor IBMX, suggesting that mGlu5 receptors stimulate the rate of cAMP formation coupled to adenosine A(2A) receptors. The action of mGlu5 receptors was attenuated by inhibitors of extracellular signal-regulated kinase, but not by inhibitors of phospholipase C, p38, casein kinase 1, or Cdk5. The results demonstrate that mGlu5 receptors potentiate adenosine A(2A)DARPP-32 signaling by stimulating the adenosine A(2A) receptor-mediated formation of cAMP in an extracellular signal-regulated kinase-dependent manner.     

Regulation of cyclin-dependent kinase 5 and casein kinase 1 by metabotropic glutamate receptors

Cyclin-dependent kinase 5 (Cdk5) is a multifunctional neuronal protein kinase that is required for neurite outgrowth and cortical lamination and that plays an important role in dopaminergic signaling in the neostriatum through phosphorylation of Thr-75 of DARPP-32 (dopamine and cAMP-regulated phosphoprotein, molecular mass 32 kDa). Casein kinase 1 (CK1) has been implicated in a variety of cellular functions such as DNA repair, circadian rhythm, and intracellular trafficking. In the neostriatum, CK1 has been found to phosphorylate Ser-137 of DARPP-32. However, first messengers for the regulation of Cdk5 or CK1 have remained unknown. Here we report that both Cdk5 and CK1 are regulated by metabotropic glutamate receptors (mGluRs) in neostriatal neurons. (S)-3,5-dihydroxyphenylglycine (DHPG), an agonist for group I mGluRs, increased Cdk5 and CK1 activities in neostriatal slices, leading to the enhanced phosphorylation of Thr-75 and Ser-137 of DARPP-32, respectively. The effect of DHPG on Thr-75, but not on Ser-137, was blocked by a Cdk5-specific inhibitor, butyrolactone. In contrast, the effects of DHPG on both Thr-75 and Ser-137 were blocked by CK1-7 and IC261, specific inhibitors of CK1, suggesting that activation of Cdk5 by mGluRs requires CK1 activity. In support of this possibility, the DHPG-induced increase in Cdk5 activity, measured in extracts of neostriatal slices, was abolished by CK1-7 and IC261. Treatment of acutely dissociated neurons with DHPG enhanced voltage-dependent Ca(2+) currents. This enhancement was eliminated by either butyrolactone or CK1-7 and was absent in DARPP-32 knockout mice. Together these results indicate that a CK1-Cdk5-DARPP-32 cascade may be involved in the regulation by mGluR agonists of Ca(2+) channels.     

Regulation of a protein phosphatase cascade allows convergent dopamine and glutamate signals to activate ERK in the striatum

Many drugs of abuse exert their addictive effects by increasing extracellular dopamine in the nucleus accumbens, where they likely alter the plasticity of corticostriatal glutamatergic transmission. This mechanism implies key molecular alterations in neurons in which both dopamine and glutamate inputs are activated. Extracellular signal-regulated kinase (ERK), an enzyme important for long-term synaptic plasticity, is a good candidate for playing such a role. Here, we show in mouse that d-amphetamine activates ERK in a subset of medium-size spiny neurons of the dorsal striatum and nucleus accumbens, through the combined action of glutamate NMDA and D1-dopamine receptors. Activation of ERK by d-amphetamine or by widely abused drugs, including cocaine, nicotine, morphine, and Delta(9)-tetrahydrocannabinol was absent in mice lacking dopamine- and cAMP-regulated phosphoprotein of M(r) 32,000 (DARPP-32). The effects of d-amphetamine or cocaine on ERK activation in the striatum, but not in the prefrontal cortex, were prevented by point mutation of Thr-34, a DARPP-32 residue specifically involved in protein phosphatase-1 inhibition. Regulation by DARPP-32 occurred both upstream of ERK and at the level of striatal-enriched tyrosine phosphatase (STEP). Blockade of the ERK pathway or mutation of DARPP-32 altered locomotor sensitization induced by a single injection of psychostimulants, demonstrating the functional relevance of this regulation. Thus, activation of ERK, by a multilevel protein phosphatase-controlled mechanism, functions as a detector of coincidence of dopamine and glutamate signals converging on medium-size striatal neurons and is critical for long-lasting effects of drugs of abuse.     

