The physiological state of the cell is controlled by signal transduction mechanisms which regulate the balance between protein kinase and protein phosphatase activities. Here we report that a single protein can, depending on which particular amino-acid residue is phosphorylated, function either as a kinase or phosphatase inhibitor. DARPP-32 (dopamine and cyclic AMP-regulated phospho-protein, relative molecular mass 32,000) is converted into an inhibitor of protein phosphatase 1 when it is phosphorylated by protein kinase A (PKA) at threonine 34. We find that DARPP-32 is converted into an inhibitor of PKA when phosphorylated at threonine 75 by cyclin-dependent kinase 5 (Cdk5). Cdk5 phosphorylates DARPP-32 in vitro and in intact brain cells. Phospho-Thr 75 DARPP-32 inhibits PKA in vitro by a competitive mechanism. Decreasing phospho-Thr 75 DARPP-32 in striatal slices, either by a Cdk5-specific inhibitor or by using genetically altered mice, results in increased dopamine-induced phosphorylation of PKA substrates and augmented peak voltage-gated calcium currents. Thus DARPP-32 is a bifunctional signal transduction molecule which, by distinct mechanisms, controls a serine/threonine kinase and a serine/threonine phosphatase.

The bis-indole indirubin is an active ingredient of Danggui Longhui Wan, a traditional Chinese medicine recipe used in the treatment of chronic diseases such as leukemias. The antitumoral properties of indirubin appear to correlate with their antimitotic effects. Indirubins were recently described as potent (IC 50 : 50 -100 nM) inhibitors of cyclin-dependent kinases (CDKs). We report here that indirubins are also powerful inhibitors (IC 50 : 5-50 nM) of an evolutionarily related kinase, glycogen synthase kinase-3 ␤ (GSK-3 ␤). Testing of a series of indoles and bis-indoles against GSK-3 ␤, CDK1/cyclin B, and CDK5/p25 shows that only indirubins inhibit these kinases. The structure-activity relationship study also suggests that indirubins bind to GSK-3␤'s ATP binding pocket in a way similar to their binding to CDKs, the details of which were recently revealed by crystallographic analysis. GSK-3 ␤, along with CDK5, is responsible for most of the abnormal hyperphosphorylation of the microtubule-binding protein tau observed in Alzheimer's disease. Indirubin-3-monoxime inhibits tau phosphorylation in vitro and in vivo at Alzheimer's diseasespecific sites. Indirubins may thus have important implications in the study and treatment of neurodegenerative disorders. Indirubin-3-monoxime also inhibits the in vivo phosphorylation of DARPP-32 by CDK5 on Thr-75, thereby mimicking one of the effects of dopamine in the striatum. Finally, we show that many, but not all, reported CDK inhibitors are powerful inhibitors of GSK-3␤. To which extent these GSK-3␤ effects of CDK inhibitors actually contribute to their antimitotic and antitumoral properties remains to be determined. Indirubins constitute the first family of low nanomolar inhibitors of GSK-3 ␤ to be described.

Cocaine enhances dopamine-mediated neurotransmission by blocking dopamine re-uptake at axon terminals. Most dopamine-containing nerve terminals innervate medium spiny neurons in the striatum of the brain. Cocaine addiction is thought to stem, in part, from neural adaptations that act to maintain equilibrium by countering the effects of repeated drug administration. Chronic exposure to cocaine upregulates several transcription factors that alter gene expression and which could mediate such compensatory neural and behavioural changes. One such transcription factor is DeltaFosB, a protein that persists in striatum long after the end of cocaine exposure. Here we identify cyclin-dependent kinase 5 (Cdk5) as a downstream target gene of DeltaFosB by use of DNA array analysis of striatal material from inducible transgenic mice. Overexpression of DeltaFosB, or chronic cocaine administration, raised levels of Cdk5 messenger RNA, protein, and activity in the striatum. Moreover, injection of Cdk5 inhibitors into the striatum potentiated behavioural effects of repeated cocaine administration. Our results suggest that changes in Cdk5 levels mediated by DeltaFosB, and resulting alterations in signalling involving D1 dopamine receptors, contribute to adaptive changes in the brain related to cocaine addiction.

