Gretchen L. Snyder

Indirubins Inhibit Glycogen Synthase Kinase-3β and CDK5/P25, Two Protein Kinases Involved in Abnormal Tau Phosphorylation in Alzheimer's Disease A PROPERTY COMMON TO MOST CYCLIN-DEPENDENT KINASE INHIBITORS?

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 (IC50: 50–100 nm) inhibitors of cyclin-dependent kinases (CDKs). We report here that indirubins are also powerful inhibitors (IC50: 5–50 nm) of an evolutionarily related kinase, glycogen synthase kinase-3β (GSK-3β). Testing of a series of indoles and bis-indoles against GSK-3β, 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-3βs ATP binding pocket in a way similar to their binding to CDKs, the details of which were recently revealed by crystallographic analysis. GSK-3β, along with CDK5, is responsible for most of the abnormal hyperphosphorylation of the microtubule-binding protein tau observed in Alzheimers disease. Indirubin-3′-monoxime inhibits tau phosphorylation in vitro and in vivo at Alzheimers disease-specific sites. Indirubins may thus have important implications in the study and treatment of neurodegenerative disorders. Indirubin-3′-monoxime also inhibits the in vivophosphorylation 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-3β. To which extent these GSK-3β 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-3β to be described.

Phosphorylation of DARPP-32 by Cdk5 modulates dopamine signalling in neurons

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 (refs 2, 3). 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.

Effects of chronic exposure to cocaine are regulated by the neuronal protein Cdk5.

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 ΔFosB, 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 ΔFosB by use of DNA array analysis of striatal material from inducible transgenic mice. Overexpression of ΔFosB, 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 ΔFosB, and resulting alterations in signalling involving D1 dopamine receptors, contribute to adaptive changes in the brain related to cocaine addiction.

A Dopamine/D1 Receptor/Protein Kinase A/Dopamine- and cAMP- Regulated Phosphoprotein (M r 32 kDa)/Protein Phosphatase-1 Pathway Regulates Dephosphorylation of the NMDA Receptor

We have investigated the mechanism by which activation of dopamine (DA) receptors regulates the glutamate sensitivity of medium spiny neurons of the nucleus accumbens. Our results demonstrate that DA regulates the phosphorylation state of the NR1 subunit of NMDA-type glutamate receptors. The effect of DA was mimicked by SKF82526, a D1-type DA receptor agonist, and by forskolin, an activator of cAMP-dependent protein kinase (PKA), and was blocked by H-89, a PKA inhibitor. These data indicate that DA increases NR1 phosphorylation through a PKA-dependent pathway. DA-induced phosphorylation of NR1 was blocked in mice bearing a targeted deletion of the gene for dopamine- and cAMP-regulated phosphoprotein of Mr 32 kDa (DARPP-32), a phosphoprotein that is a potent and selective inhibitor of protein phosphatase-1, indicating that the effect of PKA is mediated, in part, by regulation of the DARPP-32/protein phosphatase-1 cascade. In support of this interpretation, NR1 phosphorylation was increased by calyculin A, a protein phosphatase-1/2A inhibitor. A model is proposed in which the ability of DA to regulate NMDA receptor sensitivity is attributable to a synergistic action involving increased phosphorylation and decreased dephosphorylation of the NR1 subunit of the NMDA receptor.

Distinct Roles of PDE4 and PDE10A in the Regulation of cAMP/PKA Signaling in the Striatum

Phosphodiesterase (PDE) is a critical regulator of cAMP/protein kinase A (PKA) signaling in cells. Multiple PDEs with different substrate specificities and subcellular localization are expressed in neurons. Dopamine plays a central role in the regulation of motor and cognitive functions. The effect of dopamine is largely mediated through the cAMP/PKA signaling cascade, and therefore controlled by PDE activity. We used in vitro and in vivo biochemical techniques to dissect the roles of PDE4 and PDE10A in dopaminergic neurotransmission in mouse striatum by monitoring the ability of selective PDE inhibitors to regulate phosphorylation of presynaptic [e.g., tyrosine hydroxylase (TH)] and postsynaptic [e.g., dopamine- and cAMP-regulated phosphoprotein of M r 32 kDa (DARPP-32)] PKA substrates. The PDE4 inhibitor, rolipram, induced a large increase in TH Ser40 phosphorylation at dopaminergic terminals that was associated with a commensurate increase in dopamine synthesis and turnover in striatum in vivo. Rolipram induced a small increase in DARPP-32 Thr34 phosphorylation preferentially in striatopallidal neurons by activating adenosine A2A receptor signaling in striatum. In contrast, the PDE10A inhibitor, papaverine, had no effect on TH phosphorylation or dopamine turnover, but instead robustly increased DARPP-32 Thr34 and GluR1 Ser845 phosphorylation in striatal neurons. Inhibition of PDE10A by papaverine activated cAMP/PKA signaling in both striatonigral and striatopallidal neurons, resulting in potentiation of dopamine D1 receptor signaling and inhibition of dopamine D2 receptor signaling. These biochemical results are supported by immunohistochemical data demonstrating differential localization of PDE10A and PDE4 in striatum. These data underscore the importance of individual brain-enriched cyclic-nucleotide PDE isoforms as therapeutic targets for neuropsychiatric and neurodegenerative disorders affecting dopamine neurotransmission.