Glutamate regulation of DARPP-32 phosphorylation in neostriatal neurons involves activation of multiple signaling cascades

Dopamine- and cAMP-regulated phosphoprotein of 32 kDa (DARPP-32) plays a central role in medium spiny neurons in the neostriatum in the integration of various neurotransmitter signaling pathways. In its Thr-34-phosphorylated form, it acts as a potent protein phosphatase-1 inhibitor, and, in its Thr-75-phosphorylated form, it acts as a cAMP-dependent kinase inhibitor. Here, we investigated glutamate-dependent signaling cascades in mouse neostriatal slices by analyzing the phosphorylation of DARPP-32 at Thr-34 and Thr-75. Treatment with glutamate (5 mM) caused a complex change in DARPP-32 Thr-34 phosphorylation. An initial rapid increase in Thr-34 phosphorylation was NMDA/alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/metabotropic glutamate-5 receptor-dependent and was mediated through activation of a neuronal nitric oxide synthase/nitric oxide/cGMP/cGMP-dependent kinase signaling cascade. A subsequent decrease in phosphorylation was attributable to activation of an NMDA/AMPA receptor/Ca2+/protein phosphatase-2B signaling cascade. This decrease was followed by rephosphorylation via a pathway involving metabotropic glutamate-5 receptor/phospholipase C and extracellular receptor kinase signaling cascade. Treatment with glutamate initially decreased Thr-75 phosphorylation through activation of NMDA/AMPA receptor/Ca2+/protein phosphatase-2A signaling. Thereafter, glutamate slowly increased Thr-75 phosphorylation through activation of metabotropic glutamate-1 receptor/phospholipase C signaling. Our analysis of DARPP-32 phosphorylation in the neostriatum revealed that glutamate activates at least five different signaling cascades with different time dependencies, resulting in complex regulation of protein kinase and protein phosphatase activities.     

Striatal dysregulation of Cdk5 alters locomotor responses to cocaine, motor learning, and dendritic morphology

Motor learning and neuro-adaptations to drugs of abuse rely upon neuronal signaling in the striatum. Cyclin-dependent kinase 5 (Cdk5) regulates striatal dopamine neurotransmission and behavioral responses to cocaine. Although the role for Cdk5 in neurodegeneration in the cortex and hippocampus and in hippocampal-dependent learning has been demonstrated, its dysregulation in the striatum has not been examined. Here we show that strong activation of striatal NMDA receptors produced p25, the truncated form of the Cdk5 co-activator p35. Furthermore, inducible overexpression of p25 in the striatum prevented locomotor sensitization to cocaine and attenuated motor coordination and learning. This corresponded with reduced dendritic spine density, increased neuro-inflammation, altered dopamine signaling, and shifted Cdk5 specificity with regard to physiological and aberrant substrates, but no apparent loss of striatal neurons. Thus, dysregulation of Cdk5 dramatically affects striatal-dependent brain function and may be relevant to non-neurodegenerative disorders involving dopamine neurotransmission.     

The B'/PR72 subunit mediates Ca2+-dependent dephosphorylation of DARPP-32 by protein phosphatase 2A