Dopamine has been shown to stimulate phosphorylation of DARPP-32, a phosphoprotein highly enriched in medium-sized spiny neurons of the neostriatum. Here, we investigated the contribution of D1-like and D2-like dopamine receptors in the regulation of DARPP-32 phosphorylation in mouse striatal slices. D1-like and D2-like receptors had opposing effects on the state of DARPP-32 phosphorylation. The D1 receptor agonist SKF82526 increased DARPP-32 phosphorylation. In contrast, the D2 receptor agonist quinpirole decreased basal as well as D1 agonist-, forskolin-, and 8-bromo-cAMP-stimulated phosphorylation of DARPP-32. The ability of quinpirole to decrease D1-stimulated DARPP-32 phosphorylation was calcium-dependent and was blocked by the calcineurin inhibitor cyclosporin A, suggesting that the D2 effect involved an increase in intracellular calcium and activation of calcineurin. In support of this interpretation, Ca 2ϩ -free/EGTA medium induced a greater than 60-fold increase in DARPP-32 phosphorylation and abolished the ability of quinpirole to dephosphorylate DARPP-32. The antipsychotic drug raclopride, a selective D2 receptor antagonist, increased phosphorylation of DARPP-32 under basal conditions and in D2 agonist-treated slices. The results of this study demonstrate that dopamine exerts a bidirectional control on the state of phosphorylation of DARPP-32.Key words: DARPP-32; dopamine; D2 receptor; phosphorylation; neostriatum; calcineurin; raclopride DARPP-32, a dopamine-and cAM P-regulated phosphoprotein of M r 32,000, is a cytosolic protein that is selectively enriched in medium-sized spiny neurons in neostriatum (Ouimet et al., 1984;Walaas and Greengard, 1984). DARPP-32 is phosphorylated by cAM P-dependent protein kinase (PK A) on a single threonine residue, thr 34 , resulting in its conversion into a potent inhibitor of protein phosphatase-1 . DARPP-32 can be dephosphorylated and inactivated in vitro by the calcium/ calmodulin-dependent protein phosphatase calcineurin (King et al., 1984). Dopamine has been shown to stimulate the phosphorylation of DARPP-32 in neostriatum by activation of a biochemical cascade involving stimulation of D1 receptors, activation of adenylyl cyclase, increased cAM P formation, and increased activity of PK A . The selective enrichment of DARPP-32 in dopaminoceptive neurons and its regulation by dopamine strongly indicate that DARPP-32, by regulating protein phosphatase-1 activity, plays a key role in mediating the effects of dopamine on these cells. The control of protein phosphatase-1 activity by DARPP-32 is likely to have a significant role in the regulation of neuronal excitability. For instance, in neostriatum, dopamine-mediated effects on the function of calcium channels (Surmeier et al., 1994), voltagedependent sodium channels (Surmeier et al., 1992;Schiffman et al., 1994), and Na ϩ ,K ϩ -ATPase (Aperia et al., 1991) are regulated directly or indirectly by protein phosphatase-1.Medium-sized spiny neurons of the neostriatum and nucleus accumbens receive dopaminergic input fro...

Dopaminergic neurons exert a major modulatory effect on the forebrain. Dopamine and adenosine 3′,5′-monophosphate–regulated phosphoprotein (32 kilodaltons) (DARPP-32), which is enriched in all neurons that receive a dopaminergic input, is converted in response to dopamine into a potent protein phosphatase inhibitor. Mice generated to contain a targeted disruption of the DARPP-32 gene showed profound deficits in their molecular, electrophysiological, and behavioral responses to dopamine, drugs of abuse, and antipsychotic medication. The results show that DARPP-32 plays a central role in regulating the efficacy of dopaminergic neurotransmission.