The Dopamine/D1 Receptor Mediates the Phosphorylation and Inactivation of the Protein Tyrosine Phosphatase STEP via a PKA-Dependent Pathway

The striatal-enriched protein tyrosine phosphatase (STEP) family is expressed within dopaminoceptive neurons of the CNS and is particularly enriched within the basal ganglia and related structures. Alternative splicing produces several isoforms that are found in a number of subcellular compartments, including postsynaptic densities of medium spiny neurons. The variants include STEP61, a membrane-associated protein, and STEP46, a cytosolic protein. The C terminals of these two isoforms are identical, whereas the N-terminal domain of STEP61 contains a novel 172 amino acid sequence that includes several structural motifs not present in STEP46. Amino acid sequencing revealed a number of potential phosphorylation sites in both STEP isoforms. Therefore, we investigated the role of phosphorylation in regulating STEP activity. Both STEP61 and STEP46 are phosphorylated on seryl residues by a cAMP-dependent protein kinase (PKA)-mediated pathway in striatal homogenates. The specific residues phosphorylated in STEP61 were identified by site-directed mutagenesis and tryptic phosphopeptide mapping as Ser160 and Ser221, whereas the major site of phosphorylation in STEP46 was shown to be Ser49. Ser160 is located within the unique N terminal of STEP61. Ser221 and Ser49 are equivalent residues present in STEP61 and STEP46, respectively, and are located at the center of the kinase-interacting motif that has been implicated in protein–protein interactions. Phosphorylation at this site decreases the activity of STEP in vitro by reducing its affinity for its substrate. In vivo studies using striatal slices demonstrated that the neurotransmitter dopamine leads to the phosphorylation of STEP via activation of D1 receptors and PKA.

Regulation of phosphorylation of the GluR1 AMPA receptor by dopamine D2 receptors

In the striatum, stimulation of dopamine D2 receptors results in attenuation of glutamate responses. This effect is exerted in large part via negative regulation of AMPA glutamate receptors. Phosphorylation of the GluR1 subunit of the AMPA receptor has been proposed to play a critical role in the modulation of glutamate transmission, in striatal medium spiny neurons. Here, we have examined the effects of blockade of dopamine D2‐like receptors on the phosphorylation of GluR1 at the cAMP‐dependent protein kinase (PKA) site, Ser845, and at the protein kinase C and calcium/calmodulin‐dependent protein kinase II site, Ser831. Administration of haloperidol, an antipsychotic drug with dopamine D2 receptor antagonistic properties, increases the phosphorylation of GluR1 at Ser845, without affecting phosphorylation at Ser831. The same effect is observed using eticlopride, a selective dopamine D2 receptor antagonist. In contrast, administration of the dopamine D2‐like agonist, quinpirole, decreases GluR1 phosphorylation at Ser845. The increase in Ser845 phosphorylation produced by haloperidol is abolished in dopamine‐ and cAMP‐regulated phosphoprotein of 32 kDa (DARPP‐32) knockout mice, or in mice in which the PKA phosphorylation site on DARPP‐32 (i.e. Thr34) has been mutated (Thr34 → Ala mutant mice), and requires tonic activation of adenosine A2A receptors. These results demonstrate that dopamine D2 antagonists increase GluR1 phosphorylation at Ser845 by removing the inhibitory tone exerted by dopamine D2 receptors on the PKA/DARPP‐32 cascade.

Role of calcineurin and protein phosphatase-2A in the regulation of DARPP-32 dephosphorylation in neostriatal neurons.