In dopaminoceptive neurons, dopamine- and cAMP-regulated phosphoprotein of 32 kDa (DARPP-32) plays a central role in integrating the effects of dopamine and other neurotransmitters. Phosphorylation of DARPP-32 at Thr-34 by protein kinase A results in inhibition of protein phosphatase 1 (PP1), and phosphorylation at Thr-75 by Cdk5 (cyclin-dependent kinase 5) results in inhibition of protein kinase A. Dephosphorylation at Thr-34 involves primarily the Ca(2+)-dependent protein phosphatase, PP2B (calcineurin), whereas dephosphorylation of Thr-75 involves primarily PP2A, the latter being subject to control by both cAMP- and Ca(2+)-dependent regulatory mechanisms. In the present study, we have investigated the mechanism of Ca(2+)-dependent regulation of Thr-75 by PP2A. We show that the PR72 (or B' or PPP2R3A) regulatory subunit of PP2A is highly expressed in striatum. Through the use of overexpression and down-regulation by using RNAi, we show that PP2A, in a heterotrimeric complex with the PR72 subunit, mediates Ca(2+)-dependent dephosphorylation at Thr-75 of DARPP-32. The PR72 subunit contains two Ca(2+) binding sites formed by E and F helices (EF-hands 1 and 2), and we show that the former is necessary for the ability of PP2A activity to be regulated by Ca(2+), both in vitro and in vivo. Our studies also indicate that the PR72-containing form of PP2A is necessary for the ability of glutamate acting at alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid and NMDA receptors to regulate Thr-75 dephosphorylation. These studies further our understanding of the complex signal transduction pathways that regulate DARPP-32. In addition, our studies reveal an alternative intracellular mechanism whereby Ca(2+) can activate serine/threonine phosphatase activity.     

Cyclin-dependent kinase 5 regulates dopaminergic and glutamatergic transmission in the striatum

Dopaminergic and glutamatergic neurotransmissions in the striatum play an essential role in motor- and reward-related behaviors. Dysfunction of these neurotransmitter systems has been found in Parkinson's disease, schizophrenia, and drug addiction. Cyclin-dependent kinase 5 (CDK5) negatively regulates postsynaptic signaling of dopamine in the striatum. This kinase also reduces the behavioral effects of cocaine. Here we demonstrate that, in addition to a postsynaptic role, CDK5 negatively regulates dopamine release in the striatum. Inhibitors of CDK5 increase evoked dopamine release in a way that is additive to that of cocaine. This presynaptic action of CDK5 also regulates glutamatergic transmission. Indeed, inhibition of CDK5 increases the activity and phosphorylation of N-methyl-d-aspartate receptors, and these effects are reduced by a dopamine D1 receptor antagonist. Using mice with a point mutation of the CDK5 site of the postsynaptic protein DARPP-32 (dopamine- and cAMP-regulated phosphoprotein, molecular mass of 32 kDa), in the absence or in the presence of a dopamine D1 receptor antagonist, we provide evidence that CDK5 inhibitors potentiate dopaminergic transmission at both presynaptic and postsynaptic locations. These findings, together with the known ability of CDK5 inhibitors to prevent degeneration of dopaminergic neurons, suggest that this class of compounds could potentially be used as a novel treatment for disorders associated with dopamine deficiency, such as Parkinson's disease.     

Distinct roles of dopamine D2L and D2S receptor isoforms in the regulation of protein phosphorylation at presynaptic and postsynaptic sites

Dopamine D2 receptors are highly expressed in the dorsal striatum where they participate in the regulation of (i) tyrosine hydroxylase (TH), in nigrostriatal nerve terminals, and (ii) the dopamine- and cAMP-regulated phosphoprotein of 32 kDa (DARPP-32), in medium spiny neurons. Two isoforms of the D2 receptor are generated by differential splicing of the same gene and are referred to as short (D2S) and long (D2L) dopamine receptors. Here we have used wild-type mice, dopamine D2 receptor knockout mice (D2 KO mice; lacking both D2S and D2L receptors) and D2L receptor-selective knockout mice (D2L KO mice) to evaluate the involvement of each isoform in the regulation of the phosphorylation of TH and DARPP-32. Incubation of striatal slices from wild-type mice with quinpirole, a dopamine D2 receptor agonist, decreased the state of phosphorylation of TH at Ser-40 and its enzymatic activity. Both effects were abolished in D2 KO mice but were still present in D2L KO mice. In wild-type mice, quinpirole inhibits the increase in DARPP-32 phosphorylation at Thr-34 induced by SKF81297, a dopamine D1 receptor agonist. This effect is absent in D2 KO as well as D2L KO mice. The inability of quinpirole to regulate DARPP-32 phosphorylation in D2L KO mice cannot be attributed to decreased coupling of D2S receptors to G proteins, because quinpirole produces a similar stimulation of [(35)S]GTPgammaS binding in wild-type and D2L KO mice. These results demonstrate that D2S and D2L receptors participate in presynaptic and postsynaptic dopaminergic transmission, respectively.     

Effect of ethanol on DARPP-32 phosphorylation in transgenic mice that express human type VII adenylyl cyclase in brain

BACKGROUND: Dopamine and cyclic adenosine monophosphate-regulated phosphoprotein of molecular weight 32 kDa (DARPP-32) is a bidirectional signaling protein found in dopaminergically innervated brain areas. The characteristics and direction of DARPP-32 effects are regulated by phosphorylation of this protein. Phosphorylation of DARPP-32 on threonine-34 (T34) is regulated through the activation of dopamine (D1) receptors and stimulation of adenylyl cyclase (AC) and protein kinase A activity and by calcineurin. Phosphorylation of DARPP-32 on threonine-75 (T75) is regulated by cyclin-dependent kinase 5 and protein phosphatase 2A. DARPP-32 has been implicated in the motivational effects of ethanol. METHODS: The authors characterized transgenic mice that overexpress an ethanol-sensitive isoform of AC (AC7) in brain by measuring basal and ethanol-modulated DARPP-32 phosphorylation. Phosphorylated and total DARPP-32 were measured by immunoblotting in brain areas associated with the motivational and anxiolytic effects of ethanol (nucleus accumbens, striatum, and amygdala). RESULTS: AC7 transgenic mice had higher basal levels of T34 DARPP-32 than wild-type mice in striatum and amygdala, whereas basal levels of T75 DARPP-32 did not differ between wild-type and transgenic mice. Ethanol administration increased T34 DARPP-32 in nucleus accumbens and amygdala (but not in the striatum) of wild-type and transgenic mice (with a greater effect in amygdala of transgenic mice than wild-type mice). Ethanol administration increased T75 DARPP-32 in amygdala of only the wild-type mice and in nucleus accumbens and striatum of both the transgenic and wild-type mice. CONCLUSIONS: The effect of ethanol on the balance of DARPP-32 phosphorylation, especially in amygdala of wild-type versus transgenic mice, may contribute to differential motivational effects of ethanol in these animals.     

Protein kinase A activates protein phosphatase 2A by phosphorylation of the B56delta subunit

Our previous studies of DARPP-32 in striatal slices have shown that activation of D1 receptors leads to cAMP-dependent dephosphorylation of Thr-75, the Cdk5 site in DARPP-32. In the current study, we have elucidated a mechanism whereby protein phosphatase 2A (PP2A) is activated by a cAMP/PKA-dependent pathway, leading to dephosphorylation of Thr-75. PP2A consists of a catalytic C subunit that associates with the scaffolding A subunit and a variety of B subunits. We have found that the A/C subunits of PP2A, in association with the B56delta (or PPP2R5D) regulatory subunit, is an active DARPP-32 phosphatase. The B56delta subunit expressed in HEK293 cells forms a heterotrimeric assembly that catalyzes PKA-mediated dephosphorylation at Thr-75 in DARPP-32 (also cotransfected into HEK293 cells). The B56delta subunit is phosphorylated by PKA, and this increases the overall activity of PP2A in vitro and in vivo. Among four PKA-phosphorylation sites identified in B56delta in vitro, Ser-566 was found to be critical for the regulation of PP2A activity. Moreover, Ser-566 was phosphorylated by PKA in response to activation of D1 receptors in striatal slices. Based on these studies, we propose that the B56delta/A/C PP2A complex regulates the dephosphorylation of DARPP-32 at Thr-75, thereby helping coordinate the efficacy of dopaminergic neurotransmission in striatal neurons. Moreover, stimulation of protein phosphatase activity by this mechanism may represent an important signaling pathway regulated by cAMP in neurons and other types of cell.     