Abstract: DARPP‐32, a dopamine‐ and cyclic AMP‐regulated phosphoprotein of Mr 32 kDa, is phosphorylated on Thr34 by cyclic AMP‐dependent protein kinase, resulting in its conversion to a potent inhibitor of protein phosphatase‐1 (PP‐1). Conversely, Thr34‐phosphorylated DARPP‐32 is dephosphorylated and inactivated in vitro by calcineurin and protein phosphatase‐2A (PP‐2A). We have investigated the relative contributions of these protein phosphatases to the regulation of DARPP‐32 dephosphorylation in mouse neostriatal slices. Cyclosporin A (5 μM), a calcineurin inhibitor, maximally increased the level of phosphorylated DARPP‐32 by 17 ± 2‐fold. Okadaic acid (1 μM), an inhibitor of PP‐1 and PP‐2A, had a smaller effect, increasing phospho‐DARPP‐32 by 5.1 ± 1.3‐fold. The effect of okadaic acid on DARPP‐32 phosphorylation was shown to be due to inhibition of PP‐2A activity. Incubation of slices in the presence of cyclosporin A plus either okadaic acid or calyculin A, another PP‐1/PP‐2A inhibitor, caused a synergistic increase in the level of phosphorylated DARPP‐32. The use of Ca2+‐free/EGTA medium mimicked the effects of cyclosporin A on DARPP‐32 phosphorylation, supporting the conclusion that the action of cyclosporin on DARPP‐32 phosphorylation was attributable to blockade of the Ca2+‐dependent activation of calcineurin. The results indicate that calcineurin and PP‐2A, but not PP‐1, act synergistically to maintain a low level of phosphorylated DARPP‐32 in neostriatal slices.

Phosphorylation of Protein Phosphatase Inhibitor-1 by Cdk5

Protein phosphatase inhibitor-1 is a prototypical mediator of cross-talk between protein kinases and protein phosphatases. Activation of cAMP-dependent protein kinase results in phosphorylation of inhibitor-1 at Thr-35, converting it into a potent inhibitor of protein phosphatase-1. Here we report that inhibitor-1 is phosphorylated in vitro at Ser-67 by the proline-directed kinases, Cdk1, Cdk5, and mitogen-activated protein kinase. By using phosphorylation state-specific antibodies and selective protein kinase inhibitors, Cdk5 was found to be the only kinase that phosphorylates inhibitor-1 at Ser-67 in intact striatal brain tissue. In vitro and in vivo studies indicated that phospho-Ser-67 inhibitor-1 was dephosphorylated by protein phosphatases-2A and -2B. The state of phosphorylation of inhibitor-1 at Ser-67 was dynamically regulated in striatal tissue by glutamate-dependent regulation ofN-methyl-d-aspartic acid-type channels. Phosphorylation of Ser-67 did not convert inhibitor-1 into an inhibitor of protein phosphatase-1. However, inhibitor-1 phosphorylated at Ser-67 was a less efficient substrate for cAMP-dependent protein kinase. These results demonstrate regulation of a Cdk5-dependent phosphorylation site in inhibitor-1 and suggest a role for this site in modulating the amplitude of signal transduction events that involve cAMP-dependent protein kinase activation.

Regulation of Na+, K+ ‐ATPase Isoforms in Rat Neostriatum by Dopamine and Protein Kinase C

Abstract : Our previous studies showed that dopamine inhibits Na+, K+ ‐ATPase activity in acutely dissociated neurons from striatum. In the present study, we have found that in this preparation, dopamine inhibited significantly (by ~25%) the activity of the α3 and/or α2 isoforms, but not the α1 isoform, of Na+, K+ ‐ATPase. Dopamine, via D1 receptors, activates cyclic AMP‐dependent protein kinase (PKA) in striatal neurons. Dopamine is also known to activate the calcium‐ and phospholipid‐dependent protein kinase (PKC) in a number of different cell types. The PKC activator phorbol 12,13‐dibutyrate reduced the activity of Na+, K+ ‐ATPase α3 and/or α2 isoforms (by ~30%) as well as the α1 isoform (by ~15%). However, dopamine‐mediated inhibition of Na+, K+ ‐ATPase activity was unaffected by calphostin C, a PKC inhibitor. Dopamine did not affect the phosphorylation of Na+, K+ ‐ATPase isoforms at the PKA‐dependent phosphorylation site. Phorbol ester treatment did not alter the phosphorylation of α2 or α3 isoforms of Na+, K+ ‐ATPase in neostriatal neurons but did increase the phosphorylation of the α1 isoform. Thus, in rat neostriatal neurons, treatment with either dopamine or PKC activators results in inhibition of the activity of specific (α3 and/or α2) isoforms of Na+, K+ ‐ATPase, but this is not apparently mediated through direct phosphorylation of the enzyme. In addition, PKC is unlikely to mediate inhibition of rat Na+, K+ ‐ATPase activity by dopamine in neostriatal neurons.