Cyclin-dependent kinase 5 modulates nociceptive signaling through direct phosphorylation of transient receptor potential vanilloid 1

Transient receptor potential vanilloid 1 (TRPV1), a ligand-gated cation channel highly expressed in small-diameter sensory neurons, is activated by heat, protons, and capsaicin. The phosphorylation of TRPV1 provides a versatile regulation of intracellular calcium levels and is critical for TRPV1 function in responding to a pain stimulus. We have previously reported that cyclin-dependent kinase 5 (Cdk5) activity regulates nociceptive signaling. In this article we report that the Cdk5-mediated phosphorylation of TRPV1 at threonine-407 can modulate agonist-induced calcium influx. Inhibition of Cdk5 activity in cultured dorsal root ganglia neurons resulted in a significant reduction of TRPV1-mediated calcium influx, and this effect could be reversed by restoring Cdk5 activity. Primary nociceptor-specific Cdk5 conditional-knockout mice showed reduced TRPV1 phosphorylation, resulting in significant hypoalgesia. Thus, the present study indicates that Cdk5-mediated TRPV1 phosphorylation is important in the regulation of pain signaling.     

Cyclin-dependent kinase 5 activity regulates pain signaling

Several molecules and cellular pathways have been implicated in nociceptive signaling, but their precise molecular mechanisms have not been clearly defined. Cyclin-dependent kinase 5 (Cdk5) is a proline-directed serine/threonine kinase implicated in the development and disease of the mammalian nervous system. The precise role of this kinase in sensory pathways has not been well characterized. Here we report a molecular role for Cdk5 in nociception. We identified the expression of Cdk5 and its activator p35 in nociceptive neurons, which is modulated during a peripheral inflammatory response. Increased calpain activity in sensory neurons after inflammation resulted in the cleavage of p35 to p25, which forms a more stable complex with Cdk5 and, consequently, leads to elevation of Cdk5 activity. p35 knockout mice (p35(-/-)), which exhibit significantly decreased Cdk5 activity, showed delayed responses to painful thermal stimulation compared with WT controls. In contrast, mice overexpressing p35, which exhibit elevated levels of Cdk5 activity, were more sensitive to painful thermal stimuli than were controls. In conclusion, our data demonstrate a role for Cdk5/p35 activity in primary afferent nociceptive signaling, suggesting that Cdk5/p35 may be a target for the development of analgesic drugs.     

Involvement of striatal and extrastriatal DARPP-32 in biochemical and behavioral effects of fluoxetine (Prozac)

Fluoxetine (Prozac) is the most widely prescribed medication for the treatment of depression. Nevertheless, little is known about the molecular basis of its clinical efficacy, apart from the fact that fluoxetine increases the synaptic availability of serotonin. Here we show that, in vivo, fluoxetine, given either acutely or chronically, regulates the phosphorylation state of dopamine- and cAMP-regulated phosphoprotein of M(r) 32,000 (DARPP-32) at multiple sites in prefrontal cortex, hippocampus, and striatum. Acute administration of fluoxetine increases phosphorylation of DARPP-32 at the protein kinase A site, Thr-34, and at the casein kinase-1 site, Ser-137, and decreases phosphorylation at the cyclin-dependent kinase 5 site, Thr-75. Each of these changes contributes, through distinct signaling pathways, to increased inhibition of protein phosphatase-1, a major serine/threonine protein phosphatase in the brain. Fluoxetine also increases phosphorylation of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunit GluR1 at Ser-831 and Ser-845. Both the fluoxetine-mediated increase in AMPA receptor phosphorylation at Ser-845-GluR1 and the beneficial responsiveness to fluoxetine in an animal test of antidepressant efficacy were strongly reduced in DARPP-32 knockout mice, indicating a critical role for this phosphoprotein in the antidepressant actions of fluoxetine. Mice chronically treated with fluoxetine had increased levels of DARPP-32 mRNA and protein and a decreased ability to increase phospho-Ser-137-DARPP-32 and phospho-Ser-831-GluR1. These chronic changes may be relevant to the delayed onset of therapeutic efficacy of fluoxetine.     

Antagonistic effects of dopaminergic signaling and ethanol on protein kinase A-mediated phosphorylation of DARPP-32 and the NR1 subunit of the NMDA receptor

BACKGROUND: This article extends our initial investigation of the interactions between dopamine and glutamate receptor systems after acute exposure to ethanol. DARPP-32 (dopamine and cyclic adenosine monophosphate-regulated phosphoprotein of approximate molecular weight 32 kDa) is an important regulator of protein phosphatase-1 that in turn regulates a large number of effectors, including the NMDA receptor. METHODS: We measured the protein kinase A (PKA)-mediated phosphorylation of DARPP-32 and the NR1 subunit of the NMDA receptor. Initially, corpus striatum was assayed after intraperitoneal treatment of mice with the D1 agonist SKF82958, the D2 agonist and anticraving drug bromocriptine, or ethanol. In other experiments we blocked D1 receptors with the selective D1 antagonist SCH23390 or blocked D2 receptors with the selective D2 antagonist eticlopride. Finally, we examined combinations of some dopaminergic drugs with and without ethanol. RESULTS: SKF82958 alone significantly increased PKA-mediated phosphorylation of both DARPP-32 and NR1. Bromocriptine alone had no effect on pDARPP-32 or on pNR1. However, when D1 receptors were blocked, bromocriptine reduced the PKA-mediated phosphorylation of both DARPP-32 and NR1. Coincident treatment with bromocriptine and ethanol reversed both of these effects with D1 receptors blocked. The combination of eticlopride (D2 blocker) and SF82958 (D1 agonist) did not significantly alter either pDARPP-32 or pNR1. CONCLUSIONS: These data demonstrate antagonistic effects of acute ethanol exposure on D1 signaling in vivo and the potential of acute in vivo challenge protocols to help fill gaps in the understanding of ethanol's effects on protein phosphorylation.     

Dopamine- and cAMP-regulated phosphoprotein DARPP-32: phosphorylation of Ser-137 by casein kinase I inhibits dephosphorylation of Thr-34 by calcineurin.

Although protein phosphatases appear to be highly controlled in intact cells, relatively little is known about the physiological regulation of their activity. DARPP-32, a dopamine- and cAMP-regulated phosphoprotein of apparent M(r) 32,000, is phosphorylated in vitro by casein kinase I, casein kinase II, and cAMP-dependent protein kinase on sites phosphorylated in vivo. DARPP-32 phosphorylated on Thr-34 by cAMP-dependent protein kinase is a potent inhibitor of protein phosphatase 1 and an excellent substrate for calcineurin, a Ca2+/calmodulin-dependent protein phosphatase. Here we provide evidence, using both purified proteins and brain slices, that phosphorylation of DARPP-32 on Ser-137 by casein kinase I inhibits the dephosphorylation of Thr-34 by calcineurin. This inhibition occurs only when phospho-Ser-137 and phospho-Thr-34 are located on the same DARPP-32 molecule and is not dependent on the mode of activation of calcineurin. The results demonstrate that the inhibition is due to a modification in the properties of the substrate which alters its dephosphorylation rate. Thus, casein kinase I may play a physiological role in striatonigral neurons as a modulator of the regulation of protein phosphatase 1 via DARPP-32.     

Nitric oxide/cGMP pathway stimulates phosphorylation of DARPP-32, a dopamine- and cAMP-regulated phosphoprotein, in the substantia nigra.

DARPP-32, a dopamine- and cAMP-regulated phosphoprotein of M(r) 32,000, has been shown to be phosphorylated on threonine-34, both in vitro with high efficiency by cAMP-dependent and cGMP-dependent protein kinases and in vivo by dopamine acting through cAMP-dependent protein kinase. In the present study, we investigated the nitric oxide (NO)/cGMP pathway for its ability to regulate the state of phosphorylation of DARPP-32 in slices of rat substantia nigra. DARPP-32 was phosphorylated on threonine-34 in these slices by sodium nitroprusside (SNP), an NO donor. The effect of SNP was abolished by preincubation of the slices with hemoglobin, indicating that the effect of SNP was due to released NO. The same concentration of SNP produced a 4-fold elevation of the cGMP level but did not alter the level of cAMP. The effect of SNP on DARPP-32 phosphorylation was mimicked by low concentrations of 8-bromo-cGMP and 8-(4-chlorophenylthio)-guanosine 3',5'-cyclic monophosphate, activators of cGMP-dependent protein kinase, but not by low concentrations of 8-bromo-cAMP, an activator of cAMP-dependent protein kinase. The data indicate a physiological role for the NO/cGMP pathway in the regulation of DARPP-32 phosphorylation in nerve terminals of striatonigral neurons. The results provide further evidence that the state of phosphorylation of DARPP-32 represents an important mechanisms for integration of information arriving at striatonigral neurons via a variety of neuronal pathways.     

Regulation of the phosphorylation of the dopamine- and cAMP-regulated phosphoprotein of 32 kDa in vivo by dopamine D1, dopamine D2, and adenosine A2A receptors

Dopamine D(1), dopamine D(2), and adenosine A(2A) receptors are highly expressed in striatal medium-sized spiny neurons. We have examined, in vivo, the influence of these receptors on the state of phosphorylation of the dopamine- and cAMP-regulated phosphoprotein of 32 kDa (DARPP-32). DARPP-32 is a potent endogenous inhibitor of protein phosphatase-1, which plays an obligatory role in dopaminergic transmission. A dose-dependent increase in the state of phosphorylation of DARPP-32 occurred in mouse striatum after systemic administration of the D(2) receptor antagonist eticlopride (0.1-2.0 mg/kg). This effect was abolished in mice in which the gene coding for the adenosine A(2A) receptor was disrupted by homologous recombination. A reduction was also observed in mice that had been pretreated with the selective A(2A) receptor antagonist SCH 58261 (10 mg/kg). The eticlopride-induced increase in DARPP-32 phosphorylation was also decreased by pretreatment with the D(1) receptor antagonist SCH 23390 (0.125 and 0.25 mg/kg) and completely reversed by combined pretreatment with SCH 23390 (0.25 mg/kg) plus SCH 58261 (10 mg/kg). SCH 23390, but not SCH 58261, abolished the increase in DARPP-32 caused by cocaine (15 mg/kg). The results indicate that, in vivo, the state of phosphorylation of DARPP-32 and, by implication, the activity of protein phosphatase-1 are regulated by tonic activation of D(1), D(2), and A(2A) receptors. The results also underscore the fact that the adenosine system plays a role in the generation of responses to dopamine D(2) antagonists in vivo.     

Severe deficiencies in dopamine signaling in presymptomatic Huntington's disease mice

In Huntington's disease (HD), mutation of huntingtin causes selective neurodegeneration of dopaminoceptive striatal medium spiny neurons. Transgenic HD model mice that express a portion of the disease-causing form of human huntingtin develop a behavioral phenotype that suggests dysfunction of dopaminergic neurotransmission. Here we show that presymtomatic mice have severe deficiencies in dopamine signaling in the striatum. These include selective reductions in total levels of dopamine- and cAMP-regulated phosphoprotein, M(r) 32 kDA (DARPP-32) and other dopamine-regulated phosphoprotein markers of medium spiny neurons. HD mice also show defects in dopamine-regulated ion channels and in the D(1) dopamine/DARPP-32 signaling cascade. These presymptomatic defects may contribute to HD pathology.